Senescent preosteoclast secretome promotes metabolic syndrome associated osteoarthritis through cyclooxygenase 2
- Department of Orthopaedic Surgery, Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, United States
- Department of Orthopaedic Surgery, The Third Xiangya Hospital of Central South University, China
- Division of Orthopaedics & Traumatology, Department of Orthopaedics, Southern Medical University Nanfang Hospital, China
- Musculoskeletal Radiology, Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, United States
- University of Southern California, Dornsife College of Letters, Arts and Sciences, United States
- Department of Radiology, Boston University School of Medicine, United States
- Johns Hopkins Asthma & Allergy Center, Johns Hopkins University School of Medicine, United States
Figures
Figure 1 with 4 supplements
High-fat-diet (HFD) challenge leads to rapid subchondral bone thickening before cartilage damage occurs.
Three-month-old C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for 0.5, 1, 3, or 5 months. n=6 mice per group. (A) Safranin O-fast green staining of the tibia subchondral bone medial …
Figure 1—figure supplement 1
Mice fed a high-fat diet (HFD) develop metabolic syndrome.
Three-month-old C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for 3 months. Body weight (A), Lean mass (B), Fat mass (C), Blood glucose (D), and serum MDA (E) of the mice were …
Figure 1—figure supplement 2
Coronal and transverse view of micro-computed tomography scanning of joint subchondral bone change in high-fat-diet (HFD)-challenged mice.
Three-month-old C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for 1, 3, or 5 months. n=6 mice per group. Coronal and transverse view of micro-computed tomography scanning of knee …
Figure 1—figure supplement 3
High-fat-diet (HFD) challenge leads to rapid subchondral bone thickening in female mice.
Three-month-old female C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for 1 month. n=6 mice per group. Three-dimensional micro-computed tomography (μCT) images (A) and quantitative …
Figure 1—figure supplement 4
STR/Ort mice develop dyslipidemia and subchondral bone thickness.
Two- and 4-month-old STR/Ort mice were euthanized. Body weight (A), blood triglyceride (TG) (B), and cholesterol (C) of the mice were measured. (D–E) Safranin O-fast green staining of the tibia …
Figure 2
Subchondral osteoblast and osteoclast lineage cells change rapidly in response to a high-fat-diet (HFD) challenge.
(A–E) Three-month-old C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for 0.5 month or 1 month. n=6 mice per group. Immunohistochemical staining of knee joint tissue sections with …
Figure 3 with 2 supplements
Senescent cells accumulate in subchondral bone of high-fat-diet (HFD)-challenged mice.
(A–B) Three-month-old Cdkn2atdTom mice were fed a standard chow-food diet (CHD) or HFD for different time periods as indicated, n=6 mice per group. Fluorescence images showing tdTom+ cells (red) (A) …
Figure 3—figure supplement 1
Senescent cells accumulate in subchondral bone of high-fat-diet (HFD)-challenged mice.
Three-month-old C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for 1 or 3 months. Immunofluorescence staining of HMGB1 (green) (A) and Lamin B1 (red) (C) with quantification of the …
Figure 3—figure supplement 2
Senescent cells accumulate in subchondral bone of STR/Ort mice.
STR/Ort mice were euthanized at 2 months. SA-βGal staining (A) and quantification of SA-βGal+ cells in subchondral bone marrow area (B), Scale bar, 100 μm(up), 50 μm(down). Immunohistochemical …
Figure 4
Subchondral marrow preosteoclasts exhibit senescence-like feature in high-fat-diet (HFD)-challenged mice.
Three-month-old Cdkn2atdTom mice were fed a CHD or HFD for 0.5 month or 1 month, n=6 mice per group. Immunofluorescence staining of knee joint tissue sections with antibody against VPP3. Double …
Figure 5 with 1 supplement
Deletion of Cdkn2a in preosteoclasts attenuates subchondral bone thickness and cartilage damage in high-fat-diet (HFD) mice.
(A–H) Three-month-old Tnfrsf11aCre/+; Cdkn2aflox/flox mice (p16cko) and Cdkn2aflox/flox littermates (wild-type [WT]) were fed a standard chow-food diet (CHD) or HFD for 1 months, n=6 mice per group. …
Figure 5—figure supplement 1
Deletion of Cdkn2a in RANK+ cells efficiently prevents subchondral cellular senescence.
Three-month-old Tnfrsf11aCre/+; Cdkn2aflox/flox mice (p16cko) and Cdkn2aflox/flox littermates (wild-type [WT]) were fed a standard chow-food diet (CHD) or HFD for 1 months, n=6 mice per group. …
Figure 6 with 4 supplements
Subchondral preosteoclasts acquire a unique secretory phenotype in high-fat-diet (HFD) mice.
Three-month-old Cdkn2aflox/flox mice were fed a standard chow-food diet (CHD). Three-month-old Tnfrsf11aCre/+; Cdkn2aflox/flox mice(p16cko) and Cdkn2aflox/flox littermates (wild-type [WT]) were fed …
Figure 6—figure supplement 1
Senescence-associated secretome inhibits osteoclastogenesis.
(A) Experimental procedure: Monocytes/macrophages were incubated with osteoclastogenesis medium (macrophage colony stimulating factor [M-CSF] and receptor activator of nuclear factor kappa-B ligand …
Figure 6—figure supplement 2
Senescence of preosteoclasts was successfully induced with hydrogen peroxide.
(A) Procedure for preparation of senescent preosteoclasts. Isolated bone marrow monocytes/macrophages were treated with M-CSF and RANKL for 3 days to obtain mononuclear preosteoclasts. Cellular …
Figure 6—figure supplement 3
RNA-seq reveals aging/senescence-induced genes in senescent preosteoclasts.
RNA-seq was performed using senescent preosteoclasts and non-senescent preosteoclasts. (A) 150 aging/senescence-induced genes (ASIGs) were defined based on previously published studies (see …
Figure 6—figure supplement 4
Osteoclast differentiation-related genes are down-regulated in the senescent preosteoclasts in the RNA-seq dataset.
Figure 7
Secreted factors from preosteoclasts stimulate osteoblast differentiation through COX2-PGE2 signaling.
(A–D) Representative images (A, C) and the quantified CFU-F frequency (B), and CFU-OB frequency (D) of bone marrow stromal cells treated with control conditioned medium (Con-CM) or senescent …
Figure 8
High-fat diet (HFD) mice have increased COX2+ cells in subchondral bone.
Three-month-old C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for different time periods as indicated, n=6 mice per group. Immunofluorescence staining of knee joint tissue sections …
Figure 9
Cyclooxygenase 2 (COX2) inhibitor alleviates high-fat diet (HFD)-induced joint degeneration in mice.
Three-month-old C57BL/6 mice were fed a standard chow-food diet (CHD) or HFD for 5 months. During the last 2 months of the HFD challenge, the mice also received celecoxib (16 mg/kg–1 daily) or …
Author response image 1
Subchondral bone changes in aged mice relative to young mice.
Tables
Table 1
Longitudinal comparison of the standard knee OA outcomes and subchondral BML worsening between human participants with MetS-OA and their matched PTOA participants (MetS+ PTOA– versus PTOA+ MetS–).
| PS-matched MetS-OA versus PTOA participants | |
|---|---|
| Knee OA standard outcomes | Hazard ratio (95% Confidence Interval), p-value, Sample size, Number of events* [MetS-OA: PTOA] |
| Knee OA incidence | 0.89 (0.56–1.41), p:0.609, N:184 (92:92), Event [34:36] |
| Knee OA progression | 1.04 (0.73–1.49), p:0.822, N:316 (158:158), Event [65:62] |
| Symptomatic incidence (NASS) | 0.95 (0.54–1.67), p:0.859, N:338 (169:169), Event [28:30] |
| Subchondral BML Worsening (MOAKS) | Odds ratio (95% Confidence Interval), P-value, N:338 (169:169), Number of events* [MetS-OA: PTOA] |
| Worsening in number of affected subregions with BML | 1.37 (1.06–1.77), p:0.015 Improvement, [16:31] No change, [92:84] Worsening, [61:54] |
| Worsening in the number of affected subregions | 0.44 (0.22–0.87), p:0.018 Yes, [31:16] |
| Maximum worsening in BML score | 1.17 (0.85–1.6), p:0.337 No change, [83:91] Worsening by ≤1 grade, [59:54] by ≥2 grades, [27:24] |
| Improvement in the subregions' BML score | 0.82 (0.51–1.30), p:0.389 Yes, [90:98] |
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Standard OA outcomes (baseline to 8th year) and validated MOAKS measures of subchondral BML worsening (between baseline and 24 month visit) were assessed between knees of participants with MetS-OA and their PS-matched knees of participants with PTOA. Cox proportional hazards were used for standard OA outcomes, and participants had a mean follow-up duration of 6.9 years (median and 1st and 3rd quartiles of 8 years). and logistic mixed-effect regression models were used for subchondral BML assessments. Knees of participants were matched for confounders using the 1:1 PS matching method. All analyses were adjusted for the baseline Kellgren-Lawrence (KL) and Osteoarthritis Research Society International medial joint space narrowing (OARSI JSN) grades of knees. Standard OA outcomes included knee OA incidence defined by KL grade ≥2 in participants with KL equal to 0–1, knee OA progression defined by partial or whole grade progression in OARSI JSN grade, and knee OA symptomatic incidence measured by NASS. Subchondral BML worsening was assessed using standard MOAKS measures. N corresponds to the total number of knees included in each analysis and the number of matched knees of MetS-OA and PTOA participants in the parenthesis.
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*
Number of events for each outcome has been shown separately for participants with MetS-OA and PTOA in the brackets.
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BML: Bone marrow lesion, COX2I: Cyclooxygenase 2 inhibitor, MetS: metabolic syndrome, MOAKS: MRI Osteoarthritis Knee Score, NASS: non-acceptable symptomatic state, PS: propensity score, OA: osteoarthritis.
Table 2
Longitudinal comparison of standard knee OA outcomes between human COX2 inhibitor users vs. non-selective NSAID users according to the presence of MetS.
| COX2I users vs. non-selective NSAID usersHazard ratio (95% Confidence Interval), p-value, Sample size,Number of events* [COX2I: NSAID] | ||
|---|---|---|
| MetS-OA+ | MetS-OA– | |
| Knee OA incidence | 0.42 (0.04–4.77), p:0.487, N: 30 (15:15), Events* [2:3] | 0.62 (0.13–2.84), p:0.537, N: 118 (59:59), Events* [5:9] |
| Knee OA progression | 0.18 (0.04–0.85), p:0.030, N: 94 (47:47), Events* [2:8] | 0.96 (0.53–1.74), p:0.886, N: 266 (133:133), Events* [21:23] |
| Symptomatic incidence (NASS) | 0.8 (0.27–2.4), p:0.689, N: 108 (54:54), Events* [2:3] | 1.56 (0.63–3.89), p:0.337, N: 324 (162:162), Events* [18:12] |
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COX2I: cyclooxygenase 2 inhibitor, MetS: metabolic syndrome, NASS: non-acceptable symptomatic state, NSAID: Non-Steroidal Anti-Inflammatory Drug, OA: Osteoarthritis. Standard OA outcomes were compared between knees of COX2 inhibitor users vs. matched non-selective NSAID users. Analysis was stratified analysis for the presence of MetS-associated OA (MetS-OA+ vs. MetS-OA–). Cox proportional hazards were used. Knees of participants were matched for confounders using the 1:1 propensity-score matching method. Events are knee OA incidence defined by Kellgren-Lawrence (KL) grade ≥2 in participants with KL equal to 0–1, knee OA progression defined by partial or whole grade progression in Osteoarthritis Research Society International medial joint space narrowing grade, and knee OA symptomatic incidence measured by NASS. The mean follow-up duration for standard knee OA outcomes was 4.4 years (median: 4 years, 1st and 3rd quartiles of 1 and 8 years). N corresponds to the total number of knees included in each analysis and the number of matched knees of COX2 inhibitor users vs. non-selective NSAID users in the parentheses.
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*
Number of events for each outcome has been shown separately in the brackets for COX2 inhibitor users and non-selective NSAID users.
Table 3
Longitudinal comparison of subchondral BML worsening between human COX2 inhibitor users vs. non-selective NSAID users with MetS-OA.
| Subchondral BML Worsening (MOAKS) | COX2 inhibitor users vs. non-selective NSAID users with MetS-OA | |
|---|---|---|
| Odds ratio (95% Confidence Interval), p-value, Sample size | Number of events in each group[COX2 inhibitor: NSAID]* | |
| Worsening in number of affected subregions with BML | 0.35 (0.13–0.93), p:0.035, N: 88 (44:44) | Improvement, [3:2] No change, [14:9] Worsening, [27:33] |
| Improvement in number of affected subregions | 1.21 (0.19–7.72), p:0.839, N: 88 (44:44) | Yes, [3:2] |
| Maximum worsening in BML score | 0.45 (0.2–0.99), p:0.046, N: 88 (44:44) | No change, [18:9] Worsening by ≤1 grade, [22:27] by ≥2 grades, 4:8 |
| Improvement in the subregions' BML score | 1.28 (0.33–5.00), p:0.722, N: 88 (44:44) | Yes, [9:6] |
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Among the PS-matched COX2 inhibitor users and non-selective NSAID users with MetS-OA, participants with available baseline and 24-month follow-up MRIs were included. A musculoskeletal radiologist read and scored MRIs according to validated MOAKS measures of subchondral BML worsening. Logistic mixed-effect regression models were used for subchondral BML assessments. All analyses were adjusted for the baseline Kellgren-Lawrence (KL) and Osteoarthritis Research Society International medial joint space narrowing (OARSI JSN) grades of knees. Subchondral BML worsening was assessed using standard MOAKS measures. N corresponds to the total number of knees included in each analysis and the number of matched knees of MetS-OA and PTOA participants in the parenthesis.
-
*
Number of events for each outcome has been shown separately in the brackets for COX2 inhibitor users and non-selective NSAID users.
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BML: Bone marrow lesion, MetS: metabolic syndrome, MOAKS: MRI Osteoarthritis Knee Score, NSAID: Non-Steroidal Anti-Inflammatory Drug, OA: osteoarthritis.
Author response table 1
Result of PS-matching MetS+ and MetS- participants once with BMI inclusion in the PS-matching covariates and once without including BMI.
With BMI in the matching model, MetS+ participants have lower mean KL grades, and without its inclusion, MetS+ participants have higher mean KL grades.
| All OAI subjects | Matched subjects without BMI in matching | Matched subjects with BMI in matching | |||||||
|---|---|---|---|---|---|---|---|---|---|
| MetS– | MetS+ | MetS– | MetS+ | MetS– | MetS+ | ||||
| 7459 | 1810 | SMD | 1803 | 1803 | SMD | N: 1800 | N: 1800 | SMD | |
| Variables included in the PS matching model | |||||||||
| Age (year) [mean (SD)] | 60.38 (9.09) | 64.94 (8.49) | 0.52 | 65.22 (8.95) | 64.90 (8.48) | 0.04 | 65.12 (8.47) | 64.87 (8.46) | 0.03 |
| Sex, Female, N (%) | 4426 (59.3) | 985 (54.4) | 0.10 | 1043 (57.8) | 985 (54.6) | 0.07 | 1014 (56.3) | 983 (54.6) | 0.04 |
| Non-white race [N (%)] | 1468 (19.7) | 494 (27.3) | 0.18 | 439 (24.4) | 487 (27.0) | 0.06 | 481 (26.8) | 488 (27.1) | 0.01 |
| BMI (kg/m2) [mean (SD)] | 28.23 (4.78) | 30.71 (4.43) | 0.54 | 28.09 (4.52) | 30.72 (4.43) | 0.59 | 30.51 (4.53) | 30.69 (4.43) | 0.04 |
| Smoking, current smoker [N (%)] | 517 (6.9) | 120 (6.6) | 0.01 | 113 (6.3) | 120 (6.7) | 0.02 | 107 (5.9) | 120 (6.7) | 0.03 |
| Alcohol use, ≥1/week [N (%)] | 3250 (43.6) | 698 (38.6) | 0.10 | 689 (38.2) | 698 (38.7) | 0.01 | 733 (40.7) | 696 (38.7) | 0.04 |
| PASE score [mean (SD)] | 165.42 (83.48) | 141.00 (76.02) | 0.31 | 137.63 (72.17) | 141.25 (76.03) | 0.05 | 143.72 (78.12) | 141.42 (75.89) | 0.03 |
| Variables not included in the PS matching model | |||||||||
| KL grade, N (%) | 0.21 | 0.12 | 0.11 | ||||||
| Grade 0 | 2875 (38.5) | 540 (29.8) | 634 (35.2) | 538 (29.8) | 511 (28.4) | 539 (29.9) | |||
| Grade 1 | 1318 (17.7) | 345 (19.1) | 317 (17.6) | 342 (19.0) | 283 (15.7) | 342 (19.0) | |||
| Grade 2 | 2019 (27.1) | 515 (28.5) | 496 (27.5) | 514 (28.5) | 549 (30.5) | 511 (28.4) | |||
| Grade 3 | 1005 (13.5) | 332 (18.3) | 296 (16.4) | 331 (18.4) | 375 (20.8) | 331 (18.4) | |||
| Grade 4 | 242 (3.2) | 78 (4.3) | 60 (3.3) | 78 (4.3) | 82 (4.6) | 77 (4.3) | |||
| Medial JSN score, N (%) | 0.22 | 0.17 | 0.04 | ||||||
| Grade 0 | 4625 (65.9) | 937 (55.9) | 1068 (63.4) | 933 (55.8) | 959 (57.1) | 936 (56.0) | |||
| Grade 1 | 1503 (21.4) | 431 (25.7) | 379 (22.5) | 430 (25.7) | 409 (24.4) | 427 (25.6) | |||
| Grade 2 | 742 (10.6) | 255 (15.2) | 205 (12.2) | 254 (15.2) | 265 (15.8) | 254 (15.2) | |||
| Grade 3 | 148 (2.1) | 54 (3.2) | 32 (1.9) | 54 (3.2) | 46 (2.7) | 54 (3.2) | |||
| WOMAC pain score (mean (SD)) | 2.35 (3.28) | 2.96 (3.56) | 0.18 | 2.43 (3.27) | 2.96 (3.56) | 0.16 | 2.75 (3.62) | 2.95 (3.56) | 0.06 |
| Cardio/Cerebrovascular diseases, N (%) | 239 (3.3) | 196 (11.4) | 0.31 | 79 (4.5) | 196 (11.4) | 0.26 | 82 (4.7) | 192 (11.2) | 0.24 |
| Hypertension [N (%)] | 2704 (36.3) | 1734 (95.8) | 1.62 | 750 (41.6) | 1728 (95.8) | 1.44 | 829 (46.1) | 1724 (95.8) | 1.31 |
| Diabetes Mellitus [N (%)] | 86 (1.2) | 646 (36.7) | 1.02 | 18 (1.0) | 641 (36.5) | 1.02 | 13 (0.7) | 636 (36.3) | 1.03 |
| Dyslipidemia [N (%)] | 1010 (13.5) | 1599 (88.3) | 2.26 | 257 (14.3) | 1594 (88.4) | 2.21 | 272 (15.1) | 1591 (88.4) | 2.16 |
| Abdominal obesity [N (%)] | 6293 (84.6) | 1810 (100.0) | 0.60 | 1535 (85.4) | 1803 (100.0) | 0.58 | 1659 (92.6) | 1800 (100.0) | 0.40 |
Additional files
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Supplementary file 1
Human osteoarthritis initiative datasets used in the study.
(A) Baseline characteristics of the participants according to presence of metabolic syndrome-associated OA (MetS+ PTOA– versus PTOA+ MetS–) before and after propensity score matching.(B) Baseline characteristics of human COX2 inhibitor and non-selective NSAID users included in the study, before and after propensity score matching. Matched participants were includde in the analysis of COX2 inhibitor use association with OA outcomes, according to its phenotype.(C) Osteoarthritis Initiative (OAI) datasets used in the study. (D) Flowchart outlining the selection criteria and PS-matching process according to the presence of metabolic syndrome-associated OA (MetS-OA) and post-traumatic OA (PTOA) in Osteoarthritis initiative participants. (E) Flowchart outlining the selection criteria and PS-matching process of human COX2 inhibitor and non-selective NSAID users from the Osteoarthritis initiative dataset.
- https://cdn.elifesciences.org/articles/79773/elife-79773-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79773/elife-79773-mdarchecklist1-v2.pdf
