Browsing by Author "Kraus, Virginia Byers"

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Osteoarthritis (OA) is the most common form of arthritis in the population worldwide, resulting in significant disabilities. Currently, no treatments can prevent or reverse the development of OA. Cellular senescence has been identified as a major contributing factor to OA. Therefore, a therapy targeting senescence could be an effective treatment for OA. Several strategies have been proposed to target senescence in OA, including neutralizing agents for the senescence-associated phenotypes (SASPs), senolytics for eliminating senescent cells, and senomorphics for modifying the senescence phenotype. In fact, a senolytic, UBX0101, showed a protective effect for post-traumatic OA (PTOA) development in mouse models by reducing both OA histological grading and OA related pain. However, it failed to meet the primary endpoint of relieving symptoms in a clinical phase 2 trial. The failure of the human trial may be related to a high placebo response rate from the control group or, more likely, the heterogeneous phenotypes involved in human OA disease differ from the PTOA in the mouse model. The lack of available senescent-specific biomarkers, which could be used to refine the phenotype of the subject enrollment or to monitor the occurrence of senescence presents a challenge to evaluate a trial successfully. Alternatively, senolytics may be detrimental in tissue with a higher proportion of senescent cells. After treatment, the remaining cells may not be able to maintain the integrity of the cartilage. Therefore, in the present study, we investigated the association of cellular senescence with OA disease severity, identified a biomarker dipeptidyl peptidase-4 (DPP4) for chondrocyte senescence and OA progression, and proposed a senomorphic treatment using chromobox 4 (CBX4) for modulating cell function of the replicative senescent model WI-38 cells and human osteoarthritic chondrocytes.First, we investigated the association of OA disease severity in human knee joints with the percentage of cells expressing senescence-associated β-galactosidase activity (SA-β-gal) and p16. We assessed three regions within the tibial plateau of the knee corresponding to a gradient of disease severities in tissue procured from human medial-dominant OA. We found that SA-β-gal and p16 were positively correlated with OA severity. Our result suggested cellular senescence could be involved in OA progression, and targeting senescence could be a promising treatment for OA. Subsequently, we identified Dipeptidyl-peptidase 4 (DPP4, also known as CD26) as a potential biomarker for OA senescence. We isolated chondrocytes from knee OA cartilage and determined the association of DPP4 expression with senescence markers, SA-β-gal and p16, by flow-cytometry. We also compared the expression of anabolic and catabolic genes, senescence-related genes, and senescence-associated secretory phenotypes (SASPs) in DPP4+ and DPP4- cells, isolated by two different methods: fluorescence-activated cell sorting and magnetic-activated cell sorting. Additionally, we quantified soluble DPP4 in synovial fluid (SF) and assessed its association with radiographic knee OA. DPP4 expression was associated with higher SA-β-gal, p16 expression, senescence-related gene and catabolic gene expression, SASPs secretion, and lower anabolic gene expression in chondrocytes. In addition, SF DPP4 was significantly associated with radiographic knee OA progression (β=4.657 p=0.015). Next, we identified the senomorphic effect of CBX4 in WI-38 cells. We first observed the decrease of CBX4 protein expression and increased senescence markers and gene expression during WI-38 serial culture. We next evaluated the presence of senescence markers and expression of senescence-related genes in the CBX4 activation and CBX4 knockdown compared to controls in pre-senescent WI-38. Compared to the control group, knockdown of CBX4 increased cellular senescence, whereas activation of CBX4 decreased senescence in the pre-senescent WI-38 cells. Based on these results, we identified that CBX4 regulates replicative senescence in WI-38 cells and functions as a senomorphic and potential anti-senescence target. Additionally, we explored the mechanisms of senescence regulation from CBX4 domains by using CBX4 mutated lentiviral particles and compared them with CBX4 wildtype in WI-38 cells. CDM, SIMs, and C-box domains are all involved in the regulation of senescence by CBX4; where CDM is mainly involved in cell cycle regulation, SIMs are involved in the cell proliferation, DNA damage repair, and SASPs secretion, and C-box is related to cell proliferation and SASPs secretion. Taken together, CBX4 is a multi-functional protein, and these mutants elucidated the different non-overlapping functions in senescence regulation. Finally, we identified the CBX4 senomorphic effect in osteoarthritic chondrocytes by comparing CBX4 wildtype and control transduced cells. Compared to control, CBX4 overexpressed chondrocytes demonstrated lower DPP4 expression and higher proliferation marker EdU. Overall, our study demonstrated that cellular senescence is positively correlated with OA disease severity, identified DPP4 as a potential biomarker for cellular senescence in OA, and explored CBX4 as a potential senomorphic treatment in human WI-38 fibroblasts and OA chondrocytes.

Articular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage.

In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction.

Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well.

Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states.

Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage.

We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage.

The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.

Osteoarthritis (OA) is the most common joint disorder causing chronic disability in the world population. By the year 2030, an estimated one fifth of this population will be affected by OA. Although OA is regarded as a multi-factorial disorder with both environmental and genetic components, the exact pathogenesis remains unknown.

In this study, we hypothesize that biomarkers associated with OA can be used as quantitative traits of OA, and provide enough power to identify new genes or replicate known gene associations for OA. We established an extensive family called the CARRIAGE (CARolinas Region Interaction of Aging, Genes and Environment) family. Then, we measured and analyzed seven OA-related biomarkers (HA, COMP, PIIANP, CPII, C2C, hs-CRP and GSP) in this extensive family to evaluate their association with OA clinical phenotypes. These findings suggest that OA biomarkers can reflect hand OA in this large multigenerational family. Therefore, we performed nonparametric variance components analysis to evaluate heritability for quantitative traits for those biomarkers. Finally, based upon OA biomarkers with high heritability, we performed a genome-wide linkage scan. Our results provide the first evidence of genetic susceptibility loci identified by OA-related biomarkers, indicating several genetic loci potentially contributing to the genetic diversity of OA.

Meanwhile, we identified joint hypermobility as a factor which reduces OA risk and has an inverse association with serum COMP levels in this family. The relationship between lower serum COMP and OA have been further validated in another Caucasian GOGO (Genetics of Generalized Osteoarthritis) population. Therefore, we further hypothesize that joint hypermobility, having the characteristic of a decreased OA risk, can serve as a quantitative trait for identifying protective loci for OA. Then, we performed nonparametric variance components analysis to evaluate the heritability of joint hypermobility. The result also shows joint hypermobility has substantial heritable components in this family. Lastly, based on the same genome-wide linkage scan, we identify genetic susceptibility loci for joint hypermobility.

In conclusion, our work provides the first linkage study to identify genetic loci associated with OA using biological markers. Furthermore, we have also shown genetic susceptibility loci for joint hypermobility, possibly implying protective loci for OA.

Selenium (Se) is an essential trace element and metalloid involved in several key metabolic activities: protection against oxidative damage, regulation of immune and thyroid function, and fertility. Several recent lines of evidence from epidemiology, genetic, and transgenic animal studies suggest that Se may play a protective role in Osteoarthritis (OA). However, the exact protective mechanism of Se is still unclear.

In this study, we hypothesized that Se exerts its chondroprotective benefit via an anti-oxidative and anti-inflammatory effect mediated by specific selenoproteins that neutralize cytokine-induced inflammatory responses in chondrocytes. We established an in vitro system for studying the effect of Se in the chondrosarcoma cell line SW-1353 and in human primary chondrocytes. Selenomethionine (SeMet) induced gene expression and enzyme activity of both antioxidative enzymes glutathione peroxidase (GPX) and thioredoxin reductase (TR) in SW-1353 cells. Our data suggest that Se may be protective against oxidative stress through regulation of the activity of these antioxidative enzymes.

As IL-1β is one of the primary pro-inflammatory cytokines contributing to the progression in OA, we next investigated the effect of Se on the gene expression induced by physiological doses of IL-1β. SeMet inhibited IL-1β induced catabolic gene expression of matrix metalloproteinase 1 (MMP1) and MMP13 as well as total MMP activity in chondrocytes. Similarly, SeMet inhibited chondrocyte gene expression of IL-1β induced nitric oxide synthase (iNOS) and cyclooxygenase (COX2) with corresponding reductions in nitric oxide (NO) and prostaglandin E2 (PGE2) production. In addition, SeMet pretreatment attenuated the IL-1β induced activation of p38 MAPK but not the ERK, JNK or NFkB pathways. Taken together, our results suggest that Se inhibits IL-1β induced expression of inflammatory and catabolic genes, partly through inhibition of IL-1β cell signaling.

Since Se may function through selenoproteins, we evaluated the role of three specific major selenoproteins, GPX1, TR1 and DIO2, in modifying the inflammatory response stimulated by IL-1β in chondrocytes by RNA interference. Based on RNA interference results, DIO2 and TR1 mediated the inhibitory effect of SeMet on IL-1β induced COX2 gene expression, while GPX1 did not show a significant inhibitory effect on Se. Depletion of DIO2 increased the IL-1β induced COX2 gene expression. This suggests that DIO2 may negatively modulate the IL-1β response. Our data also suggest that part of this inhibitory effect of DIO2 could be through regulation of IL-1β gene expression itself. These results highlight a potential new role of DIO2 in modulating the inflammatory response in chondrocytes

In summary, the result of this study suggests that Se may exert its chondroprotective effect through specific selenoproteins which neutralize oxidative stress and modify the inflammatory response in chondrocytes.

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