Richard Loeser - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

DESCRIPTION (Verbatim from the Applicant): The most important risk factor for the development of osteoarthritis (OA) is age but the exact mechanism by which aging contributes to the development of OA is not known. The overall objectives of this proposal are to ascertain the role of IGF-I as a chondrocyte survival factor and establish a model for how an age-related decline in chondrocyte responsiveness to IGF-I contributes to the increased prevalence of OA with age. A decline in the number of chondrocytes in cartilage due to loss of cells from cell death has been noted with aging. Increased cell death has also been noted in OA cartilage. The factors normally responsible for keeping chondrocytes alive are not known. Based on new preliminary studies, the primary working hypothesis for this proposal is that IGF-I functions as a key autocrine survival factor for chondrocytes. A second hypothesis is that signals from the matrix mediated through the integrin family of cell-matrix receptors work in synergy with IGF-I to promote survival in cartilage. In the proposed studies, the link between aging, cell death and IGF-I action will be explored using human ankle cartilage from tissue donors of various ages. Since age-related development of OA in the ankle is uncommon, studies of this tissue can be used to separate effects of aging from effects of degeneration and OA, a problem in studies utilizing cartilage from hips or knees. In selected studies, results will be compared to those obtained using knee chondrocytes to determine if differences exist between cells from the two joints which may relate to the greater propensity for OA to develop in the knee. The Specific Aims are to: 1) Determine if an aging-related reduction in the response to IGF-I results in an increased susceptibility of chondrocytes to cell death; 2) Determine if IGF-I acts in synergy with signals generated from the extracellular matrix via integrins to promote chondrocyte survival and 3) Determine if IGF-I cell survival signaling is impaired in chondrocytes from older adults. The results of these studies will provide important new information needed to better understand the link between aging and osteoarthritis, the most common cause of chronic disability in older adults.

DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the basic mechanisms by which signals generated through integrin receptors regulate chondrocyte function. The overall hypothesis driving this work is that changes in the cartilage extracellular matrix (ECM), including production of ECM protein fragments, are recognized by chondrocyte integrins and initiate a cascade of events intended to remodel the ECM but which in arthritis result in further matrix destruction (chondrocytic chondrolysis). The focus of this proposal is to determine the basic cellular mechanisms that control signals generated through the ?5?1 integrin which regulate production of catabolic mediators including cytokines and matrix metalloproteinases (MMPs). During the previous funding period, we have defined the signaling pathways that mediate MMP-13 production in response to fibronectin fragment (FN-f) stimulation of the ?5?1integrin and discovered reactive oxygen species (ROS) are necessary second messengers. Using an innovative proteomics approach we found that the MAP kinase family member JNK2 is oxidized in FN-f stimulated cells forming a Cys-SOH (sulfenic acid) intermediate. Sulfenic acid formation serves as a major mechanism by which ROS regulate cell signaling but its role in chondrocyte signaling has not been investigated. In his competitive renewal, we propose to determine the mechanism by which sulfenic acid formation regulates JNK2 activity in chondrocytes. We will determine the role of JNK2 activation in OA in vivo by studying the development of surgically-induced OA in JNK2-/- mice. Finally, we will determine if HB-EGF, upregulated and released when chondrocytes are stimulated by FN-f, promotes Rac activity to activate a co-signaling pathway that augments MMP-13 expression and cartilage matrix destruction. These studies will have significant impact on the field by defining key hubs in a signaling network that mediates cartilage matrix destruction. By discovering novel mechanisms by which ROS regulate this signaling network, the information can be used to develop a unique approach to altering redox-regulated catabolic signaling networks in arthritis that targets specific protein modifications. This represents a significant advance over the general inhibition of ROS production which has not proven successful in treating conditions promoted by excessive ROS, including arthritis. PUBLIC HEALTH RELEVANCE: Osteoarthritis is the most common cause of chronic disability in older adults but treatments to slow the progression of the disease are lacking. The results from this project will provide new information about basic mechanisms relevant to cartilage breakdown in osteoarthritis. This information is needed in order to discover new targets and develop new therapies for slowing or stopping the progression of the disease.

The most important risk factor for the development of osteoarthritis (OA) is age but the mechanisms by which aging contributes to OA susceptibility are poorly understood. A key feature of OA is the progressive destruction and loss of articular cartilage resulting from an imbalance in chondrocyte anabolic and catabolic activity. The long-term objective of this project is to determine the mechanisms by which aging contributes to this imbalance. Maintenance of the integrity of articular cartilage requires a properly orchestrated response of the chondrocyte to cell signals generated by growth factors, cytokines, and the extracellular matrix. This project focuses on the specific cellular and molecular mechanisms responsible for an age-related decline in the chondrocyte response to a key cartilage growth factor, IGF-I. Recent studies provide novel evidence that age-related oxidative stress may play a key role in reducing the chondrocyte response to IGF-I and, furthermore, could alter the activity of cell signaling pathways resulting in an imbalance in anabolic and catabolic activity as well as reduced cell survival. It has also been found that chondrocytes express RAGE (receptor for advanced glycation end-products). RAGE signaling is known to increase ROS production and activate NF B, initiating a pro-inflammatory state. An age-related increase in oxidative stress has been found to be a key contributor to aging processes in a number of tissues but its role in cartilage aging has received little attention. Therefore, the overall hypothesis to be tested in the continuation of this project is that an age-related chondrocyte resistance to IGF-I stimulation is due to dysregulated cell signaling resulting from the chronic and accumulated effects of oxidative stress and a resultant "pro-inflammatory" state. The specific aims will be to: 1) Measure the response of human articular chondrocytes, isolated from donors of different ages, to IGF-I when the cellular redox status has been modulated;2) Determine the redox sensitive cell signaling mechanisms which regulate the chondrocyte response to IGF-I;and 3) Determine the contribution of the receptor for advanced glycation end-products (RAGE) to chondrocyte redox signaling and inhibition of the IGF-I response. The results from this project should continue to provide new information needed to establish the basic cellular and molecular mechanisms which link aging to the development of OA in humans.

Advancement of biomedical knowledge is more rapid when translation of research findings can easily and efficiently span across different biological levels of organization. This process entails conversion of fundamental molecular insights (basic research) into novel and applied clinical therapies ("forward translation"), as well as discovery of molecular mechanisms for specific clinical observations at the population level ("reverse translation"). As outlined in the Leadership and Administrative Core, one of the primary goals of the WFU OAIC is to develop new tools and implement research strategies and infrastructure for fostering translational research. The main goal of this Research Development Project is to expand the ability of the Molecular Sciences Resource Core (MSRC) to facilitate reverse translational research by developing the infrastructure to generate, analyze, and integrate encompassing data on changes in expression levels of genes in human tissues in response to clinical interventions. These data will lead to the generation of novel hypotheses regarding the molecular adaptations and mechanisms underlying improvements in physical performance/disability in response to such interventions, and will provide a tool by which OAIC investigators can incorporate investigation of biological mechanisms into their existing protocols. The proposed developmental project will accomplish this overall goal by expanding upon a unique clinical finding from our recently completed OAlC-supported randomized, controlled pilot study ("Optimizing body composition for function in older adults;OPTIMA"). The OPTIMA study was designed to assess the effects of combining caloric restriction with the peroxisome proliferator-activated receptor (PPARy) agonist pioglitizone and/or resistance training on physical function and body composition in 88 community-dwelling older (65-79 yrs) overweight/obese (BMI>27 kg/m2) adults randomized to a 4-month intervention of: 1) hypocaloric diet alone (DIET);2) DIET plus 30 mg daily pioglitizone/Actos[unreadable](PIO);3) DIET plus resistance training (RT);or 4) DIET plus PIO and RT. We discovered a remarkable interaction of pioglitizone with resistance training that resulted in significant improvements in muscle power, but not muscle strength, (see section f) such that the participants randomized to DIET+PIO+RT experienced a two-fold greater improvement in knee extensor maximal torque (muscle power) than those randomized to DIET+RT only. The mechanism for this novel observation is unknown, but could be related to altered gene expression in muscle in response to stimulation of PPARy by pioglitizone. PPARs are members of a nuclear hormone receptor superfamily that act to regulate gene transcription, which in turn regulates a number of diverse processes including lipid and carbohydrate metabolism, as well as certain inflammatory pathways.1 The effects of PPARy ligands such as pioglitizone can be tissue specific, are not completely understood, and can potentially include effects independent of PPARy stimulation. Therefore, we propose to utilize stored biopsy samples of the vastus lateralis muscle (taken before and after the interventions) to conduct gene expression microarrays, followed by pathway analysis to begin to develop an understanding of the underlying mechanism for this interaction. Dr. Loeser's previous experience in the use of microarray technology, which provides a gene expression profile of a given tissue, coupled with a systems biology analytical approach (contributed by Dr. Fetrow), are ideal tools for the task of identifying previously unknown genes (and ultimately proteins) that may be causally linked to a specific clinical observation. The array data and pathway analysis will be combined with clinical measures of physical function, fat distribution (by DXA and CT), histologic measures of muscle (already in progress), and with examination of the expression of selected candidate genes, in order to gain a complete picture of the effect of the intervention. Integration of physical function outcomes with this systems approach to examining mechanisms represents the current gold standard approach to providing comprehensive information and additional insight into the mechanisms underlying aging-related loss of physical function.

DESCRIPTION (provided by applicant): A growing body of evidence suggests that vitamin K insufficiency is associated with several age-related diseases, including osteoarthritis (OA). Several vitamin K-dependent proteins are found in joint tissue, including matrix gla protein (MGP), which functions as a calcification inhibitor. In order for MGP to function it must be carboxylated, which requires vitamin K. Recently assays that measure plasma uncarboxylated MGP (ucMGP) in humans have been developed, and plasma ucMGP is reported to be elevated (reflective of low vitamin K status) in patients with arterial calcification. Since MGP is present n both vascular tissue and cartilage and the vitamin K-dependent mechanisms underlying arterial calcification and cartilage calcification are similar, it is plausible that plasma ucMGP also reflets OA pathology. However, plasma ucMGP has not been measured in persons well-characterized for OA progression. To advance our understanding of vitamin Ks role in joint health, the investigators propose to measure circulating ucMGP and vitamin K1 in archived samples from the Health, Aging and Body Composition (Health ABC) Knee OA Study, a cohort of 1141 black and white well-functioning community-dwelling men and women aged 70-79 yrs at baseline. The following specific aims will be addressed: Specific aim 1a: To determine the association between low vitamin K status (measured by plasma ucMGP and vitamin K1) and symptomatic knee OA in older adults. Specific aims 2a and 2b: To determine the association between vitamin K status and knee OA progression assessed radiographically and by MRI in older adults over 3 years of follow-up. Specific aim 3: To determine the cross-sectional and longitudinal association between vitamin K status and lower extremity function in older adults. Participants in the Health ABC Knee OA study underwent repeated bilateral knee x-rays and MRIs and are well characterized for lower-extremity function, making this study an ideal study to determine the associations between plasma ucMGP, vitamin K1 and OA. Our results will provide innovative evidence of the mechanisms underlying vitamin Ks role in OA in a cost-effective and efficient manner, and will be used to develop future proposals to carry out follow-up trials to clarify the vitamin Ks role in OA progression and related disability.