Endothelial Dysfunction Contributes to Chondrocyte Senescence Associated With Insulin‐Like Growth Factor‐Binding Protein‐6 in a Spontaneously Hypertensive Rat Model

Abstract

Background

Given an epidemiological association between hypertension and osteoarthritis, we aim to elucidate how vascular pathology triggers avascular articular cartilage loss by comparing vascular and joint phenotypes between spontaneously hypertensive rats and normotensive Wistar Kyoto rats at tissue, cellular, and molecular levels.

Methods

Systemic and local endothelial function were assessed via blood pressure monitoring and photoacoustic imaging of knee joints, respectively (n=5–6). Tissue and cellular damages in articular cartilage, subchondral bone, and synovium were systemically evaluated by radiological and histological examination (n=6). Endothelial transcriptional changes were delineated via bulk RNA‐seq (n=3). The secretome of human endothelial cells under oxidative stress was analyzed via proteomics to identify key factors in chondrocyte senescence (n=4). Captopril, an antihypertensive medication, was adopted as a tool to rescue chondrocyte senescence and osteoarthritis both in vitro (n=3) and in vivo (n=6).

Results

Compared with Wistar Kyoto rats, spontaneously hypertensive rats exhibited higher blood pressure and local joint hypoxia with increased endothelial oxidative stress as early as 3 months old. Notably, these endothelial dysfunctions preceded the onset of structural joint damage. By 9 months of age, spontaneously hypertensive rats developed osteoarthritis‐like pathology characterized by senescent chondrocyte accumulation and cartilage degradation, which could be mitigated by captopril. Transcriptomic and proteomic analyses of rat and human endothelial cells revealed upregulated IGFBPs (insulin‐like growth factor‐binding proteins). Among them, IGFBP6 was identified to induce chondrocyte senescence in vitro. This effect can be modified by captopril treatment.

Conclusions

Systemic vascular dysfunction may induce local joint damage with upregulation of endothelial IGFBPs secretion such as IGFBP6. This study highlights a potential therapeutic target for preventing joint structural damage by addressing endothelial dysfunction.

Nonstandard Abbreviations and Acronyms

CM

conditioned medium

DOCA

deoxycorticosterone acetate

ECGS

endothelial cell growth supplement

FC

fold change

FMO

flavin‐containing monooxygenase

HUVEC

human umbilical vein endothelial cell

IGF

insulin‐like growth factor

IGFBP

insulin‐like growth factor‐binding protein

ROI

regions of interest

SA‐β‐gal

senescence‐associated beta‐galactosidase

SHR

spontaneously hypertensive rats

WKY

Wistar Kyoto rats

Research Perspective.

What Is New?

• Systemic vascular pathology, particularly high blood pressure, accelerates joint aging via secretion of insulin‐like growth factor‐binding proteins to induce chondrocyte senescence, which is modifiable by antihypertensive medication, captopril.

What Question Should Be Addressed Next?

• Interventions targeting vascular pathologies, including Food and Drug Administration‐approved antihypertensive medications, could be exploited as antiaging agents and repurposed for treating metabolic osteoarthritis.

• Insulin‐like growth factor‐binding proteins emerge as mediators for joint‐vasculature crosstalk and await further investigations as therapeutic targets for comorbid osteoarthritis.

Hypertension, a prevalent age‐related vascular pathology, emerges as an independent risk factor for knee osteoarthritis, the leading cause of chronic pain and physical disability in older adults. ¹ Vascular dysfunction—high systolic blood pressure and pulse pressure—has been associated with an increased risk of radiographic knee osteoarthritis in multiple patient cohorts. ² , ³ Recently, it was also linked with subchondral bone microstructural damage in human knee osteoarthritis samples. ⁴ However, the exact pathomechanism underlying how vascular dysfunction triggers avascular cartilage damage in osteoarthritis remains to be elucidated. Hypertension and osteoarthritis share multiple common molecular pathways, including the IGF (insulin‐like growth factor) system and IGFBPs (IGF‐binding proteins). ⁵ , ⁶

Accumulation of senescent chondrocytes induces joint damage, whereas removal of senescent cells could attenuate the severity of posttraumatic osteoarthritis. ⁷ Overexpression of chondrocytic p16Ink4a, a senescence marker, induces the expression of major matrix remodeling enzymes, MMP‐1 (matrix metalloproteinase‐1) and ‐13 for cartilage degradation. ⁸ Chondrocytic SA‐β‐gal (senescence‐associated beta‐galactosidase) activity was elevated close to sites of traumatic injuries and associated with cartilage degradation, whereas no staining of SA‐β‐gal was observed in intact cartilage. ⁹ IGF‐1 has been found to be involved in normal aging processes and lifespan extension. ¹⁰ In addition to trauma, hypertension has been reported to contribute to senescent cell accumulation in a variety of organs and tissues, such as kidneys and hearts, in a deoxycorticosterone acetate (DOCA)‐induced hypertensive rat model. ¹¹ Later on, we further demonstrated accumulation of senescent chondrocytes and cartilage degradation in knee joints in the same animal model as the previous study, specifically 6‐week‐old male Sprague–Dawley rats. ¹² Apart from secondary hypertension induced by DOCA, we are motivated to investigate the impact of primary hypertension on joint homeostasis and disease.

Given the genetic association between primary hypertension and osteoarthritis via Mendelian randomization analysis, ¹³ our study addresses the research gap by elucidating how genetically predisposed hypertension contributes to knee osteoarthritis. Local vascular dysfunction was linked with chondrocyte senescence and followed destabilization of the medial meniscus in posttraumatic osteoarthritis development. ¹⁴ Thus, we hypothesize that systemic endothelial dysfunction alters local blood supply and accelerates senescent chondrocyte accumulation in knee joints of spontaneously hypertensive rats (SHR), which ultimately causes osteoarthritis development. Compared with normotensive Wistar Kyoto rats (WKY) as the closest genetic control, we found that systemic endothelial cell dysfunction occurs at the age of 3 months, followed by premature chondrocyte aging and loss of proteoglycans in articular cartilage at the age of 9 months in SHR. We further demonstrated that the secretome of human endothelial cells under oxidative stress could trigger chondrocyte senescence, possibly associated with upregulation of IGFBPs in vitro. Notably, the antihypertensive medication, captopril, could lower the levels of IGFBP6 both in vitro and in vivo, and mitigate chondrocyte senescence and cartilage degradation in the spontaneously hypertensive rat model.

METHODS

The designed animal study follows the Animal Research: Reporting of In Vivo Experiments guidelines. The data that support the findings of this study, methods used in the analysis, and materials used to conduct the experiments are available from the corresponding author upon reasonable request. Raw data for transcriptomic analysis have been deposited at Gene Expression Omnibus repository with accession number GSE302827. The source codes and processed data for this article can be viewed at github.com/MZ1808/302827.

Animals

Sample size was determined using the online statistical tool Statulator, ¹⁵ based on preliminary experiments of blood pressure measurement. To observe a difference between WKY and stroke‐prone SHR (from 160.0 in WKY to 204.5 in SHR in females; 165.7 in WKY to 221.5 in SHR in males; common SD 12.8), this study requires a sample size of 3 in each group to achieve 80% power at a 5% significance level (P<0.05). To allow for ineligibility, we selected a sample size of 5‐6 in each group. A total of 22 WKY (n=10, 5 female, 5 male) and SHR (n=12, 6 female, 6 male) were included for continuous monitoring of blood pressure. In total, 12 WKY (3 female, 9 male) and 12 SHR (3 female, 9 male) were deployed for radiographic and histological assessment at different time points (n=3 per group at 3 months and 6 months, n=6 per group at 9 months) (Figure 1A). Captopril, an antihypertensive medication, was repurposed as a drug candidate to rescue osteoarthritis. A total of 12 SHR were evenly allocated to the saline‐treated group or the captopril treatment group. Animals were drawn in a random sequence to receive treatment or measurement. The randomization sequence was determined with a computer random number generator. The captopril group included 3 male and 3 female SHR treated with captopril (50 mg/kg) ¹⁶ by oral gavage ¹⁷ , ¹⁸ every day starting at 5 months old. Saline was administrated by oral gavage to another 3 male and 3 female SHR as control. All rats were euthanized at the age of 9 months. No rats were excluded from the analysis. The Department of Health of the Government of Hong Kong and Animal Subjects Ethics Sub‐Committee of the Hong Kong Polytechnic University approved all the experimental procedures of this study (20–21/103‐BME‐R‐GRF). The procedures followed were in accordance with institutional guidelines. All rats were housed in the Centralised Animal Facilities in the Hong Kong Polytechnic University, in a controlled environment with a 12‐hour light/dark cycle, a constant room temperature (23  ± 1 °C), and unrestricted access to food and water. Each group has the same feeding environment and is clearly marked by the experimenters.

Figure 1. Endothelial dysfunction in spontaneously hypertensive rats developed by 3 months of age without knee joint microstructure changes.

A, Flow diagrams on grouping and experimental design comparing WKY and SHR. B, Systolic blood pressure measured by tail‐cuff method in male and female WKY and SHR from 3 to 9 months. (N=5‐6). C, Carotid artery stiffness in 3‐month‐old WKY and SHR. (N=6, male). D through E, Photoacoustic images and measurement of oxygen saturation in joint tissue in 3‐month‐old WKY and SHR. (N=6, male). F, H&E staining of joint synovium in WKY and SHR at 3 months. G and H, H&E staining and quantification of joint cartilage in WKY and SHR at 3 months. (N=3, female; WKY: 263.0±3.99 μm; SHR: 261.5±9.22 μm, P=0.88). I, H&E staining of joint bone in WKY and SHR at 3 months. All data were presented as mean±SE. Two‐tailed Student’s t test (unpaired) was performed between WKY and SHR. For Figure 1A analysis of time‐course changes in blood pressure, we use repeated measures factorial ANOVA to compare blood pressure at different times. *P<0.05; **P<0.005; ***P<0.001. H&E indicates hematoxylin and eosin; SHR, spontaneously hypertensive rats; and WKY, Wistar Kyoto rats.

Blood Pressure Measurement

Blood pressure was longitudinally measured by the tail‐cuff method every 2 weeks using BP‐2000 Blood Pressure Analysis System (Visitech System, Inc., Apex, NC, USA).

Ultrasound and Photoacoustic Imaging

Photoacoustic imaging adopts near‐infrared lasers to exploit the light absorption properties of endogenous hemoglobin in blood vessels, measuring signals at 2 wavelengths: 750 nm (for deoxygenated hemoglobin) and 850 nm (for oxygenated hemoglobin). The red signal represents a ratio of these 2 measurements. Photoacoustic imaging has been applied to noninvasively measure joint tissue oxygen content in vivo without the need for any contrast agents. ¹⁹

Animals were anesthetized with 3% isoflurane and imaged with Vevo 2100 high‐frequency micro‐imaging system (VisualSonics, Toronto, Ontario, Canada). The entire knee joint was scanned in 3 dimensions by linear transposition of the transducer perpendicular to a single sagittal plane for 2‐dimensional imaging. A triangular region of interest (ROI) was manually drawn in the frame of the best represented triangular region defined by the patellar tendon, proximal tibia, and distal femur. During 3‐dimensional data analysis, the same ROI was automatically isolated in all 2‐dimensional slices by a custom‐designed script on Matlab (Vessel Analysis v1.4). ⁴ , ⁵ Vascular volume (%) was quantified by counting the number of colored pixels in the ROI at a resolution of 0.010 mm×0.010 mm on each Power Doppler and photoacoustic slice, then multiplying by the slice thickness and then dividing by the total number of voxels in the volume of interest. Similarly, the mean SO₂ (%) was converted from the mean photoacoustic signal intensity of the voxels in the volume of interest. LZ250 detectors were used for knee joint imaging. For the photoacoustic mode, an ultrasound transducer emitted a pulse with a center frequency of 21 MHz, photoacoustic gain 35 dB, depth 9 mm, and width 14.04 mm, wavelength 750/850 nm. The probe was placed parallel to the joint until a triangular area of knee joint formed by patellar tendon, distal femur, and proximal tibia was identified. LZ400 detectors were used for the carotid artery. For B mode, an ultrasound transducer emitted a pulse with a center frequency of 30 MHz, gain 28 dB, depth 12 mm, and width 13.36 mm. As a surrogate marker of arterial stiffness, pulse wave velocity quantification was typically performed at the carotid artery. Measurements were performed once in 3‐month‐old and 9‐month‐old rats before euthanization.

Micro‐Computed Tomography

The rat knees were fixed in 10% neutral buffered formalin for 24 hours and then stored in 70% ethanol. Later, the entire knee joints were scanned with a scan width of 35 mm and pixel size of 18 μm in a micro‐computed tomography imaging system (Skyscan1276, Belgium), in which X‐ray voltage, current, and filter used were set at 88 kV, 200 mA and 1.0 mm, respectively. The imaging data sets were analyzed using built‐in software DataViewer (v1.4.4.0) and CTvol (v2.3.2.0). The same volume of interests and same ROIs were selected for each sample. For tibial subchondral bone, we selected a total of 150 slides, including 50 slides above and 100 slides below the critical point defined by the end of 2 menisci, for analysis. The thresholding of gray levels in the binarized computed tomography images was set at 90 to 225. The bone mineral density, bone volume fraction, trabecular separation, trabecular thickness, trabecular number, and the structure model index were calculated for each sample. Measurements were performed once in 9‐month‐old rats after euthanization.

Histology Analysis and Immunostaining

Rat knee samples were fixed by 4% paraformaldehyde and decalcified with 10% Ethylenediaminetetraacetic acid for 1 month and then embedded in wax blocks for histological analysis. Samples were cut into 5‐μm‐thick sagittal sections. The medial compartment of rat tibiofemoral joint showing both anterior and posterior horns of medial meniscus was chosen for analysis. The ROI was defined at tibial articular cartilage and subchondral bone. In addition to routine hematoxylin and eosin and Safranin O staining, immunostaining was also performed with heat‐mediated antigen retrieval in sodium citrate buffer of pH 6.0 when deemed necessary. Primary antibodies included p16 (1:500, Abcam, ab54210), p53 (1:100, Abcam, ab131442), 4HNE (1:1000, Invitrogen, MA5‐27570), CD31 (1:500, Abcam, ab182981), MMP13 (1:500; Abcam, ab39012), IGFBP6 (insulin‐like growth factor‐binding protein 6; 1:250, Thermo Fisher, PA5115397), α‐SMA (alpha smooth muscle actin;1:1500; Abcam, ab124964), COL10 (collagen type X) antibodies (1:200, Abcam, ab49945) and Masson’s trichrome kit (Abcam, ab150686). For immunohistochemistry staining, the sections were stained with Vectastain ABC kit and 3,3’‐Diaminobenzidine peroxidase substrate kit (Vector Labs, USA) before counterstaining with hematoxylin. For immunofluorescent staining, secondary antibodies conjugated with Alexa Fluor 488 (1:500, Thermo Fisher, A11008) or Alexa Fluor 594 (1:500, Thermo Fisher, A11005) were used. SA‐β‐gal activities were detected with the Senescence β‐Galactosidase Staining Kit (Cell Signaling Technology, 9860). All images (n=6 joints/group, 3 sections/joint) were taken with Nikon Eclipse 80i microscope (Nikon, Japan) and analyzed with ImageJ (National Institutes of Health, USA). Grading of osteoarthritic cartilage damage was conducted using an established protocol. ¹⁴ , ²⁰

RNA‐Sequencing Analysis of Rat Aortic Endothelial Cells

Primary endothelial cells were isolated from the proximal segment of the aorta adjacent to the heart. Briefly, aortas were dissected from the hearts of 3‐month‐old rats and cut into pieces, then placed with the endothelium side down in 12‐well plates coated with rat tail collagen I (Gibco, A1048301). Isolated endothelial cells were cultured in endothelial cell medium (ScienCell) supplemented with 10% fetal bovine serum (v/v), 1% penicillin–streptomycin (v/v), and 1% endothelial cell growth supplement (v/v). Cells were incubated at 37 °C with 5% CO₂.

Total RNA was extracted from primary endothelial cells from the aorta of 3‐month‐old WKY and SHR (n=3 per group) with E.Z.N.A. Total RNA Kit (Omega Biotek) according to the manufacturer’s protocols. Library preparation and sequencing were outsourced to BGI Genomics (BGI, China). Briefly, samples were sequenced using DNBSEQ platform with a paired‐end read length of 100 bp (PE100). Reads of low quality, with adaptor sequences, or high levels of N base were filtered with SOAPnuke (v1.5.6). ²¹ Bowtie2 (v2.4.4) was employed to align the clean reads to the reference genes in genome GCF_000001895.5_Rnor_6.0. ²² Gene expression was quantified using RSEM (v1.2.28). ²³ Normalization and differential analysis of gene expression were performed with DESeq2 (v1.42.1) and corrected using the Benjamini–Hochberg procedure to control the false discovery rate. ²⁴ Gene Ontology enrichment analysis was performed on differentially expressed genes with adjusted P value <0.05 using GOstats (v2.68.0). ²⁵ The most prominent pathways in molecular function were compared between WKY and SHR in endothelial cells. Images were generated with ggplot2 (v3.5.0) and ComplexHeatmap (v2.15.4) in RStudio.

Cell Experiments and Secretomics Analysis

Human umbilical vein endothelial cells (HUVECs) were cultured at a density of 4.5 × 10⁵ cells per T‐75 flask and always maintained in 6 mL of media. Conditioned medium (CM) was collected from the same population of cells at different time points. Fresh medium was replenished and collected as “normal CM” 24 hours after passaging. Cells were then incubated in 150 μM of H₂O₂ for 24 hours, and the medium was retrieved as “H₂O₂ CM.” To harvest “captopril CM,” cells were treated with either 100 μmol/L or 1 mM of captopril (#62571–86‐2, Aladdin) for 1 day after H₂O₂ induction. All CM was centrifuged at 200 g for 5 minutes to remove cells and debris. They were aliquoted into small vials and kept at −20 °C for up to 2 weeks. CM was mixed with fresh DMEM/F12 medium in a 1:2 ratio for C28/I2 (human chondrocyte cell line) culturing. Comparison of cellular phenotypes was always done between chondrocytes cultured in the same batch of CM (harvested from the same flask of HUVECs).

Cell‐secreted proteins were collected by acetone precipitation of CM and resuspended in SDS‐containing resuspension buffer, before being reduced, alkylated, and digested following a revised filter aided sample preparation method as reported. ²⁶ Then 10 μg of tryptic‐digested peptides from each sample from 4 replicates of all treatment groups were collected and iTRAQ labeled. A pooled sample was prepared by mixing an equal number of peptides as the quantitation base. iTRAQ‐labeled peptides were then mixed and fractionated by high‐PH reverse phase column (Thermo), before being analyzed on a TimsTof mass spectrometer (Bruker) in DDA‐PASEF mode. Data obtained were analyzed on Mascot 2.6 engine against human Uniprot database plus built‐in contaminant database. Exogenous bovine serum proteins supplied in medium were identified and excluded from further analysis. Proteins with an average fold change (FC) >50% and a Student t test P value <0.05 between any 2 treatments were regarded as significantly changed (proteins of interest).

Human IGFBP6 protein (1 μg/mL, HY‐P73141, MCE) was administered to C28/I2 cell culture for 6 hours to induce cellular senescence.

Statistical Analysis

During randomized allocation and experimental procedures, only the experimenter knew the specific grouping. Data analysis is conducted under unknown grouping conditions, and the specific grouping is known only after obtaining the results. Statistical analysis was performed using GraphPad Prism (Version 8.0, CA, USA). All data were presented as mean ± SEM. The Shapiro–Wilk test was performed to assess the normality of data. All data were normally distributed. Two‐tailed independent t test was performed to compare means between 2 groups with normally distributed data. The comparison of mRNA expression or SA‐β‐gal activities among chondrocytes subject to different treatments was performed using one‐way ANOVA.

RESULTS

Systemic Vascular Dysfunction in 3‐Month‐Old SHR Characterized by an Increase of Oxidative Stress and IGFBPs

Higher blood pressure was observed in SHR compared with WKY as early as 3 months old in both males and females (Figure 1B). Male SHR demonstrated a statistically significant increase starting from 4 months (201.17 ± 3.03 mm Hg) (P=0.012) until 9 months (224.83 ± 1.38 mm Hg) (P<0.001) when comparing to the time point at 3 months (190.92 ± 1.42 mm Hg). The blood pressure stabilized and remained relatively constant from 6 to 9 months compared with 6 months (217.17 ± 3.16 mm Hg) (P=0.050). In female SHR, blood pressure also increased in 6 months (197.83 ± 3.97 mm Hg) (P=0.012) compared with 3 months (184 ± 2.22 mm Hg), continuing to rise until 7 months (193.50±4.51 mm Hg) (P=0.088), after which it stabilized and remained relatively constant up to 9 months (207.5±3.69 mm Hg) (P<0.001). Whether male or female, SHR’s blood pressure at 3 months old (male: 190.92 ± 1.42 mm Hg; female: 184 ± 2.22 mm Hg) is higher than WKY’s (male: 162.20±2.80 mm Hg; female: 153.70±1.14 mm Hg). Afterwards, the difference in blood pressure gradually increased. Carotid artery stiffness in 3‐month‐old WKY and SHR did not yet show statistically significant differences (0.225 [95% CI, −0.5746 to 1.025]; WKY: 2.88±0.15 mm/s; SHR: 3.10±0.37 mm/s; P=0.53) (Figure 1C). In addition, SHR also showed local vascular changes in joints at 3 months compared with WKY rats. Joint oxygen saturation was significantly lower in young SHR compared with WKY (WKY: 8.325±1.561%; SHR: 3.254±0.867%; P=0.018) (Figure 1D and 1E). However, there were no prominent histological changes observed in SHR joints when compared with WKY. Hematoxylin and eosin staining at 3 and 6 months revealed similar cartilage, synovium, and bone microarchitecture in both strains including in cartilage thickness (3 months: −1.570 [95% CI, [−29.45 to 26.31]]; 6 months: −5.150 [95% CI, [−19.57 to 9.273]]) (Figure 1F through 1I; Figure S1A through S1D).

To further decipher cellular changes in the vasculature of SHR, primary endothelial cells were isolated from the aorta of the 3‐month‐old animals (Figure 2A). SHR endothelial cells demonstrated distinct gene expression profiles from WKY endothelial cells (Figure 2B). To describe the differences in endothelial function, pathway enrichment analysis was performed using the Gene Ontology terms for molecular function. Enrichment in growth factor binding, oxidoreductase activities, and IGF signaling‐associated gene was noted (Figure 2C). Expression of Fmo1 (log2FC=4.28, P<0.0001), Fmo2 (log2FC=5.34, P=0.0005), and Fmo3 (log2FC=5.76, P<0.0001) was significantly higher in SHR endothelial cells (Figure 2D and 2E). Fmo1, one of the top upregulated genes in SHR endothelial cells, is involved in H₂O₂ accumulation induced by DNA damage. ²⁷ , ²⁸ Mammalian FMOs (flavin‐containing monooxygenases) are a source of H₂O₂ production. Elevated oxidative stress was thus implied in 3‐month‐old SHR (Figure 2F and 2G). IGF‐binding proteins, Igfbp3 (log2FC=2.12, P=0.0373), Igfbp4 (log2FC=1.99, P=0.0053), and Igfbp6 (log2FC=1.41, P=0.053) were also elevated in SHR endothelial cells compared with WKY counterparts, whereas there was no statistically significant difference between 2 strains in terms of Igfbp2 (P=0.3905), Igfbp5 (P=0.9856), and Igfbp7 (P=0.6237). We performed correlation analyses between IGFBP3, IGFBP4, IGFBP6, and von Willebrand factor and found that IGFBP4 (Pearson r=0.749, P=0.087) and IGFBP6 (Pearson r=0.806, P=0.053) showed stronger correlations (Figure S2). IGF signaling is implicit in cellular senescence and oxidative injury and contributes to the pathogenesis of hypertension. ²⁹ Roles of the IGFBP family have been discussed in osteoarthritis. Prior studies focused on IGFBP3 (anti‐inflammatory and proapoptotic) ³⁰ , ³¹ and IGFBP‐7 (proapoptotic) ³² in osteoarthritis. The role of IGFBP4 in aging is controversial. Some studies have shown that it has prosenescence ³³ and antisenescence ¹³ properties. There are relatively few studies related to IGFBP6, so we chose IGFBP6 as the research interest for downstream experiments.

Figure 2. Elevated IGF binding proteins in vasculature of 3‐month‐old spontaneously hypertensive rats compared with Wistar Kyoto rats.

A, Schematic for isolation of primary endothelial cells for RNA‐sequencing. B, Volcano plot of differentially expressed genes between WKY and SHR endothelial cells. Genes are considered significantly different when adjusted P value is <0.05 and fold change is >1. Red: higher expression in SHR. Blue: higher expression in WKY. Green: not significantly different. Top upregulated and downregulated genes were annotated. Note that FMO1 is among the annotated upregulated genes. C, Top upregulated Gene Ontology terms pertaining to molecular function in SHR compared with WKY. (n=3). D and E, Heatmap of genes involved in monooxygenase activity and the normalized expression of selected genes, FMO1, FMO2, and FMO3. F through G, Heatmap of genes in insulin‐like growth factor system and the normalized expression of selected genes, IGFBP6. Gene expression was normalized using R package DESeq2’s median of ratios. Adjusted P value was shown for selected genes of interest. Likelihood‐ratio test included in DESeq2 was used to compute the adjusted P value. All data were presented as mean±SE. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. EC indicates endothelial cells; FMO, flavin‐containing monooxygenase; IGFBP6, insulin‐like growth factor‐binding protein 6; ns, not significant; SHR, spontaneously hypertensive rats; and WKY, Wistar Kyoto rats.

Local Joint Senescence, Fibrosis and Degeneration in 9‐Month‐Old SHR

4HNE was higher in SHR aorta compared with WKY (WKY: 8.33±1.42; SHR: 28.34±3.44; P<0.001) (Figure 3A and 3B). Senescent endothelial cell accumulation was found in the aorta of 9‐month‐old SHR (27.52 [95% CI [19.1–35.94]]; WKY: 14.95±1.37%; SHR: 42.47±3.52%; P<0.001) (Figure 3C and 3D). Unlike at 3 months, the vascular stiffness of SHR at 9 months old was significantly higher than that of WKY (WKY: 8.09±1.030 mm/s; SHR: 3.966±0.290 mm/s; P=0.002) (Figure 3E). Consistent with our findings in aortic endothelial cells, there was a higher expression of oxidative stress marker 4HNE in SHR synovial vasculature, colocalized with CD31, a marker of blood vessels (Figure 3F). SHR joints displayed osteoarthritis‐like changes from 9 months. With increasing age, both SHR and WKY exhibited a reduction in joint oxygen saturation, and no statistically significant difference was observed between the 2 groups (WKY: 0.64±0.14%; SHR: 0.63±0.19%; P=0.9606) (Figure 3G). There was more IGFBP6 in the synovium of SHR than WKY (WKY: 55.3±2.97; SHR: 105.2±7.63; P<0.001) (Figure 3H and 3I). The synovium of 9‐month‐old SHR appeared to be more fibrotic than age‐matched WKY, as indicated by alpha smooth muscle actin (α‐SMA) (WKY: 86.1±2.79; SHR: 137.2±3.00; P<0.001) and Masson’s Trichrome staining (blue) (WKY: 0.81±0.36; SHR: 11.78±1.99; P=0.002) (Figure 3J and 3M). There were also more p16^INK4a‐positive senescent cells in the synovium of SHR than WKY (WKY: 8.64±1.58%; SHR: 25.19±2.21%; P<0.001) (Figure 3N and 3O). The expression level of IGFBP6 in the bone tissue of SHR was significantly higher compared with that in WKY (128 [95% CI, 51.05–205]; WKY: 89.20±4.79; SHR: 209.9±12.71; P<0.001) (Figure 3P and 3Q). Bone loss in subchondral bone was also observed in SHR compared with WKY (WKY: 0.4651±0.0056 g/cm³; SHR: 0.3525±0.0129 g/cm³; P<0.001) (Figure 3R and 3S, Figure S3). In cartilage, we did not observe the expression of IGFBP6 (Figure 3T). Therefore, IGFBP6 is derived from the synovium and bone. In the articular cartilage, SHR showed lower proteoglycan content and higher Osteoarthritis Research Society International score compared with WKY (1.667 [95% CI, 1.197–2.136]; WKY: 0.50±0.18; SHR: 2.17±0.11; P<0.001) (Figure 3U and 3V, ²⁰ ). At the cellular level, accumulation of p16^INK4a‐positive cells was observed at the articular cartilage in SHR (27.6 [95% CI, 16.91–38.28]; WKY: 13.89±2.91%; SHR: 41.45±4.41%; P<0.001) (Figure 3W and 3X). This was accompanied by a higher level of MMP13 in the extracellular matrix of SHR chondrocytes (21.67 [95% CI, 15.17–28.16]; WKY: 16.42±1.97%; SHR: 38.08±2.15%; P<0.001) (Figure 3Y and 3Z).

Figure 3. Joint structural and functional deterioration in spontaneously hypertensive rats at 9 months old.

A and B, Representative immunofluorescent staining and quantification of 4HNE in aortas in 9‐month‐old rats. (N=6, male). C and D, Representative images and quantification of p16ink4a staining in aorta in 9‐month‐old animals. Scale bar 50 μm. (N=6, male). E, Carotid artery stiffness in 9‐month‐old WKY and SHR. F, Representative immunofluorescent staining of 4HNE and CD31 in synovial tissue in 9‐month‐old WKY and SHR. (N=6, male). G, Photoacoustic images and measurement of oxygen saturation in joint tissue in 9‐month‐old WKY and SHR. (N=6, male). H and I, Immunohistochemistry staining and quantification of IGFBP6 in the synovium of 9‐month‐old WKY and SHR. (N=6, male). J and M, Representative images and quantification of α‐SMA and Masson’s Trichrome (blue) staining in synovium of 9‐month‐old WKY and SHR. (N=6, male). N and O, Immunohistochemistry staining and quantification of p16ink4a in synovium of 9‐month‐old WKY and SHR. (N=6, male). P and Q, Immunohistochemistry staining and quantification of IGFBP6 in subchondral bone of 9‐month‐old WKY and SHR. (N=6, male). R and S, Representative images and quantification of micro‐computed tomography result for subchondral bone of WKY and SHR joint for 9‐month‐old WKY and SHR. (N=6, male) T, IGFBP6 staining of cartilage in 9‐month‐old WKY and SHR. U, Representative images of Safranin‐O/Fast Green staining for 9‐month‐old WKY and SHR joints. (N=6, male) V, OARSI score grading system was adopted from Waldstein et al. ²⁰ (N=6, male) W and X, Immunohistochemistry staining and quantification of p16ink4a in cartilage of 9‐month‐old WKY and SHR. (N=6, male). Y and Z, Immunohistochemistry staining and quantification of MMP13 in cartilage of 9‐month‐old WKY and SHR. (N=6, male) All data were presented as mean±SE. Two‐tailed Student’s t test (unpaired) was performed between WKY and SHR. * P<0.05; ** P<0.005; *** P<0.001. α‐SMA indicates alpha smooth muscle actin; BMD, bone mineral density; IGFBP6, insulin‐like growth factor‐binding protein 6; MMP13, matrix metalloproteinase 13; OARSI, Osteoarthritis Research Society International; SHR, spontaneously hypertensive rats; and WKY, Wistar Kyoto rats.

Overall, SHR displayed phenotypes of joint deterioration in both synovial tissue and articular cartilage, including accumulation of fibrotic tissues, high oxidative stress, cellular senescence, and matrix decomposition. These changes are often associated with osteoarthritis, ³⁴ indicating that SHR showed premature joint aging.

Captopril Rescues Chondrocyte Senescence Induced by Endothelial Cells Under Oxidative Stress Associated With IGFBP6 In Vitro

The addition of 150μM H₂O₂ efficiently induced the upregulation of p16 ^INK4a and p21 in HUVEC at the transcriptional level (p16 ^INK4a : FC=2.29, P<0.001; p21: FC=1.53, P=0.005) (Figure 4A). The changes were accompanied by overexpression of p16^INK4a protein (control: 6.33±0.57; H₂O₂: 15.29±1.49; P=0.002) and upregulated SA‐β‐gal activity (control: 20.18±0.08%; H₂O₂: 57.00±7.18%; P<0.001) (Figure 4B through 4D). Captopril treatment could reduce the expression of p16INK4a protein (H₂O₂ + 100μM captopril: 5.93±1.07, P=0.002; H₂O₂+1 mmol/L captopril: 4.23±1.14, P<0.001) and downregulate SA‐β‐gal activity (H₂O₂ + 100 μM captopril: 22.30±1.76%, P<0.001; H₂O₂+1 mmol/L captopril: 21.89±1.92%, P<0.001).

Figure 4. Endothelial senescence induced senescence in chondrocytes.

A, Relative mRNA level of p16 ^INK4a and p21 in HUVECs under various conditions (n=3). B and C, Immunofluorescent and quantification of p16^INK4a (red) on HUVECs with DAPI (blue) (n=3). Scale bar 25 μm. D, Representative images and quantification of SA‐β‐gal assay in HUVECs (n=4). Scale bar 50 μm. E, Schematic diagram of CM coculture model. F, Relative mRNA level of COL10, MMP13, IHH, p16 ^INK4a , p21, and SOX9 in each group (n=3). Statistical comparison was performed between the experimental groups and the CM group. G and H, Representative images of immunofluorescent and quantification of COL10 (green) and p16^INK4a (red) with DAPI (blue) in C28/I2 after coculture (n=3). I, Representative images and quantification of SA‐β‐gal assay on C28/I2 after coculture (n=5). Scale bar 50 μm. J and K, Immunofluorescent and quantification of COL10 (green) and p16^INK4a (red) with DAPI (blue) on C28/I2 under direct challenge of H₂O₂ and captopril (n=3). L, Relative mRNA level of COL10, p16 ^INK4a , and p21 in each group (n=3). Statistical comparison was performed between experimental groups and the control. All data are shown as means±SE, and each data point represents an individual experiment. One‐way ANOVA with Tukey’s multiple‐comparisons test was employed for statistical analysis to compare between each experimental group and the control group. *P<0.05; **P<0.005; ***P<0.001; ****P<0.0001. CM indicates conditioned medium; Ctrl, Control; ECM, extracellular matrix; HUVEC, human umbilical vein endothelial cell; and SA‐β‐gal, senescence‐associated beta‐galactosidase.

We cocultured conditioned medium from HUVECs with chondrocytes to study the effect on chondrocyte homeostasis. To examine the impact of endothelial senescence on chondrocytes, H₂O₂ CM, was collected, filtrated, and diluted before being used to culture the chondrogenic C28/I2 cells. A coculture system with CM was employed to assimilate the avascular environment in articular cartilage layer (Figure 4E). With H₂O₂ CM incubation, C28/I2 demonstrated elevated expression of p21 and p16 at the mRNA level (p16 ^INK4a : FC=2.56, P=0.003; p21: FC=2.38, P=0.006) (Figure 4F), as well as p16^INK4a protein (CM: 8.64±0.88; H₂O₂ CM: 22.81±2.77; P<0.001) and SA‐β‐gal activities (CM: 18.30±0.98%; H₂O₂ CM: 41.55±3.76%; P<0.001) (Figure 4B through 4D; Figure 4G and 4I), implying senescent phenotypes in cells. There was also an increasing trend of hypertrophic markers, MMP13 and COL10, expression (MMP13: FC=1.90, P=0.20; COL10: FC=1.897, P=0.074). No alteration in SOX9 and IHH expression was observed (SOX9: FC=1.85, P=0.12; IHH: FC=1.03, P=0.99), implying that CM treatment did not affect the chondrogenic property of cells (Figure 4F).

Remarkedly, captopril CM seemed to clear senescent cells in the coculture system. Cells that received 100 μM or 1 mmol/L captopril CM treatment had diminished COL10 (H₂O₂ CM: 16.03±1.34; H₂O₂+100 μmol/L captopril CM: 7.65±1.80, P=0.013; H₂O₂+1 mmol/L captopril CM: 6.04±2.00, P=0.004). The expression of p16^INK4a was also lowered (H₂O₂+100 μmol/L captopril CM: 4.19±0.65, P<0.001; H₂O₂+1 mmol/L captopril CM: 4.90±0.32, P<0.001). SA‐β‐gal assay demonstrated population decline of senescent cells (H₂O₂+100 μmol/L captopril CM: 13.13±0.64%, P<0.001; H₂O₂+1 mmol/L captopril CM: 12.85±1.18%, P<0.001) (Figure 4G and 4I). Expression levels of p16 ^INK4a and p21 in both groups dropped to comparable levels as the control group (H₂O₂+100 μmol/L captopril CM: p16 ^INK4a : FC=0.39, P=0.003; p21: FC=0.42, P=0.006; H₂O₂+1 mmol/L captopril CM: p16 ^INK4a : FC=0.40, P=0.003; p21: FC=0.44, P=0.007) (Figure 4F). Direct addition of captopril to chondrocyte culture failed to elicit the same effects (H₂O₂+100 μmol/L captopril CM: COL10: FC=0.89, P=0.94; p16 ^INK4a : FC=0.73, P=0.70; p21: FC=0.64, P=0.11; H₂O₂+1 mmol/L captopril CM: COL10: FC=0.90, P=0.95; p16 ^INK4a : FC=1.02, P=0.99; p21: FC=0.85, P=0.70) (Figure 4J through 4L). The result suggested that the captopril treatment not only restored HUVECs’ normal state but also their secretome, and thereby attenuated chondrocyte senescence.

Conditioned media used in the in vitro experiment were further analyzed by proteomics to understand the changes in the secretome of endothelial cells upon stress and treatment. H₂O₂ CM showed the most prominent changes in secretome (9 downregulated, 11 upregulated proteins) compared with normal CM, whereas captopril CM almost completely rescued its effect (1 down‐ and 3 upregulated compared with control) (Figure S4A through S4C). H₂O₂ treatment‐elevated secreted proteins mainly enriched in functions related to extracellular matrix, growth factor binding, and mechano‐sensing (integrin binding and laminin binding), whereas the treatment repressed secretion of proteins related to energy metabolism (glycolysis and NADH regeneration) as well as plasminogen activation (Figure 5A). A heatmap of the selected 17 proteins of interest showed a distinguishable pattern as regulated by H₂O₂ challenge and drug rescue (Figure 5B). The results partly resembled the findings in RNA sequencing data, such as upregulation of IGFBP6 in early SHR endothelial cells.

Figure 5. Insulin‐like growth factor‐binding protein 6 plays a role in crosstalk between endothelial cells and chondrocytes in a human cell line model.

A, Divided the 17 proteins of interest into 2 groups to analyze their functional enrichment according to their response to H₂O₂ challenge. B, Heatmap showing relative expression of proteins across groups. (n=4). C and D, SA‐β‐gal staining in C28/I2 cells with or without IGFBP6. (n=3). E, Relative mRNA level of p16 ^INK4a and p21 in C28/I2 cells with or without IGFBP6. (n=3) (F and G) SA‐β‐gal staining in C28/I2 cell for control, H₂O₂, IGFBP6 antibody, and H₂O₂+IGFBP6 antibody group. (n=3). H, Relative mRNA level of p16 ^INK4a for control, H₂O₂, IGFBP6 antibody, and H₂O₂ + IGFBP6 antibody group. (n=3) All data are shown as means±SE, and each data point represents an individual experiment. One‐way ANOVA with Tukey’s multiple‐comparisons test was employed for statistical analysis to compare between each experimental group and control group. *P<0.05; **P<0.005; ***P<0.001. ECM indicates extracellular matrix; IGFBP6, insulin‐like growth factor‐binding protein 6; and SA‐β‐gal, senescence‐associated beta‐galactosidase.

Both transcriptomic and secretomic analyses indicated that endothelial cells expressed a higher level of IGFBP6. After stimulating C28/I2 cells with 1μg/ml IGFBP6 protein, cells had increased SA‐β‐gal activities (control: 22.53±4.21%; stimulated: 63.07±11.27%; P=0.028) and elevated expression of p16 ^INK4a mRNA (p16 ^INK4a : FC=2.29, P=0.036; p21: FC=1.95, P=0.180) (Figure 5C through 5E). We collected the culture medium of endothelial cells with the addition of H₂O₂ and IGFBP6 antibody to culture chondrocytes. The results showed that compared with the case where only H₂O₂ (72.07±4.97%) was added, the addition of IGFBP6 antibody (31.10±4.02%) can effectively reduce the generation of senescent cells (SA‐β‐gal: P=0.003; p16 ^INK4a : FC=0.52, P=0.046) (Figure 5F through 5H). This implied that IGFBP6 may play a role in crosstalk between endothelial cells and chondrocyte senescence.

Captopril Rescues Chondrocyte Senescence Induced by Endothelial Cells Under Oxidative Stress Associated With IGFBP6 In Vivo

Captopril is a Food and Drug Administration‐approved antihypertensive drug frequently prescribed to patients. To validate the effect of captopril in vivo, SHR were administered captopril at 50 mg/kg (Figure 6A). The treated group showed statistically significant reductions in blood pressure (at 14 weeks: SHR: 258.80±17.20 mm Hg; captopril: 182.50±3.33 mm Hg; P=0.011) (Figure 6B). Meanwhile, aortic senescence was alleviated after captopril treatment (−14.7 [95% CI, −23.68 to −5.723]; SHR: 39.95±3.72%; captopril: 25.25±1.56%; P=0.0045) (Figure 6C and 6D). This result suggested that captopril improved vascular health both functionally and phenotypically. Immunostaining with 4HNE and CD31 also revealed reduced vascular oxidative stress in SHR following treatment with captopril (Figure 6E). The expression level of IGFBP6 in synovium was lower in the captopril‐treated group compared with the saline‐treated group (Figure 6F and 6G) (SHR: 90.10±10.10; captopril: 38.85±7.00; P=0.002). The synovium of captopril‐treated SHR showed lower staining intensity of fibrotic content (SHR: 12.80±1.75%; captopril: 2.48±1.13%; P<0.001) and α‐SMA (SHR: 137.0±3.08; captopril: 113.3±5.83; P=0.0048), indicating lessened fibrosis (Figure 6H through 6K). A reduction in senescent cell accumulation was observed in the synovial tissue (SHR: 21.43±2.48%; captopril: 9.10±1.63; P=0.002) (Figure 6L and 6M). The expression level of IGFBP6 in bone tissue was lower in the captopril‐treated group compared with the saline‐treated group (SHR: 174.9±20.35; captopril: 124.2±6.41; P=0.04) (Figure 6N and 6O). Interestingly, captopril was able to rescue the bone mineral density of the subchondral bone (0.03694 [95% CI, 0.001– 0.07335]; SHR: 0.3595±0.0098 g/cm³; captopril: 0.3964±0.0131 g/cm³; P=0.047) (Figure 6P and 6Q). However, bone volume fraction, trabecular thickness, and trabecular number were not restored (Figure S5). Following captopril treatment, the proteoglycan content in cartilage was significantly higher, and Osteoarthritis Research Society International score decreased compared with the saline‐treated group (−0.8333 [95% CI, −1.165 to −0.5012]; SHR: 2.17±0.11; captopril: 1.33±0.11; P<0.001) (Figure 6R and 6S). The clearance of senescent cells was not only limited in vasculature but also noticeable in cartilage (SHR: 47.24±3.44; captopril: 22.47±3.86; P<0.001) (Figure 6T through 6U). Expression of MMP13 in articular cartilage was significantly attenuated (−14.56 [95% CI, −22.03 to −7.089]; SHR: 28.94±2.97; captopril: 14.38±1.56; P=0.0015) (Figure 6V through 6W). These data suggested captopril also exerted chondroprotective effect apart from its antihypertensive function.

Figure 6. Captopril attenuated joint structural and functional deterioration in spontaneously hypertensive rats at 9 months old.

A, Flow diagrams on grouping and experimental design for evaluation of the therapeutic effects of captopril treatment. B, Systolic blood pressure in SHR and SHR after captopril treatment. C and D, Immunohistochemistry staining and quantification of p16ink4a in aorta of captopril‐treated and saline‐treated 9‐month‐old SHR. (N=6, 3 male and 3 female). E, Representative immunofluorescent staining of 4HNE and CD31 in synovial vasculature in 9‐month‐old SHR and SHR after captopril treatment. F and G, Immunohistochemistry staining and quantification of IGFBP6 in synovium of captopril‐treated and saline‐treated 9‐month‐old SHR. (N=6, 3 male and 3 female). H and K, Representative images and quantification of α‐SMA and Masson’s Trichrome staining in synovium of 9‐month‐old SHR and SHR after captopril treatment. (N=6, 3 male and 3 female). L and M, Immunohistochemistry staining and quantification of p16ink4a in synovium of captopril‐treated and saline‐treated 9‐month‐old SHR. (N=6, 3 male and 3 female). N through O, Immunohistochemistry staining and quantification of IGFBP6 in bone of captopril‐treated and saline‐treated 9‐month‐old SHR and SHR after captopril treatment. (N=6, 3 male and 3 female). P and Q, Representative images and quantification of micro‐computed tomography result for subchondral bone of joint for 9‐month‐old SHR and SHR after captopril treatment. (N=6, N=6, 3 male and 3 female). R, Representative images of Safranin‐O/Fast Green staining for 9‐month‐old joints of SHR and SHR after captopril treatment. (N=6, 3 male and 3 female). S, OARSI score for treated and saline‐treated 9‐month‐old SHR and SHR after captopril treatment. (N=6, 3 male and 3 female). T and U, Immunohistochemistry staining and quantification of p16ink4a in cartilage of captopril‐treated and saline‐treated 9‐month‐old SHR. (N=6, 3 male and 3 female). V and W, Immunohistochemistry staining and quantification of MMP13 in cartilage of captopril‐treated and saline‐treated 9‐month‐old SHR. Scale bar 50 μm. (N=6, 3 male and 3 female) All data were presented as mean±SE. Two‐tailed Student’s t test (unpaired) was performed for staining intensity and percentage of positively stained cells between SHR and captopril‐treated group. *P<0.05; **P<0.005; ***P<0.001. α‐SMA indicates alpha smooth muscle actin; BMD, bone mineral density; IGFBP6, insulin‐like growth factor‐binding protein 6; MMP13, matrix metalloproteinase 13; OARSI, Osteoarthritis Research Society International; and SHR, spontaneously hypertensive rats.

DISCUSSION

Our study elucidates a mechanistic link between systemic vascular endothelial dysfunction and osteoarthritis progression, mediated by IGFBP6‐dependent insulin‐like growth factor signaling. In SHR, elevated aortic oxidative stress and reduced joint oxygen saturation at 3 months, markers of early vascular dysfunction, preceded accelerated chondrocyte senescence and whole‐joint degeneration by 9 months. In vitro, H₂O₂‐induced endothelial dysfunction directly triggered chondrocyte senescence, and multiomics profiling identified oxidative stress and IGFBP6 as critical mediators of endothelial‐chondrocyte crosstalk. Therapeutically, captopril‐mediated blood pressure reduction ameliorated endothelial dysfunction, attenuating joint senescence and structural damage. Our finding positions vascular pathology as a modifiable driver of osteoarthritis and highlights IGFBP6 as a potential therapeutic target.

Although SHR are the well‐established models for studying cardiovascular diseases, hypertension‐driven joint damage was less investigated.³⁵ Previous studies employed Sprague–Dawley controls rather than the genetically matched WKY strain. ³⁵ This critical methodological distinction limits mechanistic interpretation, as Sprague–Dawley rats differ from SHR in hypertension‐unrelated genetic and physiological traits. Our work resolves this by comparing stroke‐prone SHR to WKY controls, directly isolating hypertension‐specific effects. ³⁶ We demonstrated that stroke‐prone SHR develop spontaneous osteoarthritis changes without surgical induction in prior models. Notably, we have identified endothelial dysfunction beginning at 3 months, significantly earlier than joint degeneration reported in 9‐month‐old SHR, ³⁶ suggesting hypertension‐related genes may drive osteoarthritis pathogenesis via vascular mechanisms. This timeline aligns with our multiomics data implicating oxidative stress and IGFBPs in endothelial‐chondrocyte crosstalk, revealing a preclinical time window for vascular‐targeted interventions.

Our previous study investigated the effects of DOCA‐salt, a secondary hypertension model induced by exogenous mineralocorticoid activation, on joint tissues. However, the potential confounding influence, notably DOCA’s direct off‐target effects on joint tissues, could not be ruled out. In contrast, the SHR model develops hypertension through mechanisms mimicking human essential hypertension, representing a distinct form of hypertension. To our knowledge, no prior studies have systematically examined the role of primary hypertension in joint aging, leaving this a critical unanswered question that our research now addresses. Although our earlier work demonstrated that DOCA‐induced hypertension leads to senescence‐associated phenotypic changes (eg, p16 expression) in joints, it did not explore the upstream molecular drivers. In the present study, we identified Igfbp6 as a key mediator in hypertension‐associated joint senescence. Furthermore, other studies show mechanosensitive pathways (eg, PIEZO1) and senescence‐associated secretory phenotype factors are implicated in this process, ³⁷ offering new avenues for mechanistic exploration.

Our findings highlighted oxidative stress as a critical mediator of endothelial‐cartilage crosstalk in osteoarthritis. SHR exhibited elevated oxidative stress in aortic and knee synovial vasculature, consistent with our prior work demonstrating that vascular dysfunction and hypoxia preceded joint damage in posttraumatic osteoarthritis models. ¹⁴ This aligns with broader evidence implicating oxidative stress in vascular aging. ³⁸ To decipher this mechanism, we investigated endothelial dysfunction in vitro using H₂O₂‐induced senescence, a strategy that recapitulated the oxidative stress‐driven chondrocyte senescence observed in vivo. These results underscored oxidative stress as a systemic precursor to joint degeneration, offering a mechanistic basis for vascular‐targeted osteoarthritis interventions.

Beyond oxidative stress, multiomics profiling identified IGFBP6 as a key mediator of endothelial‐chondrocyte communication. Both the results of animal endothelial cell RNA‐seq and the secretome analysis of HUVECs found that the levels of IGFBP6 increased. Our work hinted at the involvement of IGFBP6 in endothelial dysfunction‐associated osteoarthritis. We observed robust upregulation of IGFBP6 in SHR vasculature and joints, particularly under H₂O₂ stimulation. IGFBP6, a selective IGF‐2 inhibitor, holds dual relevance: its elevation in rheumatoid arthritis and osteoarthritis patient sera ³⁹ and its mechanosensitive expression in cartilage ⁴⁰ , ⁴¹ suggest context‐dependent roles. Critically, we demonstrate that IGFBP6 exacerbates chondrocyte senescence, a finding distinct from its reported reduction in mechanically overloaded cartilage. ⁴⁰ This paradox highlights the complex regulation of IGFBP6, potentially modulated by vascular‐derived signals in hypertension.

Our study also demonstrated that captopril, an angiotensin‐converting enzyme inhibitor, reduces endothelial oxidative stress and senescent cell burden in SHR, thereby attenuating chondrocyte senescence and joint degeneration. Although captopril’s antihypertensive ⁴² and antioxidant ⁴³ properties have been reported, we uniquely showed its indirect chondroprotective effects via rescue of endothelial senescence, underscoring endothelial‐chondrocyte crosstalk as the therapeutic target. Our findings are consistent with clinical observations of antihypertensive medicine. For example, a combination of amlodipine and celecoxib was employed to treat hypertension and osteoarthritis pain. ⁴⁴ Another group of antihypertensive drugs, beta blockers, also showed potency in symptom relief for patients with knee osteoarthritis with relatively low pain scores. ⁴⁵ Our work provides mechanistic evidence to position antihypertensives as systemic senotherapuetics for comorbid osteoarthritis.

Our study has several major limitations to be taken into consideration in data interpretation. A key limitation is our limited investigation of sex differences in joint degeneration and disease. Although preliminary data showed both male and female SHR developing elevated blood pressure by 3 months of age (Figure 1A), male SHR maintained higher pressures and had shorter lifespans. Based on these observations, we focused on male SHR for subsequent analyses. Future studies should include female SHR to elucidate potential sex‐specific variations in disease progression and outcomes. Second, our study focused solely on radiographic osteoarthritis, but the association between hypertension and symptomatic osteoarthritis should not be overlooked. Notably, emerging evidence suggests that hypertension may protect against symptomatic osteoarthritis joint pain, ¹³ even though it exacerbates radiographic osteoarthritis. Our unpublished data have shown that SHRs exhibit lower pain sensitivity than WKY controls, implying a dissociation between structural joint damage and pain perception in hypertensive models. We are going to investigate hypertension‐associated analgesia and its implications for symptomatic osteoarthritis in future study. Last but not least, our study is limited by the H₂O₂‐induced senescence model in HUVECs, which, although well established and preserving key pathways relevant to hypertension, ⁴⁶ , ⁴⁷ , ⁴⁸ does not fully simulate chronic hypertensive conditions. To better capture the pathophysiological microenvironment, future work could employ advanced systems such as organ‐on‐a‐chip or microfluidic platforms to deliver more physiologically relevant mechanical and biochemical cues.

CONCLUSIONS

In conclusion, our study elucidates vascular dysfunction as a systemic driver of osteoarthritis, linking endothelial oxidative stress in earlier age to chondrocyte senescence and proteoglycan loss in later life of SHR. We identify IGFBP6 as a plausible mediator of endothelial‐chondrocyte crosstalk. Moreover, we demonstrate that captopril rescues joint health by targeting endothelial senescence, not through direct chondrocyte action. These findings bridge hypertension and osteoarthritis and advocate for vascular‐targeted therapies for osteoarthritis.

Sources of Funding

This work was supported by Health and Medical Research Fund Scheme (16172691#), Research Grants Council of Hong Kong General Research Fund (PolyU15100821M), National Natural Science Foundation of China/Research Grants Council Collaborative Research Scheme (N_PolyU 520/20) and the Hong Kong Polytechnic University Project of Strategic Importance (ZE2C). Natural Science Foundation of Guangdong Province (Grant No. 2020A1515010451), and Science and Technology Program of Guangzhou (Grant No. 202201010913). The authors would also like to thank the Hong Kong Polytechnic University, Hong Kong Baptist University, and The Third Affiliated Hospital of Sun Yat‐sen University for providing equipment and technical and financial support.

Disclosures

None.

Supporting information

Figures S1–S5