Previously we have identified the glycosphingolipid globotriaosylceramide (Gb3/Pk) as an inhibitor and resistance factor against HIV-1 infection in vitro. Here we show that a novel, soluble, completely synthetic Gb3 analogue, FSLGb3, inhibits infection of X4 strains of HIV-1 in the Jurkat T-cell line and both R5 and X4 strains in PBMCs. FSLGb3 absorbs into cellular plasma membranes and membrane adsorbed FSLGb3 was able to inhibit subsequent HIV-1 infection. We have also developed a mouse model to test in vivo the efficacy of soluble Gb3 analogs in the prevention of mucosal viral infection. Soluble Gb3 was incorporated into gel or alone and applied directly to the vaginal and rectal mucosal tissue of mice. We have not yet shown a statistically significant reduction in infection, although a trend towards inhibition is evident. Our studies show synthetic Gb3 to be an inhibitor of HIV-1 infection and further exploration of therapeutic strategies are warranted.

Previously we have identified the glycosphingolipid globotriaosylceramide (Gb3/Pk) as an inhibitor and resistance factor against HIV-1 infection in vitro. Here we show that a novel, soluble, completely synthetic Gb3 analogue, FSLGb3, inhibits infection of X4 strains of HIV-1 in the Jurkat T-cell line and both R5 and X4 strains in PBMCs. FSLGb3 absorbs into cellular plasma membranes and membrane adsorbed FSLGb3 was able to inhibit subsequent HIV-1 infection. We have also developed a mouse model to test in vivo the efficacy of soluble Gb3 analogs in the prevention of mucosal viral infection. Soluble Gb3 was incorporated into gel or alone and applied directly to the vaginal and rectal mucosal tissue of mice. We have not yet shown a statistically significant reduction in infection, although a trend towards inhibition is evident. Our studies show synthetic Gb3 to be an inhibitor of HIV-1 infection and further exploration of therapeutic strategies are warranted.

Shaken baby syndrome is a clinicopathologic syndrome characterized by a triad of findings: subdural hemorrhage, retinal hemorrhage and axonal injury. Although shaking is widely believed to cause the triad, it is not yet entirely clear if shaking without head impact can produce the triad. Initial attempts to test the effect of shaking in mouse pups were unsuccessful as neither controlled continuous vibration nor pulse acceleration caused any of the components of the triad. With no other convenient modeling system available, a pilot study with three adult subhuman primates was conducted. Although a conclusive statement cannot be made, manual shaking did not immediately cause hemorrhagic injuries to the primates’ brains and eyes. Future studies should test for delayed development of axonal injury. In addition, a comparative anatomical study should also be conducted to test the validity of the adult primate as a model system for human infant injuries.

Shaken baby syndrome is a clinicopathologic syndrome characterized by a triad of findings: subdural hemorrhage, retinal hemorrhage and axonal injury. Although shaking is widely believed to cause the triad, it is not yet entirely clear if shaking without head impact can produce the triad. Initial attempts to test the effect of shaking in mouse pups were unsuccessful as neither controlled continuous vibration nor pulse acceleration caused any of the components of the triad. With no other convenient modeling system available, a pilot study with three adult subhuman primates was conducted. Although a conclusive statement cannot be made, manual shaking did not immediately cause hemorrhagic injuries to the primates’ brains and eyes. Future studies should test for delayed development of axonal injury. In addition, a comparative anatomical study should also be conducted to test the validity of the adult primate as a model system for human infant injuries.

The role of the Angiopoietin-1 (Ang1)-Tie2 pathway in pulmonary arterial hypertension (PAH) is controversial, with one group suggesting that increased Ang1-Tie2 activity may even play a causal role in PAH progression. However, Ang1 has been well-characterized as a homeostatic factor, which prevents endothelial cell (EC) activation and injury through Tie2 receptor stimulation. Furthermore, we have previously demonstrated that lung Tie2 expression and activity are significantly downregulated in experimental PAH, implicating reduced Tie2 signaling in the pathogenesis of this disease. Thus, we hypothesized that manipulations to increase Ang1-Tie2 signaling will protect against lung EC injury and onset of PAH, whereas a loss-of-function of this pathway in Tie2-deficient mice (Tie2+/-) would predispose to PAH. Approximately 13% of Tie2+/- mice developed spontaneous elevation in right ventricular systolic pressure (RVSP), although no significant difference was observed between the WT and Tie2+/- groups following chronic hypoxic exposure. Serotonin (5-HT) infusion for one week or daily interleukin-6 (IL-6) injection for two weeks produced substantial RVSP elevations in Tie2+/-, but not WT mice (P<0.05). Following one week of 5-HT or IL-6 treatment, there was a significant decrease in Ang1 protein expression in both WT and Tie2+/- mice (P<0.05). Similarly, following exposure of cultured pulmonary arterial smooth muscle cells to 5-HT or IL-6, Ang1 secretion was significantly decreased (P<0.01). Moreover, 5-HT or IL-6 exposure resulted in decreased Tie2 activity and increased apoptosis only in the lungs of Tie2+/- mice (P<0.01), and treatment with a pan-caspase inhibitor, Z-VAD, prevented PAH in 5-HT-treated Tie2-deficient mice. Together with in vitro studies showing that pulmonary arterial ECs subjected to Tie2-siRNA were more susceptible to apoptosis following 5-HT treatment, this strongly implicates EC death as a primary mechanism of PAH in the presence of reduced Tie2 activity. In addition, doxycycline-conditional, endothelial-targeted Ang1 binary transgenic mice showed no evidence of elevated RVSP under basal conditions and if anything, demonstrated a blunted response to 5-HT treatment compared to non-binary littermate controls. Therefore, our findings support the importance of EC survival-signaling via the Ang1-Tie2 pathway as a protective mechanism in PAH and emphasize the pivotal role of EC apoptosis in the onset of this disease.

The role of the Angiopoietin-1 (Ang1)-Tie2 pathway in pulmonary arterial hypertension (PAH) is controversial, with one group suggesting that increased Ang1-Tie2 activity may even play a causal role in PAH progression. However, Ang1 has been well-characterized as a homeostatic factor, which prevents endothelial cell (EC) activation and injury through Tie2 receptor stimulation. Furthermore, we have previously demonstrated that lung Tie2 expression and activity are significantly downregulated in experimental PAH, implicating reduced Tie2 signaling in the pathogenesis of this disease. Thus, we hypothesized that manipulations to increase Ang1-Tie2 signaling will protect against lung EC injury and onset of PAH, whereas a loss-of-function of this pathway in Tie2-deficient mice (Tie2+/-) would predispose to PAH. Approximately 13% of Tie2+/- mice developed spontaneous elevation in right ventricular systolic pressure (RVSP), although no significant difference was observed between the WT and Tie2+/- groups following chronic hypoxic exposure. Serotonin (5-HT) infusion for one week or daily interleukin-6 (IL-6) injection for two weeks produced substantial RVSP elevations in Tie2+/-, but not WT mice (P<0.05). Following one week of 5-HT or IL-6 treatment, there was a significant decrease in Ang1 protein expression in both WT and Tie2+/- mice (P<0.05). Similarly, following exposure of cultured pulmonary arterial smooth muscle cells to 5-HT or IL-6, Ang1 secretion was significantly decreased (P<0.01). Moreover, 5-HT or IL-6 exposure resulted in decreased Tie2 activity and increased apoptosis only in the lungs of Tie2+/- mice (P<0.01), and treatment with a pan-caspase inhibitor, Z-VAD, prevented PAH in 5-HT-treated Tie2-deficient mice. Together with in vitro studies showing that pulmonary arterial ECs subjected to Tie2-siRNA were more susceptible to apoptosis following 5-HT treatment, this strongly implicates EC death as a primary mechanism of PAH in the presence of reduced Tie2 activity. In addition, doxycycline-conditional, endothelial-targeted Ang1 binary transgenic mice showed no evidence of elevated RVSP under basal conditions and if anything, demonstrated a blunted response to 5-HT treatment compared to non-binary littermate controls. Therefore, our findings support the importance of EC survival-signaling via the Ang1-Tie2 pathway as a protective mechanism in PAH and emphasize the pivotal role of EC apoptosis in the onset of this disease.

Introduction: Activation of Runt Related Transcription Factor 2 (RUNX2) is required for transdifferentiation of Vascular Smooth Muscle Cells (VSMCs) into a calcifying osteoblast-like phenotype. Our lab showed that deletion of Discoidin Domain Receptor 1 (Ddr1), decreased atherosclerotic vascular calcification in the Ldlr-/- mouse. Hypothesis: DDR1 regulates RUNX2 activity by affecting microtubule organization during VSMC mediated calcification. Results: Ddr1-/- VSMCs show reduced RUNX2 activity when compared to Ddr1+/+ VSMCs. Addition of the microtubule-destabilizing agent nocodazole inhibited both RUNX2 activity and nuclear localization in Ddr1+/+ VSMCs. Addition of the microtubule-stabilizing agent taxol rescued RUNX2 nuclear localization in Ddr1-/- VSMCs. Despite this, Taxol was unable to rescue RUNX2 activity as it eliminated activity in both genotypes. Conclusion: These findings indicate that under osteogenic conditions, Ddr1 deletion impedes the dynamic instability required for the maintenance of microtubule architecture. This prevents RUNX2 nuclear localization and transcriptional activation in VSMCs.

Introduction: Activation of Runt Related Transcription Factor 2 (RUNX2) is required for transdifferentiation of Vascular Smooth Muscle Cells (VSMCs) into a calcifying osteoblast-like phenotype. Our lab showed that deletion of Discoidin Domain Receptor 1 (Ddr1), decreased atherosclerotic vascular calcification in the Ldlr-/- mouse. Hypothesis: DDR1 regulates RUNX2 activity by affecting microtubule organization during VSMC mediated calcification. Results: Ddr1-/- VSMCs show reduced RUNX2 activity when compared to Ddr1+/+ VSMCs. Addition of the microtubule-destabilizing agent nocodazole inhibited both RUNX2 activity and nuclear localization in Ddr1+/+ VSMCs. Addition of the microtubule-stabilizing agent taxol rescued RUNX2 nuclear localization in Ddr1-/- VSMCs. Despite this, Taxol was unable to rescue RUNX2 activity as it eliminated activity in both genotypes. Conclusion: These findings indicate that under osteogenic conditions, Ddr1 deletion impedes the dynamic instability required for the maintenance of microtubule architecture. This prevents RUNX2 nuclear localization and transcriptional activation in VSMCs.

High Grade Serous Carcinoma is the most aggressive tubal/ovarian histotype. The Rb pathway, which functions to preserve cell cycle regulation and genomic stability, is frequently deregulated in HGSC. The aim of this study was to identify non-redundant mechanisms of Rb pathway deregulation with clinical relevance. Immunohistochemistry analysis of P16 and RB1 expression identified a P16 homogeneous/RB1+ subgroup with the shortest recurrence free survival, while P16 heterogeneous/RB1+ and P16 homogenous/RB1- subgroups showed longer recurrence free survival. These P16/RB1 categories were shared between precursor lesions and the associated HGSC. Characterization of the Rb pathway in the three subgroups revealed CCNE1 amplification and highest protein expression in the P16 homogeneous/RB1+, high cyclin D1 protein expression in P16 heterogeneous/RB1+, and E2F3 amplification and highest Ki67 protein expression in P16 homogeneous/RB1- subgroup. Rb pathway deregulation is common in HGSC, occurs early in tumor development, and results from multiple non-redundant mechanisms that correlate with clinical outcome.

High Grade Serous Carcinoma is the most aggressive tubal/ovarian histotype. The Rb pathway, which functions to preserve cell cycle regulation and genomic stability, is frequently deregulated in HGSC. The aim of this study was to identify non-redundant mechanisms of Rb pathway deregulation with clinical relevance. Immunohistochemistry analysis of P16 and RB1 expression identified a P16 homogeneous/RB1+ subgroup with the shortest recurrence free survival, while P16 heterogeneous/RB1+ and P16 homogenous/RB1- subgroups showed longer recurrence free survival. These P16/RB1 categories were shared between precursor lesions and the associated HGSC. Characterization of the Rb pathway in the three subgroups revealed CCNE1 amplification and highest protein expression in the P16 homogeneous/RB1+, high cyclin D1 protein expression in P16 heterogeneous/RB1+, and E2F3 amplification and highest Ki67 protein expression in P16 homogeneous/RB1- subgroup. Rb pathway deregulation is common in HGSC, occurs early in tumor development, and results from multiple non-redundant mechanisms that correlate with clinical outcome.