Increased vascular endothelial growth factor expression is associated with cruciate ligament degeneration in patients with osteoarthritis of the knee

Abstract

Background

This study aimed to investigate the expression of vascular endothelial growth factor (VEGF) in cruciate ligaments from patients with osteoarthritis (OA). It was hypothesized that the expression level of VEGF is associated with the extent of degeneration of the cruciate ligaments.

Methods

Remnants of anterior cruciate ligaments (ACLs) from patients with acute ACL injury due to trauma, and ACLs and posterior cruciate ligaments (PCLs) from patients with primary OA were assessed histologically. Samples were immunohistochemically stained with VEGF and tenomodulin, and immunopositive cells were quantitatively assessed by the histological grades of ligament degeneration.

Results

Histological analysis showed significant degeneration of the ACLs from OA patients compared with trauma patients, with increased expression of VEGF correlating with higher grades of degeneration. Conversely, tenomodulin expression was lower in more degenerated cruciate ligaments. The percentage of VEGF-positive cells was correlated inversely with that of tenomodulin-positive cells.

Conclusions

Increased VEGF expression is associated with degeneration of cruciate ligaments in patients with osteoarthritis of the knee.

Introduction

Osteoarthritis (OA) of the knee is a degenerative joint disease affecting the whole joint organ, including articular cartilage, subchondral bone, synovial tissues, and menisci. In addition, deterioration of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) is also seen in OA knees. Severe degeneration of the cruciate ligaments is often observed in patients with high-grade OA [1], and loss of joint stability is a risk factor for the development of OA [2]. Although the suppression of degenerative change in cruciate ligaments seems to be one strategy to prevent OA progression, the detailed biological mechanisms underlying ligament degeneration have not been well investigated.

Histopathological changes in degenerative human ligaments have been characterized by disorganization of the collagen fiber arrangement, mucoid changes, chondroid metaplasia, and cystic changes [1, 3]. Although ligaments are poorly vascularized tissues compared with other tissues such as bone and skin [4], a recent study suggested that inflammatory processes of knee OA lead to increased vascularization, as well as calcification and formation of fibrocartilage-like tissue in the ACL [5]. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor, and its involvement in OA pathogenesis has been widely investigated. A meta-analysis suggested that VEGF expression levels in multiple disease tissues of OA patients are associated with the pathogenesis of OA [6]. However, VEGF expression levels in the cruciate ligaments of OA patients are not known and need to be elucidated.

The purpose of this study was to investigate the expression of VEGF in the cruciate ligaments of OA patients. It was hypothesized that the expression level of VEGF is associated with the extent of degeneration of cruciate ligaments.

Materials and methods

Participants

Ten ACL remnants were harvested from 10 patients with acute ACL injury due to trauma (mean age: 28 years; range: 16–55 years; 3 females, 7 males) who underwent ACL reconstruction (mean time: 70.2 days after the injury), and 10 ACLs and 10 PCLs were harvested from 10 patients with primary OA (all K-L grade 4; mean age: 75 years; range: 61–84 years; 5 females, 5 males) who underwent posterior stabilized total knee arthroplasty. Patients who had a history of previous knee surgery, inflammatory arthritis, joint infection, or immunosuppressive therapy were excluded from the study. The present study was approved by the Institutional Review Board at the hospital where the surgeries were performed, and informed consent was obtained from all patients. For minors (age under 18 years), informed assent from patients and informed consent from their parents were obtained.

Sample preparation for histological analysis

Sample preparation was conducted according to the previously described method [7]. Briefly, the mid portions of ligaments were excised and preserved immediately in 10% neutral buffered formalin. The samples were then dehydrated in an ascending series of ethanol and embedded in paraffin. Paraffin-embedded samples were sectioned longitudinally (thickness: 5 μm). The sections were stained with haematoxylin/eosin according to the standard protocols.

Immunohistochemical staining

The sections were deparaffinized and treated with citrate buffer for antigen activation. Endogenous peroxidase was quenched with 0.3% H₂O₂ in methanol. Nonspecific binding was blocked with 10% sheep serum in phosphate-buffered saline (PBS). Anti-VEGF (Catalog Number:19003-1-AP, Proteintech, Rosemont, IL, USA) or anti-tenomodulin antibodies (Catalog Number: LS-B8193, LSBio, Seattle, WA, USA) were incubated at room temperature for 60 min. Slides were washed with PBS and incubated with N-Histofine Simple Stain MAX PO(MULTI) (Nichirei Biosciences, Tokyo, Japan) for 30 min at room temperature. The slides were washed with PBS, and the reaction was visualized by colorization with 3,3’-Diaminobenzidine tetrahydrochloride (DAB). Finally, sections were contrast-stained with haematoxylin and mounted on coverslips.

Evaluation of degenerative changes in cruciate ligaments

Five high-power fields per section were randomly captured using an all-in-one microscope (BZ-X800, Keyence, Osaka, Japan), which was equipped with a digital camera (CFI 60, Nikon Corporation, Tokyo, Japan). Each field was assessed for cellular arrangement and morphological changes of cell nuclei according to the previously described method [7]. Briefly, the angles between the long axis in each cell and the direction of collagenous fibres were measured, and the cellular arrangement was scored as follows: 0 points, alignment less than 10 degrees in all cells examined; 1 point, alignment of 11–45 degrees in less than 50% cells and less than 10 degrees in the rest; and 2 points, alignment greater than 10 degrees in more than 50% cells or the existence of cells with alignment over 45 degrees. The morphological changes of the cell nuclei were scored as follows: 0 points, no round nuclei; 1 point, round nuclei < 50%; and 2 points, round nuclei ≥ 50%. The round-shaped nuclei were defined as those with a length-to-width ratio less than 2:1 (long axis/short axis < 2). When both variables scored 0 points, or either variable scored 1 point, the field was defined as grade 1. When both variables showed abnormal changes (1 or 2 points), and at least one variable scored 2 points, the field was defined as grade 3. Consequently, each field was graded by the total score: total score 0 or 1 = grade 1; total score 2 = grade 2; and total score 3 or 4 = grade 3 (Fig. 1). For the assessment of degenerative grade, at least five different high-power fields were examined in each ligament, and each parameter was calculated. Two independent observers assessed degenerative grade. The kappa coefficient value in the grading score was 0.67, indicating good agreement for inter-observer reproducibility.

Fig. 1

Photomicrographs showing assessment of cellular arrangement (upper panels) and morphological changes of cell nuclei (lower panels) in each degenerative grade. Upper panels: the angles between the long axis in each cell (dotted line) and the direction of collagenous fibres (arrow) are noted. Lower panels: round nuclei are circled. Left panels represent total score 0 (grade 1), center panels represent total score 2 (grade 2), and right panels represent total score 4 (grade 3)

Quantification of labelled cells

The numbers of total cells and VEGF- and tenomodulin-positive cells were counted in each field, and then the positive cell ratio was calculated in five different high-power fields per section. The results were compared between trauma and OA cases, or by the different degeneration grades of ligaments in OA patients.

Statistical analysis

Statistical analysis was carried out using BellCurve for Excel version 4.07 (Social Survey Research Information, Tokyo, Japan). All data are presented as mean and standard deviation values. Since normal distribution was confirmed in all dataset using Shapiro-Wilk test and Levene test (P > 0.05), Student’s t-test and one way analysis of variance with Tukey-Kramer post-hoc test were used for significant differences among groups. A p value < 0.05 was considered significant.

Results

Histological features of cruciate ligaments from trauma and OA cases are shown in Fig. 2, and degenerative changes in OA patients were graded in terms of cellular alignment and cellular shape (i.e., spindle or round) (Table 1). The ACLs from trauma patients showed a parallel fibre arrangement with spindle-shaped cell nuclei. In OA patients, most ACL samples were classified as advanced degeneration grade, characterized by irregular fibre arrangement with increased round-shaped cell nuclei, whereas most PCL samples showed mostly regular fiber arrangement with fewer round-shaped cell nuclei.

Fig. 2

Histological features of cruciate ligaments from patients with trauma and osteoarthritis (OA). The areas stained with pink represent the cytoplasm and extracellular matrix, and those stained with blue-purple represent the nuclei. Scale bar = 25 μm. ACL, anterior cruciate ligament. PCL, posterior cruciate ligament

Table 1 Classification of the grade of degeneration of cruciate ligaments from patients with trauma and osteoarthritis

Immunohistochemical expressions of VEGF and tenomodulin in ACLs were compared between trauma and OA cases (Fig. 3). VEGF-positive cells were mainly observed in round-shaped cells. Samples from OA cases showed significantly higher expression of VEGF than those from trauma cases (P < 0.01). Tenomodulin-positive cells were observed in both round-shaped cells and spindle-shaped cells. Samples from OA cases showed significantly lower expression of tenomodulin than those from acute injury cases (P < 0.01).

Fig. 3

Immunohistochemical expressions of VEGF (A) and tenomodulin (B) in anterior cruciate ligaments from patients with trauma and osteoarthritis (OA). Upper panels: low magnification (scale bar = 100 μm). Lower panels: high magnification (scale bar = 25 μm). Brown colour (arrowheads) represents immunopositive cells of VEGF (A) or tenomodulin (B), and blue-purple colour represents the nuclei. Graphs represent mean percentages of cells expressing VEGF (A) or tenomodulin (B) with standard deviations. *P < 0.01

Immunohistochemical expressions of VEGF and tenomodulin in ACLs and PCLs from OA cases were assessed in each degeneration grade (Fig. 4). The rate of VEGF-positive cells was significantly higher in grade 3 than in grade 1 and 2 (P < 0.05). In contrast, the rate of tenomodulin-positive cells was significantly lower in grade 3 than in grade 1 and 2 (P < 0.05).

Fig. 4

Immunohistochemical expressions of VEGF (A) and tenomodulin (B) in each degeneration grade of cruciate ligaments from patients with osteoarthritis. Scale bar = 25 μm. Brown colour (arrowheads) represents immunopositive cells of VEGF (A) or tenomodulin (B), and blue-purple colour represents the nuclei. Graphs represent mean percentages of cells expressing VEGF (A) or tenomodulin (B) with standard deviations. *P < 0.05

The rate of VEGF-positive cells in the cruciate ligaments was negatively correlated with the rate of tenomodulin-positive cells (r=-0.59, P < 0.01) (Fig. 5).

Fig. 5

Correlation of VEGF and tenomodulin expressions. r=-0.55, P < 0.01

Discussion

The most important findings of this study were that increased expression of VEGF and decreased expression of tenomodulin were seen in cruciate ligaments of patients with OA, and they were associated with degeneration grade. The results supported the initial hypothesis that the expression level of VEGF is associated with the extent of degeneration in cruciate ligaments.

The role of endogenous VEGF expression during degenerative processes has been less clear in cruciate ligaments than in tendons. The molecular and morphological features overlap in tendons and ligaments [8, 9], and the mechanism of tendon degeneration may be helpful to some extent for understanding that of ligament degeneration. VEGF is minimally expressed in healthy tendons, but its expression increases with degeneration [10]. Numerous potential elements, such as hypoxia, inflammatory cytokines, nerve signals, and mechanical load, are believed to increase VEGF expression in both acute and chronic tendon injuries [11]. Since VEGF has the ability to trigger the generation of matrix metalloproteinases while inhibiting the production of tissue inhibitors of matrix metalloproteinases (TIMPs) across various cell types such as endothelial cells, fibroblasts, and chondrocytes, it could have a substantial impact on the degenerative processes involved in tendons and ligaments [10]. In the present study, ACLs from OA patients showed greater expression of VEGF than ACLs from trauma patients, and their expression was significantly higher in advanced degeneration grades. Thus, increased expression of VEGF has the potential to be involved in the progression of degenerative changes in the cruciate ligaments of OA patients.

Tenomodulin is predominantly expressed in tendons and ligaments, and it is a well-accepted gene marker for the mature tendon/ligament lineage [12,13,14]. Tenomodulin is essential for maintaining the proper microenvironment and cellular behaviour necessary for tendon homeostasis [15]. Depletion of tenomodulin leads to scar formation with abnormal extracellular matrix (ECM) composition and accumulation of blood vessels in the injured tendon [16]. Although, to date, the details of changes in the expression of tenomodulin in cruciate ligaments associated with OA were not well known, the present study showed that expression of tenomodulin decreased as cruciate ligament degeneration progressed. Furthermore, this study showed a negative correlation between the expressions of tenomodulin and VEGF. Tenomodulin has an anti-angiogenic function [17], and local absence of tenomodulin in the injured tendinous tissue is associated with increased angiogenesis and strong expression of VEGF [18]. It is assumed that the interaction between tenomodulin and VEGF, that is decreased expression of tenomodulin promotes increased expression of VEGF, or increased expression of VEGF induces further suppressed expression of tenomodulin, but further studies to explore the direct causal relationships and mechanisms underlying these changes are needed.

The role of VEGF in OA has been extensively recognized. Higher levels of VEGF are linked to OA progression, and its effects are primarily found in cartilage [19]. Various studies have shown that VEGF signalling directly increases pre-catabolic mediators in chondrocytes [20, 21], and the addition of external VEGF raises levels of proinflammatory cytokines in these cells [21]. Inflammation, which often features synovitis of joints, plays a central role in OA progression. The production of VEGF in the synovium, which leads to synovitis, is driven by increased angiogenesis [22, 23], and this process of angiogenesis further enhances inflammation, creating a cycle in which each process exacerbates the other [24]. The consistent increase in proinflammatory mediators and VEGF in OA joints is thought to impact various intra-articular tissues, including the cruciate ligaments. Preclinical trials using animal models for anti-VEGF treatments in OA have shown positive effects on several tissues such as articular cartilage, subchondral bone, and synovial tissue [25,26,27,28]. The direct effects of anti-VEGF agents have been validated by their ability to promote anabolic activity and reduce catabolic processes in human OA articular cartilage [29]. Given these insights, anti-VEGF therapy holds potential for mitigating degeneration of cruciate ligaments, as well as degeneration of cartilage in OA patients, and it needs further investigation.

One potential limitation of this study could be the lack of a healthy control group without any rupture or degenerative changes. However, the aim of this study was to examine the differences in cellular activities associated with degeneration, and the findings reported are adequate to support the conclusions. Another limitation could be the lack of comparison between ACL and PCL. The observed differences in expressions of VEGF and tenomodulin among different degenerative grades may be ligament-specific since degeneration grades in ACL and PCL are very different.

Conclusions

This study demonstrated that increased expression of VEGF is associated with degeneration of cruciate ligaments in patients with osteoarthritis of the knee.