In clinical medicine, aging may be best defi ned by the words Supreme Court Justice Potter Stewart used to describe pornography: “I know it when I see it” (JACOBELLIS v. OHIO, 378 U.S. 184 (1964)). Every physician has witnessed the effects of aging, both in individual patients followed over a period of time and in their collective patient population base. Nevertheless, a number of defi nitions of aging have been offered, and these have been elegantly summarized in Carrington’s review entitled: “Aging bone and cartilage: cross-cutting issues” (1). In the clinical care setting, aging is generally associated with the loss of a wide range of physiological processes (1) (Table 2.1). These include decreased fertility (2), decreased resilience in response to environmental stressors such as infections, surgery, or physical attack (3), and decreased physical strength. Inevitably, aging is also associated with end of life and death (2,4). At the cellular level, several fundamental and interconnected processes accompany aging in vitro and in vivo (1) (Table 2.1). Hayfl ick fi rst defi ned the process of cellular senescence, demonstrating that “normal” diploid cells can undergo a limited number of cell doublings in vitro (5). These pioneering observations set the framework within which much of our understanding of aging is now predicated. Consequently, the Hayfl ick model for replicative senescence has been employed in biogerontology research to unravel mechanisms of age-related cellular defects (6). Using this model, several investigators have reported an inverse relationship between the donor age and maximal proliferative potential of the cells in vitro (7). The Kassem laboratory has characterised a Hayfl ick model for replicative senescence of human osteoblasts (8,9). During continuous culture in vitro, human osteoblasts exhibited typical senescence-related phenotype including senescent-associated decrease in osteoblast marker production (alkaline phosphatase [AP], osteocalcin, collagen type I), decrease in mean telomere fragment length, and increase in the number of senescence-associated β-galactosidase (SA β-gal) positive cells (10,11). With each progressive mitotic cycle, each telomere, located at the ends of individual chromosomes, decreases in length; and this has been associated with senescence (12). It has been postulated that the telomere length acts as a “mitotic clock,” and it is known that the overexpression of telomerase, the enzyme responsible for maintaining telomere length, leads to cell immortalization (13). The Kassem laboratory has further examined the effect of donor age on the maximal proliferative potential of bone marrow stem cells (BMSC). An age-related decline in the maximal life span from

Throughout life, a balance exists within the marrow cavity between adipose tissue and bone. Each tissue derives from a common progenitor cell known both as a “bone marrow-derived multipotent stromal cell” and as a “mesenchymal stem cell” (BMSC). The majority of in vitro and in vivo data suggest that BMSCs differentiate into adipocytes or osteoblasts in a reciprocal manner. For example, while ligand induction of the transcription factors peroxisome proliferator-activated receptor γ initiates BMSC adipogenesis, it suppresses osteogenesis. Nevertheless, this hypothesis may oversimplify a complex regulatory paradigm. The picture may be further complicated by the systemic impact of extramedullary adipose depots on bone via the secretion of protein adipokines and lipid metabolites. This review focuses on past and current literature examining the mechanisms governing the adipose–bone interface.

OBJECTIVES:The authors have set out to evaluate the literature relevant to the dynamic regulation of adipogenesis and osteogenesis. DESIGN AND METHODS:A detailed search of the past and recent literature was conducted on Pubmed using a combination of keywords including: adipogenesis, bone marrow, hematopoiesis, mesenchymal stromal/stem cell, and osteogenesis. RESULTS:Throughout one's lifespan, the bone marrow microenvironment provides a unique niche for mesenchymal stromal/stem cells (BMSCs) and hematopoietic stem cells (HSCs). The marrow changes as a function of biological age and pathophysiology. Historically, clinical biochemistry has observed these changes from an HSC and hematological perspective. Nevertheless, these changes also reflect the balance between BMSC adipogenic and osteogenic processes which can display an inverse or reciprocal relationship. Multiple hormonal factors and nuclear hormone receptor ligands and drugs are responsible for BMSC lineage selection. Data from a number of laboratories now implicates endocrine feedback loops between extramedullary adipose depots and the central nervous system. CONCLUSIONS:This concise review provides a perspective on the mechanisms regulating BMSC differentiation in the context of biological aging, obesity, and osteoporosis.

Information relating to the biology, culture expansion, and mechanisms relating to adipose-derived cells has advanced significantly in the past decade. Both the heterogeneous stromal vascular fraction (SVF) and more homogeneous adipose-derived stem cells (ASC) offer unique opportunities as novel cell-based therapeutics and as traditional pharmaceutical discovery tools. This review highlights the cytokine secretory functions of ASC and SVF cells as well as their potential use as immunomodulators and gene delivery vehicles. These functions make it feasible to exploit adipose-derived cells in the treatment of ischemic, musculoskeletal, and oncological disorders. With appropriate commercial development and in vivo validation, ASC and SVF cells will have a significant therapeutic impact in the future.

SUMMARY: Mesenchymal stem cells (MSCs) represent a class of multipotent progenitor cells that have been isolated from multiple tissue sites. Of these, adipose tissue and bone marrow offer advantages in terms of access, abundance, and the extent of their documentation in the literature. This review focuses on the in vitro differentiation capability of cells derived from adult human tissue. Multiple, independent studies have demonstrated that MSCs can commit to mesodermal (adipocyte, chondrocyte, hematopoietic support, myocyte, osteoblast, tenocyte), ectodermal (epithelial, glial, neural), and endodermal (hepatocyte, islet cell) lineages. The limitations and promises of these studies in the context of tissue engineering are discussed.

In clinical medicine, aging may be best defi ned by the words Supreme Court Justice Potter Stewart used to describe pornography: “I know it when I see it” (JACOBELLIS v. OHIO, 378 U.S. 184 (1964)). Every physician has witnessed the effects of aging, both in individual patients followed over a period of time and in their collective patient population base. Nevertheless, a number of defi nitions of aging have been offered, and these have been elegantly summarized in Carrington’s review entitled: “Aging bone and cartilage: cross-cutting issues” (1). In the clinical care setting, aging is generally associated with the loss of a wide range of physiological processes (1) (Table 2.1). These include decreased fertility (2), decreased resilience in response to environmental stressors such as infections, surgery, or physical attack (3), and decreased physical strength. Inevitably, aging is also associated with end of life and death (2,4). At the cellular level, several fundamental and interconnected processes accompany aging in vitro and in vivo (1) (Table 2.1). Hayfl ick fi rst defi ned the process of cellular senescence, demonstrating that “normal” diploid cells can undergo a limited number of cell doublings in vitro (5). These pioneering observations set the framework within which much of our understanding of aging is now predicated. Consequently, the Hayfl ick model for replicative senescence has been employed in biogerontology research to unravel mechanisms of age-related cellular defects (6). Using this model, several investigators have reported an inverse relationship between the donor age and maximal proliferative potential of the cells in vitro (7). The Kassem laboratory has characterised a Hayfl ick model for replicative senescence of human osteoblasts (8,9). During continuous culture in vitro, human osteoblasts exhibited typical senescence-related phenotype including senescent-associated decrease in osteoblast marker production (alkaline phosphatase [AP], osteocalcin, collagen type I), decrease in mean telomere fragment length, and increase in the number of senescence-associated β-galactosidase (SA β-gal) positive cells (10,11). With each progressive mitotic cycle, each telomere, located at the ends of individual chromosomes, decreases in length; and this has been associated with senescence (12). It has been postulated that the telomere length acts as a “mitotic clock,” and it is known that the overexpression of telomerase, the enzyme responsible for maintaining telomere length, leads to cell immortalization (13). The Kassem laboratory has further examined the effect of donor age on the maximal proliferative potential of bone marrow stem cells (BMSC). An age-related decline in the maximal life span from

PPARγ plays a central role in the formation of fat. Regulation of PPARγ activity depends on numerous factors ranging from dietary ligands to nuclear hormone coactivators and corepressors to oxygen-sensing mechanisms. In addition, the interplay of PPARγ with other nuclear hormone receptors has implications for the balance between adipogenesis and osteogenesis in mesenchymal stem cells of the bone marrow stroma. This review will explore a range of factors influencing PPARγ activity and how these interactions may affect osteogenesis.

Whereas continuous PTH infusion increases bone resorption and bone loss, intermittent PTH treatment stimulates bone formation, in part, via reactivation of quiescent bone surfaces and reducing osteoblast apoptosis. We investigated the possibility that intermittent and continuous PTH treatment also differentially regulates osteogenic and adipocytic lineage commitment of bone marrow stromal progenitor/mesenchymal stem cells (MSC). The MSC were cultured under mildly adipogenic conditions in medium supplemented with dexamethasone, insulin, isobutylmethytxanthine and troglitazone (DUT), and treated with 50 nM human PTH(1-34) for either I h/day or continuously (PTH replenished every 48 h). After 6 days, cells treated with PTH for 1 h/day retained their normal fibroblastic appearance whereas those treated continuously adopted a polygonal.. irregular morphology. After 12-18 days numerous lipid vacuole and oil red O-positive adipocytes had developed in cultures treated with DIIT alone, or with DIIT and continuous PTH. In contrast, adipocyte number was reduced and alkaline phosphatase staining increased in the cultures treated with DIIT and 1 h/day PTH, indicating suppression of adipogenesis and possible promotion of early osteoblastic differentiation. Furthermore, intermittent but not continuous PTH treatment suppressed markers of differentiated adipocytes such as mRNA expression of lipoprotein lipase and PPAR-gamma as well as glycerol 3-phosphate dehydrogenase activity. All of these effects of intermittent PTH were also produced by a 1 h/day treatment with AH3960 (30 mu M), a small molecule, non-peptide agonist of the PTH1 receptor. AH3960, like PTH, activates both the cAMP and calcium signaling pathways. Treatment with the adenylyl cyclase activator forskolin for 1 h/day, mimicked the anti-adipogenic effect of intermittent PTH, whereas pretreatment with the protein kinase-A inhibitor H89 prior to intermittent PTH resulted in almost complete conversion to adipocytes. In contrast, the MAP kinase inhibitor PD 98059 failed to prevent the anti-adipocytic effect of intermittent PTH, suggesting that the inhibitory effect of PTH on adipocyte differentiation is predominantly cAMP-dependent. These results demonstrate a differential effect of PTH1 receptor agonists on the adipocytic commitment and differentiation of adult human bone marrow mesenchymal stem cells. This response may represent an additional mechanism that contributes to the overall bone anabolic action of intermittent PTH. (c) 2006 Elsevier Inc. All rights reserved.

Until recently, adipose tissue was considered to serve only as a triglyceride reservoir and was relegated to a passive endocrine role. With the discovery of leptin and other adipokines, adipose tissue is now recognized as an active participant in systemic metabolism. This review focuses on the complex relationship existing between adipose tissue and bone metabolism and differentiation. It explores the paradigms that have shaped the past decade’s research and what these findings forecast for the future. Particular attention is given to the multipotent adult stem cell populations that reside within bone and fat. These adult stem cells have critical importance to the emerging field of tissue engineering and regenerative medicine.

The increase in marrow adipogenesis associated with osteoporosis and age-related osteopenia is well known clinically. However, we are only now beginning to understand the mechanisms that control the differentiation of mesenchymal stem cells to either osteoblasts or adipocytes. Recent work with gene silencing and overexpression has provided insight into critical pathways that determine the fate of these multipotential cells. One of these pathways — that of the nuclear hormone receptor peroxisome proliferator activated receptor-γ — when activated, promotes adipogenesis and inhibits osteogenesis. This in vitro mechanism of action has been confirmed in vivo using ligands to this receptor. Discovery of this and other targets and pathways, such as Wnt signaling, notch/delta/jagged ligands and receptors, and RhoA gene expression, provides new insights into mesenchymal stem cell differentiation. These pathways provide exciting future pharmacological targets with which to enhance bone formation and therefore reduce the risk of fracture.

Until recently, adipose tissue was considered to serve only as a triglyceride reservoir and was relegated to a passive endocrine role. With the discovery of leptin and other adipokines, adipose tissue is now recognized as an active participant in systemic metabolism. This review focuses on the complex relationship existing between adipose tissue and bone metabolism and differentiation. It explores the paradigms that have shaped the past decade's research and what these findings forecast for the future. Particular attention is given to the multipotent adult stem cell populations that reside within bone and fat. These adult stem cells have critical importance to the emerging field of tissue engineering and regenerative medicine.

Advances in genomics and proteomics have revolutionised the drug discovery process and target validation. Identification of novel therapeutic targets for chronic skeletal diseases is an extremely challenging process based on the difficulty of obtaining high-quality human diseased versus normal tissue samples. The quality of tissue and genomic information obtained from the sample is critical to identifying disease-related genes. Using a genomics-based approach, novel genes or genes with similar homology to existing genes can be identified from cDNA libraries generated from normal versus diseased tissue. High-quality cDNA libraries are prepared from uncontaminated homogeneous cell populations harvested from tissue sections of interest. Localised gene expression analysis and confirmation are obtained through in situ hybridisation or immunohistochemical studies. Cells overexpressing the recombinant protein are subsequently designed for primary cell-based high-throughput assays that are capable of screening large compound banks for potential hits. Afterwards, secondary functional assays are used to test promising compounds. The same overexpressing cells are used in the secondary assay to test protein activity and functionality as well as screen for small-molecule agonists or antagonists. Once a hit is generated, a structure–activity relationship of the compound is optimised for better oral bioavailability and pharmacokinetics allowing the compound to progress into development. Parallel efforts from proteomics, as well as genetics/transgenics, bioinformatics and combinatorial chemistry, and improvements in high-throughput automation technologies, allow the drug discovery process to meet the demands of the medicinal market. This review discusses and illustrates how different approaches are incorporated into the discovery and validation of novel targets and, consequently, the development of potentially therapeutic agents in the areas of osteoporosis and osteoarthritis. While current treatments exist in the form of hormone replacement therapy, antiresorptive and anabolic agents for osteoporosis, there are no disease-modifying therapies for the treatment of the most common human joint disease, osteoarthritis. A massive market potential for improved options with better safety and efficacy still remains. Therefore, the application of genomics and proteomics for both diseases should provide much needed novel therapeutic approaches to treating these major world health problems.

Purpose: There is a need to find novel oestrogen receptor (ER) ligands that antagonize oestrogen action in the reproductive tissues and would therefore have therapeutic potential in oestrogen-dependent tumours. We tested novel ER ligands in both breast and endometrial cells to profile agonism/antagonism in these oestrogen target reproductive tissues. Methods: Novel analogues of the ER antagonist ICI 182,780 were synthesized and tested for their ability to inhibit gene expression dependent on oestrogen response elements (ERE) in human breast (MCF-7) and endometrial (Ishikawa) cell lines. This activity was correlated with inhibition of oestrogen-induced cell proliferation and ER binding. Results: The sulphide analogue (compound 1) and sulphone analogue (compound 2) had no intrinsic ERE-dependent agonism in either breast cancer or endometrial cells in culture. All three compounds dose-dependently inhibited ERE-mediated oestrogen agonism. Moreover, these ER ligands inhibited oestrogen-stimulated proliferation of breast cancer and endometrial cells. ICI 182,780, compound 1 and compound 2 were all able to bind both isoforms of the ER (ERα and ERβ). In endometrial cells, the relative binding to ERβ correlated with the ERE-dependent antioestrogenic effect of these ligands, suggesting that in this tissue this receptor is the predominant isoform that determines antioestrogenic activity. Conclusions: The ability of these analogues of ICI 182,780 to inhibit oestrogen-stimulated transcriptional activity and cell proliferation suggests that these agents, in particular the sulphone analogue, have therapeutic potential in the treatment of breast cancer without exhibiting the unwanted oestrogenic effects in the endometrium.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

Recent drug discovery has been driven largely by a genomics-based approach. This revolution in pharmaceutics is based on localized expression of either a novel gene or homologue of a known gene found in cDNA libraries made from normal versus diseased tissue. The choice and quality of cDNA library is critical for the success of this approach. Expression is normally verified at the cellular level by either immunocytochemistry or in situ hybridization. Activity of the recombinant protein in secondary cell-based assays allows highthroughput screens to be formulated to identify small-molecule effecters of this protein. More recently, a proteomics approach has also been incorporated into this process. This technology directly measures proteins whose expression is localized in disease tissue as the basis for cell-based screens to look for either activators or inhibitors, of this activity. The majority of screens are designed to look for inhibitors. Activity of small-molecules found by screening gives rise to pharmacokinetic studies and verification of activity in animal models of the disease. Structure-activity relationship (SAR) optimization of these small-molecules allows for suitable oral bioavailability and pharmacokinetics, resulting in compounds progressing from discovery to development. Based on these strategies, we have developed inhibitors of osteoclast-mediated bone resorption and are currently screening for bone anabolic agents. In addition, we have also developed small-molecule caspase inhibitors which prevent chondrocyte apoptosis and retain cell function in an attempt to find therapeutic agents to either prevent or treat osteoarthritis. These agents may well have utility in the treatment of temporomandibular joint diseases. Copyright (C) 2001 S. Karger AG, Basel.

Accelerated bone loss secondary to loss of ovarian function at menopause is well recognised as a major risk factor for osteoporotic fractures in postmenopausal women. Postmenopausal bone loss can be prevented or arrested by oestrogen replacement therapy (ERT). It has also been reported that ERT protects against cardiovascular disease by improving the serum lipid profile, however there are mixed reports concerning these benefits. Unopposed ERT causes an unacceptable increase in the risk of endometrial cancer and proliferative effects in mammary tissue resulting in an increased risk of breast cancer. While this can be counteracted by combining ERT with a low-dose of a progestin, withdrawal bleeding and the continuing uncertainty about the effect of oestrogen on the risk of breast cancer contribute to poor compliance for long-term use. Because of the known and suspected risks of oestrogen therapy it has been estimated that in the US < 40% of women on ERT will continue treatment beyond one year. An ideal therapy would retain the desirable skeletal and cardiovascular effects of oestrogen, lack oestrogenic activity on the endometrium and reduce the incidence of breast cancer. The concept of selective oestrogen receptor modulation (SERM) has been demonstrated for a number of compounds including tamoxifen, raloxifene, droloxifene, GW-5638 and levormeloxifene. However, the clinical utility of these agents will depend on the profile of tissue-specific effects and the extent to which they are translated into in vivo efficacy. A SERM is defined as a compound that has oestrogen agonism on one or more of the desired target tissues, such as bone or liver, and has antagonism and/or minimal agonism (i.e., clinically insignificant) in reproductive tissue, such as the breast or uterus. Although tamoxifen acts as a SERM, it is also associated with an increased incidence (4% gynaecological symptoms greater than placebo control) of endometrial cancer. Indeed, there have been a number of mechanistic-based studies to explain the increased incidence of endometrial carcinomas in tamoxifen treated patients, which provide an in vitro insight into the adverse clinical observations in vivo. Attempts to improve on the pharmacological profile of tamoxifen have resulted in compounds that differ in their oestrogen agonist/antagonist characteristics, including the pure oestrogen antagonists. This suggests that it may be possible to develop a molecule with a desired profile of tissue-specific agonist/antagonist activities by establishing bone and cardiovascular protective effects but having no effects (or even behaving as an antagonist) in the reproductive tissues.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive erosion of articular cartilage as well as thickening of subchondral bone. Almost 30% of the US population between the ages of 25–74 have radiographic OA of the hand and over 30% of the population aged 63–93 have radiographic OA of the knee [25, 41]. Currently, the disease is treated with anti-inflammatory or analgesic therapy because no disease-modifying drugs are yet available. Hallmarks of the disease include a reduction in chondrocyte (cartilage cell) cell number [44, 69], an increase in number of degenerative chondrocytes (fragmented cells, lipid droplets, nuclear clumping and loss of organelles) [73], uncontrolled matrix degradation and articular cartilage swelling.