Age-related increase in matrix stiffness downregulates α-klotho in cartilage and induces cartilage degeneration

      Purpose: The lack of disease-modifying treatments for knee osteoarthritis (KOA) is attributed, in part, to an incomplete understanding of the molecular mechanisms driving disease development. The pathologic cascade currently associated with KOA includes inflammation, impaired autophagy, and cellular senescence, which are hallmarks of aging. Therefore, identification of therapeutic targets that can rejuvenate aged cartilage represents a promising research direction for the treatment of age-related KOA. The longevity protein, α-Klotho, plays an important role in the attenuation of cellular senescence. Studies have shown α-Klotho decreases with aging, whereas α-Klotho supplementation counteracts age-induced cartilage degeneration. However, little is known about the underlying mechanisms driving declines in α-Klotho expression in cartilage. Such knowledge may aid the development of novel therapeutics that promote a regenerative microenvironment for chondrocytes and maintain cartilage integrity during aging. Here, we sought to elucidate mechanisms leading to age-related declines in α-Klotho in cartilage. The extracellular matrix (ECM) plays a dynamic role in directing chondrocyte phenotype and cartilage homeostasis, and undergoes extensive biophysical remodeling characterized by decreased compliance (i.e., increased ECM stiffness) during aging. This increased stiffness is due, in part, to a loss of proteoglycans and increased collagen cross-linking. We hypothesized that an aged stiff microenvironment drives a decline in chondrocyte expression of α-Klotho, but a youthful soft microenvironment increases α-Klotho expression, thereby promoting a more healthy phenotype (Fig. 1).
      Methods: First, we evaluated the trajectory of α-Klotho expression and cartilage integrity with aging using histology, isolating knee cartilage from young (4-6 mo), middle-aged (10-12 mo), and aged (21-24 mo) male and female C57/BL6 mice, as well as young and middle-aged α-Klotho+/- mice. We then microdissected cartilage from young, middle-aged, and aged mice for LC-MS/MS mass spectrometry proteomics to further explore the mechanisms of natural aging on knee cartilage, performing gene set enrichment analysis (GSEA) to evaluate pathways of interest. Next, we engineered polyacrylamide gels within a physiological range of cartilage ECM stiffness (5kPa, 20kPa, and 100kPa) and seeded young or aged primary chondrocytes onto the gel surfaces. After 3 days in culture, we evaluated: (1) chondrogenicity (type II collagen expression), and (2) α-Klotho expression using immunofluorescenct staining. To explore the role of ECM stiffness in vivo, we administered β-aminopropionitrile (BAPN), an inhibitor of lysyl oxidase (LOX) activity, daily for four weeks. We next evaluated (1) cartilage integrity and (2) α-Klotho expression in chondrocytes by histology and immunofluorescence. Finally, to probe the potential mechanotransductive mechanisms associated with the effect of biophysical ECM properties on α-Klotho expression, we extracted 53 features of chondrocyte nuclear morphology across experimental groups using Cell Profiler software and performed principal component analysis (PCA) with nuclear morphology features as input variables.
      Results: As expected, histological studies confirmed progressive cartilage degeneration with aging starting from middle-aged mice, including reduced cellularity and decreased α-Klotho expression. Notably, histological evidence of KOA with aging was recapitulated in young and middle-aged Klotho+/- mice, suggesting that an age-related loss of α-Klotho may contribute to the onset of cartilage degeneration. The molecular changes observed in middle-aged mice were supported by GSEA, with three pathways significantly upregulated at middle-age, including: (1) Cell Adhesion, (2) Extracellular Matrix, and (3) Signal Transduction Activity or Receptor Binding. Therefore, we next asked whether the ECM influences α-Klotho expression and chondrogenicity in isolated chondrocytes. Independent of age, chondrocytes seeded on stiff substrates displayed an “aged phenotype”, as indicated by reduced type II collagen and α-Klotho expression. Conversely, chondrocytes seeded on soft substrates displayed a more “youthful phenotype”, as indicated by increased type II collagen and α-Klotho expression. The changes observed when cells were cultured on soft substrates were consistent with a more youthful nuclear morphology, and specifically, decreased eccentricity (i.e., more spherical nucleus). These in vitro findings were further supported by in vivo histological analysis, which revealed aged cartilage had less spherical nuclei. Moreover, the decreased sphericity was associated with decreased α-Klotho expression. Inspired by our in vitro findings, we tested whether modulation of biophysical ECM properties in vivo could exert similar effects on α-Klotho expression and cartilage integrity (Fig. 2A). BAPN injections increased α-Klotho expression and improved cartilage integrity in aged mice. Interestingly, histological findings revealed that BAPN administration induced a more spherical nuclear morphology, mimicking young chondrocytes (Fig. 2B). Notably, PCA of nuclear morphological features revealed BAPN-treated nuclei clustered with young nuclei but segregated from aged control nuclei (Fig. 2C). Specifically, BAPN-treated aged nuclei displayed decreased nuclear eccentricity (Fig. 2D), which was again inversely correlated with α-Klotho levels.
      Conclusions: Evidence of the accelerated cartilage degeneration in Klotho+/- mice suggests age-related declines in α-Klotho may contribute to the onset of OA. In vitro and in vivo experiments further indicated that a stiffer microenvironment, as is observed with aging, decreased α-Klotho and type II collagen expression. On the other hand, a softer microenvironment restored aged chondrocytes towards a more youthful phenotype with increased α-Klotho expression. Our preliminary findings also suggest that the rejuvenating effects of a softer microenvironment may be partially attributed to altered mechanotransductive signaling, as is suggested by alterations in nuclear morphology. These findings warrant future studies investigating the role of α-Klotho as a mediator of the biophysical effects of ECM on cartilage health.
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      Figure 1Hypothesis model. We hypothesized that an aged stiff microenvironment drives a decline in chondrocyte expression of α-Klotho, thereby impairs cartilage integrity. We tested this hypothesis using (1) 2D chondrocytes culture model on the engineered polyacrylamide gels and (2) randomized controlled trial evaluating the effects of BAPN injection, an inhibitor of lysyl oxidase activity. To probe the potential mechanotransductive mechanisms associated with the effect of the biophysical properties of ECM on α-Klotho expression, we also quantified nuclear morphological changes in these experimental models and investigated the association of the nuclear morphology with α-Klotho expression as well as cartilage integrity.
      Image 47
      Figure 2BAPN injection induces spherical nuclear morphology, a more youthful phenotype. We performed daily injection of BAPN to aged male mice for a total of 28 days (A). BAPN treatment resulted in changes in nuclear morphology, including more spherical nuclei (B, nuclei were false-colored to show their morphology). PCA revealed that BAPN treatment resulted in nuclei clustering with young nuclei, but segregated from aged control nuclei (C). Nuclei in BAPN-treated group displayed significantly lower eccentricity (i.e., more spherical nucleus) than those in the saline control group (D).