Inhibition of prostaglandin-degrading enzyme 15-PGDH rejuvenates aged muscle mass and strength.

Treatments are lacking for sarcopenia, a debilitating age-related skeletal muscle wasting syndrome. We identifed increased amounts of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the prostaglandin E2 (PGE2)-degrading enzyme, as a hallmark of aged tissues, including skeletal muscle. The consequent reduction in PGE2 signaling contributed to muscle atrophy in aged mice and results from 15-PGDH-expressing myofibers and interstitial cells, such as macrophages, within muscle. Overexpression of 15-PGDH in young muscles induced atrophy. Inhibition of 15-PGDH, by targeted genetic depletion or a small-molecule inhibitor, increased aged muscle mass, strength, and exercise performance. These benefits arise from a physiological increase in PGE2 concentrations, which augmented mitochondrial function and autophagy and decreased transforming growth factor-ß signaling and activity of ubiquitin-proteasome pathways. Thus, PGE2 signaling ameliorates muscle atrophy and rejuvenates muscle function, and 15-PGDH may be a suitable therapeutic target for countering sarcopenia.

Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture.

Stem cells that naturally reside in adult tissues, such as muscle stem cells (MuSCs), exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a highly automated single-cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (approximately 10(6) kilopascals), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by noninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle-wasting diseases.

A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions.

The orphan receptor tyrosine kinase ErbB2 is activated by each of the EGFR family members upon ligand binding. However, difficulties monitoring the dynamic interactions of the membrane receptors have hindered the elucidation of the mechanism of ErbB2 activation. We have engineered a system to monitor protein-protein interactions in intact mammalian cells such that different sets of protein interactions can be quantitatively compared. Application of this system to the interactions of the EGFR family showed that ErbB2 interacts stably with the EGFR and ErbB3, but fails to spontaneously homooligomerize. The widely used anti-cancer antibody Herceptin was found to effectively inhibit the interaction of the EGFR and ErbB2 but not to interfere with the interaction of ErbB2-ErbB3. Treatment of cells expressing EGFR and ErbB2 with Herceptin results in increased EGFR homooligomerization in the presence of EGF and a subsequent rapid internalization and down-regulation of the EGFR. In summary, the protein interaction system described here enabled the characterization of ErbB2 interactions within the biological context of the plasma membrane and provides insight into the mechanism of Herceptin action on cells overexpressing ErbB2.

Laminin-induced change in conformation of preexisting alpha7beta1 integrin signals secondary myofiber formation.

Two distinct populations of myoblasts, distinguishable by alpha7 integrin expression have been hypothesized to give rise to two phases of myofiber formation in embryonic limb development. We show here that alpha7 integrin is detectable far earlier than previously reported on both "primary" and "secondary" lineage myoblasts and myofibers. An antibody (1211) that recognizes an intracellular epitope allowed detection of alpha7 integrin previously missed using an antibody (H36) that recognizes an extracellular epitope. We found that when myoblasts were isolated and cultured from different developmental stages, H36 only detected alpha7 integrin that was in direct contact with its ligand, laminin. Moreover, alpha7 integrin detection by H36 was reversible and highly localized to subcellular points of contact between myoblasts and laminin-coated 2.8-microm microspheres. Prior to secondary myofiber formation in limb embryogenesis, laminin was present but not in close proximity to clusters of primary myofibers that expressed alpha7 integrin detected by antibody 1211 using deconvolution microscopy. These results suggest that the timing of the interaction of preexisting alpha7 integrin with its ligand, laminin, is a major determinant of allosteric changes that result in an activated form of alpha7 integrin capable of transducing signals from the extracellular matrix commensurate with secondary myofiber formation.

Purification of mouse primary myoblasts based on alpha 7 integrin expression.

Fundamental insights have come from the study of myogenesis. Primary myoblasts isolated directly from muscle tissue more closely approximate myogenesis than established cell lines. However, contamination of primary muscle cultures with nonmyogenic cells can complicate the results. To overcome this problem, we previously described a method for myoblast purification based on novel culture conditions (T. A. Rando and H. M. Blau, 1994, J. Cell Biol. 125, 1275--1287). Here we report a refinement of this method that leads directly to an enriched population of mouse primary myoblasts, within significantly fewer population doublings. The method described here avoids using adhesion as a criterion for selection. This advance capitalizes on the ability of the antibody CA5.5 to recognize alpha 7 integrin, a muscle-specific cell surface antigen. Enrichment of myoblasts to greater than 95% of the cell population can be achieved by a single round of flow cytometry or magnetic bead separation. This is the first description of a mouse myoblast purification method based on a cell-type-specific antigen. The ease of this procedure for isolating primary myoblasts should expand the opportunities for (1) using these cells in cell transplantation studies in animal models of human disease, (2) isolating and characterizing mutant myoblasts from transgenic animals, and (3) allowing in vitro studies of molecules that regulate muscle cell growth, differentiation, and neoplasia.

From marrow to brain: expression of neuronal phenotypes in adult mice.

After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.

Transcriptional control: rheostat converted to on/off switch.

Individual cells translate concentration gradients of extracellular factors into all-or-none threshold responses leading to discrete patterns of gene expression. Signaling cascades account for some but not all such threshold responses, suggesting the existence of additional mechanisms. Here we show that all-or-none responses can be generated at a transcriptional level. A graded rheostat mechanism obtained when either transactivators or transrepressors are present is converted to an on/off switch when these factors compete for the same DNA regulatory element. Hill coefficients of dose-response curves confirm that the synergistic responses generated by each factor alone are additive, obviating the need for feedback loops. We postulate that regulatory networks of competing transcription factors prevalent in cells and organisms are crucial for establishing true molecular on/off switches.

Induction of angiogenesis by implantation of encapsulated primary myoblasts expressing vascular endothelial growth factor.

BACKGROUND: We previously demonstrated that intramuscular implantation of primary myoblasts engineered to express vascular endothelial growth factor (VEGF) constitutively resulted in hemangioma formation and the appearance of VEGF in the circulation. To investigate the potential for using allogeneic myoblasts and the effects of delivery of VEGF-expressing myoblasts to non-muscle sites, we have enclosed them in microcapsules that protect allogeneic cells from rejection, yet allow the secretion of proteins produced by the cells. METHODS: Encapsulated mouse primary myoblasts that constitutively expressed murine VEGF164, or encapsulated negative control cells, were implanted either subcutaneously or intraperitoneally into mice. RESULTS: Upon subcutaneous implantation, capsules containing VEGF-expressing myoblasts gave rise to large tissue masses at the implantation site that continued to grow and were composed primarily of endothelial and smooth muscle cells directly surrounding the capsules, and macrophages and capillaries further away from the capsules. Similarly, when injected intraperitoneally, VEGF-producing capsules caused significant localized inflammation and angiogenesis within the peritoneum, and ultimately led to fatal intraperitoneal hemorrhage. Notably, however, VEGF was not detected in the plasma of any mice. CONCLUSIONS: We conclude that encapsulated primary myoblasts persist and continue to secrete VEGF subcutaneously and intraperitoneally, but that the heparin-binding isoform VEGF164 exerts localized effects at the site of production. VEGF secreted from the capsules attracts endothelial and smooth muscle cells in a macrophage-independent manner. These results, along with our previous results, show that the mode and site of delivery of the same factor by the same engineered myoblasts can lead to markedly different outcomes. Moreover, the results confirm that constitutive delivery of high levels of VEGF is not desirable. In contrast, regulatable expression may lead to efficacious, safe, and localized VEGF delivery by encapsulated allogeneic primary myoblasts that can serve as universal donors.

Angiogenesis monitored by perfusion with a space-filling microbead suspension.

Numerous laboratories are focusing efforts on delivering gene products to induce or prevent the development of new blood vessels in adults, with the hope of rescuing ischemic tissues, circumventing cardiac bypass surgery, or inhibiting tumor growth. Current approaches to the assessment of vascular continuity involve the introduction of either dyes or fluorescent microspheres to track blood flow. However, dyes and dextrans are subject to leakage when vessels are hyperpermeable, a situation that may occur in studies of tumor vasculature and during efforts to stimulate therapeutic angiogenesis. Furthermore, the microspheres that are used for flow studies do not allow a comprehensive visual analysis of vascular continuity. Here we report a method for the visual assessment of microvascular continuity in mouse muscle under circumstances in which vessels are leaky. The approach involves perfusion of the vasculature with fluorescent beads that are much smaller than those used for flow studies. The suspension behaves like a fluid and completely fills the vessels, yet the beads do not leak from VEGF-permeablized capillaries and remain localized in histological sections. Use of beads with the proper fluorescence emission wavelengths allows immunofluorescent colocalization with vessel-specific markers. We compare this improved method with other methods for tracking vascular continuity involving dextrans and larger beads. This approach should aid in the dynamic study of tumor angiogenesis and the evaluation of efforts to deliver angiogenic factors.

VEGF gene delivery to myocardium: deleterious effects of unregulated expression.

BACKGROUND: Vascular endothelial growth factor (VEGF) is being investigated for therapeutic angiogenesis in ischemic myocardium. Primarily, transient delivery systems have been tested. The goal of this study was to investigate the effects of continuous expression of VEGF in myocardium by use of myoblast-mediated delivery. METHODS AND RESULTS: Primary murine myoblasts (5 x 10(5) cells in 10 microL of PBS with 0.5% BSA) expressing both the murine VEGF gene and the beta-galactosidase (beta-gal) gene from a retroviral promoter were implanted in the ventricular wall of immunodeficient mice (n=11) via a subdiaphragmatic approach. Control immunodeficient mice (n=12) were injected with the same number of myoblasts expressing only the beta-gal gene. Between days 14 and 16, surviving mice were euthanized and the hearts processed for histology. In the experimental group, 11 of 11 mice demonstrated failure to thrive by day 13; 5 deaths occurred between days 8 and 15. There were no complications in the control mice. Histochemistry documented successful implantation of myoblasts (positive beta-gal reaction product) in 6 of 6 surviving experimental mice and 12 of 12 controls. Histology disclosed intramural vascular tumors resembling hemangiomas in the VEGF-myoblast-injected myocardium in 6 of 6 surviving mice. beta-Gal-expressing cells were present at the site of the vascular tumors. Immunohistochemistry localized abundant endothelial nitric oxide synthase and CD31 (platelet and endothelial cell adhesion molecule) within the lesion, consistent with the presence of endothelial cells. CONCLUSIONS: In this model, unregulated continuous expression of VEGF is associated with (1) a high rate of failure to thrive/death and (2) formation of endothelial cell-derived intramural vascular tumors in the implantation site. These results underscore the importance of regulating VEGF expression for therapeutic angiogenesis.

A novel means of drug delivery: myoblast-mediated gene therapy and regulatable retroviral vectors.

A potentially powerful approach to drug delivery in the treatment of disease involves the use of cells to introduce genes encoding therapeutic proteins into the body. Candidate genes for delivery include those encoding secreted factors that could have broad applications ranging from treatment of inherited single-gene deficiencies to acquired disorders of the vasculature or cancer. Myoblasts, the proliferative cell type of skeletal muscle tissues, are potent tools for stable delivery of a gene of interest into the body, as they become an integral part of the muscle into which they are injected, in close proximity to the circulation. The recent development of improved tetracycline-inducible retroviral vectors allows for fine control of recombinant gene expression levels. The combination of ex vivo gene transfer using myoblasts and regulatable retroviral vectors provides a powerful toolbox with which to develop gene therapies for a number of human diseases.

Neural cell adhesion molecule (NCAM) and myoblast fusion.

Considerable evidence points to an involvement of neural cell adhesion molecule (NCAM) in myoblast fusion. Changes in the level of NCAM expression, isoform specificity, and localization in muscle cells and tissues correspond to key morphogenetic events during muscle differentiation and repair. Furthermore, anti-NCAM antibodies have been shown by others to reduce the rate of myoblast fusion, whereas overexpression of NCAM cDNAs increases the rate of myoblast fusion compared to controls. In this study we have used a novel fusion assay based on intracistronic complementation of lacZ, in combination with fluorescent X-gal histochemistry and immunocytochemistry to assess levels of NCAM expression in individual muscle cells. Our results indicate that a substantial proportion of newly fused myoblasts have NCAM expression levels unchanged from the levels of the surrounding unfused population suggesting that increased expression of NCAM is not required for wild-type myoblasts to fuse. Moreover, pure populations of primary myoblasts isolated from mice homozygous null for NCAM and therefore lacking the molecule, when placed in differentiation medium, consistently fused to form contractile myotubes with kinetics equivalent to wild-type primary myoblasts. We conclude that the increase in expression of NCAM, although typically observed during myogenesis, is not essential to myoblast fusion to form myotubes.

Epidermal growth factor receptor dimerization monitored in live cells.

We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing beta-galactosidase (beta-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as beta-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. beta-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and beta-gal complementation. These results demonstrate that beta-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.

The phosphoprotein protein PEA-15 inhibits Fas- but increases TNF-R1-mediated caspase-8 activity and apoptosis.

We have characterized a phosphoprotein protein with a death effector domain that has a novel bifunctional role in programmed cell death. The 15-kDa phosphoprotein enriched in astrocytes (PEA-15) inhibits Fas-mediated apoptosis and increases tumor necrosis factor receptor-1 (TNF-R1)-mediated apoptosis in the same cell type in a ligand-dependent manner. Phosphorylation appears to play a role in its differential effects, since point mutations at one or both phosphorylation consensus sites within PEA-15 destroy its effect on Fas-mediated, but not TNF-R1-mediated, apoptosis. Furthermore, the differential effect is evident at the level of caspase-8 activity which is inhibited via Fas activation, but increased via TNF-R1 activation upon PEA-15 expression. These results show that PEA-15 provides a potential mechanism during development for distinguishing between diverse extracellular death-inducing signals that culminate either in apoptosis or in survival.

Plasticity of cell fate: insights from heterokaryons.

Experiments with somatic cell hybrids and stable heterokaryons have demonstrated that differentiated cells exhibit a remarkable capacity to change. Heterokaryons have been particularly useful in determining the extent to which the differentiated state of a cell is plastic. Cell fate can be altered by a change in the balance of positive and negative trans-acting regulators. Although a single regulator may be sufficient in certain environments to trigger a change in cell fate, that regulator may be ineffective in other cell contexts where it encounters a different composition of regulators.