A differential pattern of gene expression in skeletal muscle of tumor-bearing rats reveals dysregulation of excitation­contraction coupling together with additional muscle alterations.

INTRODUCTION: Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass. METHODS: By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia-inducing tumor, the AH-130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor-bearing animals, as compared with their healthy counterparts. RESULTS: The results show important differences in gene expression in EDL skeletal muscle between tumor-bearing animals with cachexia and control animals. CONCLUSIONS: The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome.

Patterns of gene expression in muscle and fat in tumor-bearing rats: effects of CRF2R agonist on cachexia.

The hypothesis we tested was that administering corticotropin-releasing factor receptor agonists preserves muscle mass during cancer that is related to changes in tissue gene expression. cDNA microarrays were used to compare mRNAs from muscle and adipose tissues of non-treated and agonist-treated tumor-bearing rats. In muscle of non-tumor-bearing agonist-treated animals we observed decreased expression of genes associated with fatty acid uptake and esterification. In tumor-bearing animals, CRF2R agonist administration produced decreased mRNA content of the atrogene lipin-1. In white adipose tissue, agonist treatment of non-tumor-bearing animals induced genes typically related to muscle structure and function. The fact that this treatment decreased expression of atrogenes could have clinical application. In addition, agonist treatment changed the gene pattern of adipose tissue to render it similar to that of skeletal muscle; thus, treatment with this agonist alters the gene pattern to what could be called "muscularization of white adipose tissue."

Corticortophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice.

BACKGROUND: Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy. METHODS: Using the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression. RESULTS: We have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression. CONCLUSION: Together, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.

Efficacy of systemic administration of SDF-1 in a model of vascular insufficiency: support for an endothelium-dependent mechanism.

OBJECTIVE: Studies have reported that administration of stromal cell-derived factor-1 (SDF-1), the ligand for the G-protein coupled receptor CXCR4, increased collateral blood flow in a mouse model of vascular insufficiency via recruitment of endothelial precursor cells (EPC). The present study investigated the contribution of mature endothelial cells in the actions of SDF-1. METHODS: The regulation of SDF-1 and CXCR4 was examined in the rat cornea cauterization (CC) and aortic ring (AR) model. The functional significance of the SDF-1/CXCR4 pathway was explored in cultured endothelial cells, the AR model, and on collateral blood flow in a rat model of vascular insufficiency. RESULTS: In the present study, the CXCR4 transcript was dramatically upregulated in the rat CC and AR explants, systems containing and lacking bone marrow-derived EPCs, respectively. Addition of AMD3100, a selective CXCR4 antagonist, had no effect on vessel growth in the AR alone, but completely inhibited SDF-1 mediated increases in vascular sprouting. In cultured endothelial cells, SDF-1 alone or in combination with vascular endothelial growth factor (VEGF) significantly enhanced cell survival and migration. Finally, systemic administration of SDF-1 in a rat model of arterial insufficiency enhanced collateral blood flow above vehicle control and equal to that of VEGF after 2 weeks of treatment. CONCLUSION: These studies support activation of the SDF-1/CXCR4 axis as a means to promote blood vessel growth and enhance collateral blood flow, at least in part, via direct effects on vascular endothelial cells.

An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresis.

Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.

The c-myc x E2F-1/p21 interactive gene expression index augments cytomorphologic diagnosis of lung cancer in fine-needle aspirate specimens.

Morphological analysis of cytologic samples obtained by fine-needle aspirate (FNA) or bronchoscopy is an important method for diagnosing bronchogenic carcinoma. However, this approach has only about 65 to 80% diagnostic sensitivity. Based on previous studies, the c-myc x E2F-1/p21WAF1/CIP1 (p21 hereafter) gene expression index is highly sensitive and specific for distinguishing normal from malignant bronchial epithelial tissues. In an effort to improve sensitivity of diagnosing lung cancer in cytologic specimens, we used Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) to measure the c-myc x E2F-1/p21 index in cDNA samples from 14 normal lung samples (6 normal lung parenchyma and 8 normal bronchial epithelial cell [NBEC] biopsies), and 16 FNA biopsies from 14 suspected tumors. Based on cytomorphologic criteria, 11 of the 14 suspected tumors were diagnosed as bronchogenic carcinoma and three specimens were non-diagnostic. Subsequent biopsy samples confirmed that the three non-diagnostic samples were derived from lung carcinomas. The index value for each bronchogenic carcinoma was above a cut-off value of 7000 and the index value of all but one normal sample was below 7000. Thus the c-myc x E2F-1/p21 index may augment cytomorphologic diagnosis of bronchogenic carcinoma biopsy samples, particularly those considered non-diagnostic by cytomorphologic criteria.