Specimen-specific nonlinear finite element modeling to predict vertebrae fracture loads after vertebroplasty.

STUDY DESIGN: Vertebral fracture load and stiffness from a metastatic vertebral defect model were predicted using nonlinear finite element models (FEM) and validated experimentally. OBJECTIVE: The study objective was to develop and validate an FEM-based tool for predicting polymer-augmented lytic vertebral fracture load and stiffness and the influence of metastatic filling materials. SUMMARY OF BACKGROUND DATA: Percutaneous vertebroplasty has the potential to reduce vertebral fracture risk affected with lytic metastases by providing mechanical stabilization. However, it has been shown that the mismatch in mechanical properties between poly(methyl-methacrylate) (PMMA) and bone induces secondary fractures and intervertebral disc degeneration. A biodegradable copolymer, poly(propylene fumarate-co-caprolactone) (P(PF-co-CL)), has been shown to possess the appropriate mechanical properties for bone defect repair. METHODS: Simulated metastatic lytic defects were created in 40 cadaveric vertebral bodies, which were randomized into 4 groups: intact vertebral body (intact), simulated defect without treatment (negative), defect treated with P(PF-co-CL) (copolymer), and defect treated with PMMA (PMMA). Spines were imaged with quantitative computed tomography (QCT), and QCT/FEM-subject-specific, nonlinear models were created. Predicted fracture loads and stiffness were identified and compared with experimentally measured values using Pearson correlation analysis and paired t test. RESULTS: There was no significant difference between the measured and predicted fracture loads and stiffness for each group. Predicted fracture loads were larger for PMMA augmentation (3960 N [1371 N]) than that for the copolymer, negative and intact groups (3484 N [1497 N], 3237 N [1744 N], and 1747 N [702 N]). A similar trend was observed in the predicted stiffness. Moreover, predicted and experimental fracture loads were strongly correlated (R=0.78), whereas stiffness showed moderate correlation (R=0.39). CONCLUSION: QCT/FEM was successful for predicting fracture loads of metastatic, polymer-augmented vertebral bodies. Overall, we have demonstrated that QCT/FEM may be a useful tool for predicting in situ vertebral fracture load resulting from vertebroplasty. LEVEL OF EVIDENCE: N/A.

Role of Rab5 in insulin receptor-mediated endocytosis and signaling.

Activated insulin receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between insulin receptor signaling and endocytosis is not well understood. This study utilizes both overexpression and depletion of Rab5 proteins to show that they play a critical role in both insulin-stimulated fluid phase and receptor-mediated endocytosis. Specifically, Rab5:WT and Rab5:Q79L (a GTP-hydrolysis defective mutant) enhance both types of endocytosis in response to insulin, while Rab5:S34N (a GTP-binding defective mutant) has the opposite effect. Morphological analysis indicates that both Rab5 and insulin receptor are found on early endosomes, but not at the plasma membrane. In addition, expression of Rab5:WT and Rab5:Q79L enhance both Erk1/2 and Akt activation without affecting JN- and p38-kinase activities, while the expression of Rab5:S34N blocks both Erk1/2 and Akt activation. Consistent with these observations, DNA synthesis is also altered by the expression of Rab5:S34N. Taken together, these results demonstrate that Rab5 is required for insulin receptor membrane trafficking and signaling.

Rin1 regulates insulin receptor signal transduction pathways.

Rin1 is a multifunctional protein containing several domains, including Ras binding and Rab5 GEF domains. The role of Rin1 in insulin receptor internalization and signaling was examined by expressing Rin1 and deletion mutants in cells utilizing a retrovirus system. Here, we show that insulin-receptor-mediated endocystosis and fluid phase insulin-stimulated endocytosis are enhanced in cells expressing the Rin1:wild type and the Rin1:C deletion mutant, which contain both the Rab5-GEF and GTP-bound Ras binding domains. However, the Rin1:N deletion mutant, which contains both the SH2 and proline-rich domains, blocked insulin-stimulated receptor-mediated and insulin-stimulated fluid phase endocytosis. In addition, the expression of Rin1:delta (429-490), a natural occurring splice variant, also blocked both receptor-mediated and fluid phase endocystosis. Furthermore, association of the Rin1 SH2 domain with the insulin receptor was dependent on tyrosine phosphorylation of the insulin receptor. Morphological analysis indicates that Rin1 co-localizes with insulin receptor both at the cell surface and in endosomes upon insulin stimulation. Interestingly, the expression of Rin1:wild type and both deletion mutants blocks the activation of Erk1/2 and Akt1 kinase activities without affecting either JN or p38 kinase activities. DNA synthesis and Elk-1 activation are also altered by the expression of Rin1:wild type and the Rin1:C deletion mutant. In contrast, the expression of Rin1:delta stimulates both Erk1/2 and Akt1 activation, DNA synthesis and Elk-1 activation. These results demonstrate that Rin1 plays an important role in both insulin receptor membrane trafficking and signaling.

Rab5-activating protein 6, a novel endosomal protein with a role in endocytosis.

Rab GTPases are regulators of membrane trafficking that cycle between active (GTP-bound) and inactive (GDP-bound) states. In this study, we report the identification of a new human Rab5 guanine nucleotide exchange factor (GEF), which we have named RAP6 (Rab5-activating protein 6). RAP6 contains a Rab5 GEF and a Ras GAP domain. We show that the Vps9 domain is sufficient for the interaction of RAP6 with GDP-bound Rab5 and that RAP6 stimulates Rab5 guanine nucleotide exchange. We also find that the Ras GAP domain of RAP6 shows GAP activity for Ras. Immunofluorescence experiments reveal that RAP6 is associated with plasma membrane and small intracellular vesicles that also contain Rab5. Additionally, the overexpression of RAP6 affects both fluid phase and receptor-mediated endocytosis. This study is the first to show that RAP6 is a novel regulator of endocytosis that exhibits GEF activity specific for Rab5 and GAP activity specific for Ras.