Recombinant human platelet-derived growth factor-BB augmentation of new-bone formation in a rat model of distraction osteogenesis.

BACKGROUND: Distraction osteogenesis creates a challenging bone-healing environment with protracted demand for cells of the osteoblast lineage. Platelet-derived growth factor-BB (PDGF-BB) is an osteoblast mitogen and chemotaxin that has been shown to accelerate and/or enhance bone-healing in several preclinical studies. The purpose of the present study was to determine whether recombinant human platelet-derived growth factor-BB (rhPDGF-BB) would have a similar effect on regenerate healing after distraction osteogenesis. METHODS: Unilateral 7-mm mid-diaphyseal femoral lengthening procedures were performed in eighty-three male Sprague-Dawley rats that were separated into five experimental groups. During the distraction period (Days 7 to 28), each animal received a weekly 50-microL injection of either sodium acetate buffer, bovine collagen dissolved in sodium acetate buffer, or one of three concentrations of rhPDGF-BB (100, 300, or 1000 microg/mL) into the distraction site. Animals from each group were killed on Days 35, 42, 49, 56, and 63. Healing was assessed with biweekly serial radiographs, micro-computed tomography of the explanted bones, and histologic analysis. RESULTS: rhPDGF-BB treatment significantly increased new-bone formation at the midconsolidation time points (Days 42, 49, and 56) as well as the union rate. On Day 49 regenerate bone volume was significantly greater in each of the three rhPDGF-BB-treated groups than in the controls (p < 0.05, p = 0.0002, and p < 0.05 for the 100, 300, and 1000 microg/mL rhPDGF-BB groups, respectively), whereas on Day 42 regenerate bone volume was significantly greater in the 300 and 1000 microg/mL rhPDGF-BB groups than in the controls (p = 0.0002 and p < 0.05, respectively) and on Day 56 regenerate bone volume was significantly greater in the 100 and 300 microg/mL rhPDGF-BB groups than in the controls (p < 0.05 and p < 0.0001, respectively). The overall union rate was 40.4% (nineteen of forty-seven) in the rhPDGF-BB-treated animals, compared with 4.5% (one of twenty-two) in the controls (p = 0.01). The radiographic and histologic results were consistent with new-bone formation as quantified by micro-computed tomography, although they were less definitive. CONCLUSIONS: The administration of exogenous rhPDGF-BB into the distraction site during diaphyseal distraction enhanced bone-healing in a rat model of distraction osteogenesis as evidenced by both increased regenerate new-bone formation and a higher union rate.

Short-term arginine deprivation results in large-scale modulation of hepatic gene expression in both normal and tumor cells: microarray bioinformatic analysis.

BACKGROUND: We have reported arginine-sensitive regulation of LAT1 amino acid transporter (SLC 7A5) in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells. We hypothesized that liver cell gene expression is highly sensitive to alterations in the amino acid microenvironment and that tumor cells may differ substantially in gene sets sensitive to amino acid availability. To assess the potential number and classes of hepatic genes sensitive to arginine availability at the RNA level and compare these between normal and tumor cells, we used an Affymetrix microarray approach, a paired in vitro model of normal rat hepatic cells and a tumorigenic derivative with triplicate independent replicates. Cells were exposed to arginine-deficient or control conditions for 18 hours in medium formulated to maintain differentiated function. RESULTS: Initial two-way analysis with a p-value of 0.05 identified 1419 genes in normal cells versus 2175 in tumor cells whose expression was altered in arginine-deficient conditions relative to controls, representing 9-14% of the rat genome. More stringent bioinformatic analysis with 9-way comparisons and a minimum of 2-fold variation narrowed this set to 56 arginine-responsive genes in normal liver cells and 162 in tumor cells. Approximately half the arginine-responsive genes in normal cells overlap with those in tumor cells. Of these, the majority was increased in expression and included multiple growth, survival, and stress-related genes. GADD45, TA1/LAT1, and caspases 11 and 12 were among this group. Previously known amino acid regulated genes were among the pool in both cell types. Available cDNA probes allowed independent validation of microarray data for multiple genes. Among genes downregulated under arginine-deficient conditions were multiple genes involved in cholesterol and fatty acid metabolism. Expression of low-density lipoprotein receptor was decreased in both normal and tumor cells. CONCLUSION: Arginine-sensitive regulation appears to be an important homeostatic mechanism to coordinate cell response and nutrient availability in hepatic cells. Genes predicted as arginine-responsive in stringent microarray data analysis were confirmed by Northern blot and RT-PCR. Although the profile of arginine-responsive genes is altered and increased, a considerable portion of the "arginome" is maintained upon neoplastic transformation.

Adenoviral modulation of the tumor-associated system L amino acid transporter, LAT1, alters amino acid transport, cell growth and 4F2/CD98 expressionwith cell-type specific effects in cultured hepatic cells.

Altered expression of metabolite transporters is observed frequently in tumor cell lines and primary neoplasms. The extent to which these may to contribute to the growth autonomy associated with cancer is not clear. LAT1 is a major L-type amino acid transporter over-expressed in a variety of cancer types and a light chain component of the CD98 heterodimer. We utilized an adenoviral expression system to modulate the level of LAT1 in a hepatic in vitro model to examine phenotypic changes associated with short-term exogenous and blocked expression. LAT1 levels were increased three fold and resulted in increased L-type amino acid transport as a result of adenoviral expression in murine hepatocytes. The protein was expressed on the cell surface and complexed with the CD98 heavy chain known as 4F2. Surprisingly, levels of the total CD98 protein complex were increased 2.4-fold as a result of adenoviral expression of light chain only, suggesting coordinate regulation. Exogenous overexpression was less effective in normal rat liver cells relative to mouse. LAT1 antisense expression in hepatic tumor cells resulted in a modest though statistically significant decrease in cell number, viability and S-phase cells over a 5-day period relative to controls despite the absence of a significant decrease in L-type transport over this period. These studies are preparatory to in vivo efforts focusing on LAT1/CD98 as a potential therapeutic target.