Reversing Age-related Bone Loss by Administration of the Matricellular Protein Cyr61/CCN1

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Summary Previously, we reported that cell-free native extracellular matrix (ECM), synthesized by bone marrow (BM) stromal cells, significantly promoted the stemness of mouse and human BM-derived mesenchymal stem cells (BM-MSCs). More importantly, defects in self-renewal, differentiation, and bone formation capacity of aging BM- MSCs can be completely rescued by culture on young BM-ECM (i.e., made by cells from young donors). In contrast, old BM-ECM (i.e., made by cells from old donors) failed to support both young and old MSC self- renewal and differentiation capacity. By proteomic analysis, we found that the matricellular protein Cyr61 was abundant in young ECM but absent in old ECM. Subsequently, we used genetic methods to down- or up-regulate the incorporation of Cyr61 during synthesis of young or old BM-ECM and found that Cyr61-depleted young ECM lost its ability, while Cyr61-replenished old ECM gained its ability, to retain MSC properties. In addition, we showed that low bone mineral density (BMD) was associated with decreased amounts of Cyr61 in bone tissue of old mice. More interestingly, the presence of Cyr61 in bone is required for the anabolic effect of intermittent PTH that by itself up-regulates Cyr61 expression in old mice. The goal of the current proposal is to reverse age- related bone loss by replenishing a critical matricellular protein component, Cyr61/CCN1, to the aging bone microenvironment (i.e., stem cell niche). We hypothesize that replenishing Cyr61 to the aging bone matrix will restore the ability of the MSC microenvironment (niche) to support MSC self-renewal, differentiation, and bone formation capacity, resulting in an attenuation of bone loss and improvement in bone anabolic response to intermittent PTH treatment. We are uniquely positioned to test this hypothesis since, in addition to our strong preliminary data, we have established a unique animal model, bone-specific Cyr61 knockout out (Cyr61‒) mice, which displays osteopenia at an early age. To test the hypothesis, we will determine the effect of restoring Cyr61 to BM-ECM, produced by cells from Cyr61‒ or old wild type (wt) mice, on its ability to retain/rescue the quantity and quality of young-/old-BM-MSCs (Aim 1). Then we will determine the efficacy of recombinant Cyr61 (rCyr61), carried by multi-walled carbon nanotubes (MWCNTs), in reversing both stem cell quantity/quality and bone loss in Cyr61‒ and old wt mice (Aim 2). Finally, we will compare the efficacy of administering rCyr61 followed by intermittent PTH versus PTH alone at reversing bone loss in Cyr61‒ or old mice (Aim 3). The proposed study is highly innovative by testing our novel hypothesis via: 1) novel animal models, bone-specific Cyr61‒ and old wt mice (natural Cyr61 deficiency with aging), to determine if replenishing Cyr61 to the bone ECM can reverse BM- MSC function and bone loss; 2) a novel vehicle, MWCNTs, that specifically delivers rCyr61 to the bone matrix; and 3) testing the synergistic effects of Cyr61 administration prior to intermittent PTH on bone anabolic metabolism. The proposed study is significant because current osteoporosis treatments mainly focus on balancing osteoclast/osteoblast activity and have limited efficacy and considerable side effects. The studies have great potential to benefit both the veteran and general population by providing a new approach for treating osteoporosis that focuses on restoring the aging stem cell niche, which has not been explored previously.