David A. Lipschitz

Aging activates adipogenic and suppresses osteogenic programs in mesenchymal marrow stroma/stem cells: the role of PPAR-γ2 transcription factor and TGF-β/BMP signaling pathways

Osteoblasts and adipocytes originate from a common progenitor, which arises from bone marrow mesenchymal stroma/stem cells (mMSC). Aging causes a decrease in the number of bone‐forming osteoblasts and an increase in the number of marrow adipocytes. Here, we demonstrate that, during aging, the status of mMSC changes with respect to both their intrinsic differentiation potential and production of signaling molecules, which contributes to the formation of a specific marrow microenvironment necessary for maintenance of bone homeostasis. Aging causes a decrease in the commitment of mMSC to the osteoblast lineage and an increase in the commitment to the adipocyte lineage. This is reflected by changes in the expression of phenotype‐specific gene markers. The expression of osteoblast‐specific transcription factors, Runx2 and Dlx5, and osteoblast markers, collagen and osteocalcin, is decreased in aged mMSC. Conversely, the expression of adipocyte‐specific transcription factor PPAR‐γ2, shown previously to regulate osteoblast development and bone formation negatively and to regulate marrow adipocyte differentiation positively, is increased, as is a gene marker of adipocyte phenotype, fatty acid binding protein aP2. Furthermore, production of an endogeneous PPAR‐γ activator(s) that stimulates adipocyte differentiation and production of autocrine/paracrine factor(s) that suppresses the osteoblastic phenotype are also increased. In addition, expression of different components of TGF‐β and BMP2/4 signaling pathways is altered, suggesting that activities of these two cytokines essential for bone homeostasis change with aging.

Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARγ2

Cells of the bone marrow stroma can reversibly convert among different phenotypes. Based on this and on evidence for a reciprocal relationship between osteoblastogenesis and adipogenesis, we have isolated several murine bone marrow‐derived clonal cell lines with phenotypic characteristics of osteoblasts or adipocytes, or both. Consistent with a state of plasticity, cell lines with a mixed phenotype synthesized osteoblast markers like type I collagen, alkaline phosphatase, osteocalcin, as well as the adipocyte marker lipoprotein lipase, under basal conditions. In the presence of ascorbic acid and β‐glycerophosphate—agents that promote osteoblast differentiation—they formed a mineralized matrix. In the presence of isobutylmethylxanthine, hydrocortisone, and indomethacin—agents that promote adipocyte differentiation—they accumulated fat droplets, but failed to express adipsin and aP2, markers of terminally differentiated adipocytes. Furthermore, they were converted back to matrix mineralizing cells when the adipogenic stimuli were replaced with the osteoblastogenic ones. A prototypic cell line with mixed phenotype (UAMS‐33) expressed Osf2/Cbfa1—a transcription factor required for osteoblast differentiation, but not PPARγ2—a transcription factor required for terminal adipocyte differentiation. Stable transfection with a PPARγ2 expression construct and activation with the thiazolidinedione BRL49653 stimulated aP2 and adipsin synthesis and fat accumulation, and simultaneously suppressed Osf2/Cbfa1, α1(I) procollagen, and osteocalcin synthesis. Moreover, it rendered the cells incapable of forming a mineralized matrix. These results strongly suggest that PPARγ2 negatively regulates stromal cell plasticity by suppressing Osf2/Cbfa1 and osteoblast‐like biosynthetic activity, while promoting terminal differentiation to adipocytes. J. Cell. Biochem. 74:357–371, 1999.

Increased adipogenesis and myelopoiesis in the bone marrow of SAMP6, a murine model of defective osteoblastogenesis and low turnover osteopenia

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. However, little is known about the interrelationship between these two processes. We have previously shown that the senescence accelerated mouse‐P6 (SAMP6) exhibits decreased osteoblastogenesis in the bone marrow that is temporally linked with a low rate of bone formation and decreased bone mineral density. Here we report that in contrast to decreased osteoblastogenesis, ex vivo bone marrow cultures from SAMP6 mice exhibited an increase in the number of colony‐forming unit adipocytes, as well as an increase in the number of fully differentiated marrow adipocytes, compared with SAMR1 (nonosteopenic) controls. Further, long‐term bone marrow cultures from SAMP6 produced an adherent stromal layer more rapidly, generated significantly more myeloid progenitors and produced more IL‐6 and colony‐stimulating activity. Consistent with this, the number of myeloid cells in freshly isolated marrow from SAMP6 mice was increased, as was the number of granulocytes in peripheral blood. The evidence that SAMP6 mice exhibit decreased osteoblastogenesis, and increased adipogenesis and myelopoiesis, strongly suggests that a switch in the differentiation program of multipotential mesenchymal progenitors may underlie the abnormal phenotype manifested in the skeleton and other tissues of these animals. Moreover, these observations support the contention for the existence of a reciprocal relationship between osteoblastogenesis and adipogenesis that may explain the association of decreased bone formation and the resulting osteopenia with the increased adiposity of the marrow seen with advancing age in animals and humans.

Activation of an adipogenic program in adult myoblasts with age

Myoblasts isolated from mouse hindlimb skeletal muscle demonstrated increased adipogenic potential as a function of age. Whereas myoblasts from 8-month-old adult mice did not significantly accumulate terminal markers of adipogenesis regardless of culture conditions, myoblasts from 23-month-old mice accumulated fat and expressed genes characteristic of differentiated adipocytes, such as the fatty acid binding protein aP2. This change in differentiation potential was associated with a change in the abundance of the mRNA encoding the transcription factor C/EBPalpha, and in the relative abundance of PPARgamma2 to PPARgamma1 mRNAs. Furthermore, PPARgamma activity appeared to be regulated at the level of phosphorylation, being more highly phosphorylated in myoblasts isolated from younger animals. Although adipogenic gene expression in myoblasts from aged animals was activated, presumably in response to PPARgamma and C/EBPalpha, unexpectedly, myogenic gene expression was not effectively repressed. The Wnt signaling pathway may also alter differentiation potential in muscle with age. Wnt-10b mRNA was more abundantly expressed in muscle tissue and cultured myoblasts from adult compared with aged mice, resulting in stabilization of cytosolic beta-catenin, that may potentially contribute to inhibition of adipogenic gene expression in adult myoblasts. The changes reported here, together with those reported in bone marrow stroma with age, suggest that a default program may be activated in mesenchymal cells with increasing age resulting in a more adipogenic-like phenotype. Whether this change in differentiation potential contributes to the increased adiposity in muscle with age remains to be determined.

Arm length measurement as an alternative to height in nutritional assessment of the elderly.

The use of long bone measurements as an alternative to height in nutritional assessment of the elderly was investigated in 100 young and 63 aged individuals. Highly significant correlations between height and total arm length, humerus length, forearm length, and proximal phalanx length were found. Compared to young subjects, values for the elderly were shifted significantly, demonstrating a reduction in height not paralleled by decreases in arm length measurements. In subsequent studies, lean body mass was evaluated by creatinine excretion in groups of young and elderly male and female volunteers. In both males and females, when creatinine excretion was related to height, ideal body weight, or total arm length, significantly lower values were found in the elderly as compared to the young. In all groups highly significant correlations between the use of arm length and the other two methods of expressing creatinine excretion were found. This validates the use of arm length measurement as an alternative to height for nutritional assessment. Since arm length is less affected than height by the aging process and more easily obtained in the bedfast patient, it should provide a more accurate reflection of stature when performing nutritional assessments of the elderly. These preliminary data emphasize the need for appropriate standards for subjects of different ages when evaluating lean body mass.

Patterns of care: an analysis of the quality of nutritional care routinely provided to elderly hospitalized veterans.

To assess the adequacy of the nutritional care provided, a prospective noninterventional study was carried out on 250 randomly selected patients aged 65 and over who were admitted to a Veterans Administration Hospital. Of this group, 97 patients (39%) were found to be at high risk of having clinically significant protein-energy malnutrition. In 43 cases (17%), an assessment of the patients nutritional status was not possible because the available data were inadequate. No patient had a diagnosis of malnutrition recorded on the problem list. Only 13 percent of the high-risk patients received some form of nutrition support therapy; 10 (10%) received oral supplements, and four (4%) were started on enteral feedings. Patients who received enteral feedings experienced a high rate of complications resulting from use of the feeding tubes. Over all, none of the high risk study patients received optimal nutrition support therapy. These findings indicate that elderly patients hospitalized in the Veterans Administration hospital in this study are usually not screened appropriately for protein-energy malnutrition, the diagnosis is frequently missed or ignored, and nutrition support therapy is underutilized and often ineffectually managed.

Cancer in the Elderly: Basic Science and Clinical Aspects

The incidence of cancer increases progressively with age. Rearrangements of genomes have been found to accompany cellular aging. These factors, in concert with age-dependent alterations in immune function and host defense, may help to explain the increased risk of malignant disease in aged persons. The clinical presentation and natural history of neoplasia are also affected by aging. This conference reviews recent developments in these areas, examines the effects of drug use in the elderly and implications for management, and discusses current information on how age may influence the response of cancer to therapy.

In vitro culture of proerythroblasts: characterization of proliferative response to erythropoietin and steroids

Summary This study characterized variables affecting the in vitro liquid culture of proerythroblasts. When bone marrow from mice depleted of haemoglobin containing cells, was cultured in vitro in the presence of human urinary erythropoietin (Ep) a significant degree of erythroid cell proliferation and maturation occurred as measured directly by 3H‐thymidine (3H‐TdR) incorporation into DNA (autoradiographical measurement). Proliferation increased indirect proportion to the dose of Ep added to the culture. We also demonstrated a highly significant positive correlation between proliferation measured directly by 3H‐TdR incorporation into DNA or indirectly by 59Fe incorporation into haem. Ep was a potent stimulator of proerythroblast proliferation. We also examined the role of the androgenic and non‐androgenic steroids on in vitro proliferation. All the hormones tested were stimulatory but only in the presence of Ep. The androgenic steroids primarily affected the more mature erythroid precursors whereas the glucocorticoids were more general growth promoters. Their addition in physiologic concentration to liquid culture reduced Ep requirements. Thus when both testosterone and hydrocortisone were added to culture the Ep concentration that produced the same degree of proliferation as a culture containing Ep alone was decreased by 90%. This finding is important as it indicates that in vitro culture conditions can be created that more closely mimic in vivo erythropoiesis where Ep requirements are far less.

Age and the Hematopoietic System

The aging process is characterized by a decline in the function of many organ systems. Changes that occur in cardiovascular, endocrine, and immune function with aging have been described extensively. The bone marrow clearly plays an important role in normal physiology, being responsible for oxygen delivery to tissues as well as participating in normal hemostasis and host defense. There is evidence that anemia is common in older persons and that the increased morbidity and susceptibility to infection that occurs in the elderly is in part related to altered granulocyte function. This review will discuss normal bone marrow function and describe the most recent evidence implicating alterations in hematopoiesis with age.

Effect of age on marrow macrophage number and function

Employing flow cytometry and a monoclonal antibody against the murine macrophage antigen, Mac- 1, we found a significant increase in the number of marrow macrophages in aged mice. This was reflected as significant increase with age in the number of α- naphthyl acetate esterase positive cells, as well as in colony forming unit- macrophage (CFU- M) progenitor cells. Macrophages from the marrow of old mice generated significantly less tumor necrosis factor α (TNFα) than did macrophages from young mice, either spontaneously or when activated by granulocyte- macrophage colony stimulating factor (GM- CSF). Furthermore, conditioned medium (CM) derived from either marrow or peritoneal macrophages of old mice caused less suppression of burst forming unit- erythroid (BFU- E) colony growth than did CM obtained from young mice. Aging, therefore, is associated with an increase in the number of marrow macrophages that have an impaired ability to generate or release cytokines. The increase in macrophage number may reflect a compensation for their reduced function. Altered macrophage number and function may contribute to the age- related decline in hematopoietic reserve capacity.