Faculty Bibliography

In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by the Liver X Receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRalpha serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRalpha at S198 is non-phosphorylated. In bone marrow derived-macrophages (BMDMs) we also observe induction of CCR7 by ligands that promote non-phosphorylated LXRalpha S198 and this is lost in LXR deficient BMDMs. LXRalpha occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing non-phosphorylated (RAW-LXRalphaS198A) compared to phosphorylated LXRalpha (RAW-LXRalphaWT). Expression profiling from ligand treated RAW-LXRalphaS198A compared to RAW-LXRalphaWT cells revealed induction of cell migratory and anti-inflammatory genes, and repression of pro-inflammatory genes. Modeling of LXRalpha S198 in non-phosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with sites for protein interaction. Therefore, gene transcription is regulated by LXRalpha S198 phosphorylation including anti-atherogenic genes like CCR7.

BACKGROUND: Cell-assisted lipotransfer has shown much promise as a technique to improve fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown. METHODS: Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction (SVF) cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 mul of fat. Fat graft volume retention was assessed via CT scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity. RESULTS: Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 mul of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, while fat grafts supplemented with 10 million cells showed significant lipodegeneration compared to fat alone CONCLUSIONS:: Our study demonstrates dose dependence in the number of SVF cells that can be added to a fat graft to enhance retention. While cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the post-graft period.

Genome-wide association studies have implicated PLEXIN D1 (PLXND1) in body fat distribution and type 2 diabetes. However, a role for PLXND1 in regional adiposity and insulin resistance is unknown. Here we use in vivo imaging and genetic analysis in zebrafish to show that Plxnd1 regulates body fat distribution and insulin sensitivity. Plxnd1 deficiency in zebrafish induced hyperplastic morphology in visceral adipose tissue (VAT) and reduced lipid storage. In contrast, subcutaneous adipose tissue (SAT) growth and morphology were unaffected, resulting in altered body fat distribution and a reduced VAT:SAT ratio in zebrafish. A VAT-specific role for Plxnd1 appeared conserved in humans, as PLXND1 mRNA was positively associated with hypertrophic morphology in VAT, but not SAT. In zebrafish plxnd1 mutants, the effect on VAT morphology and body fat distribution was dependent on induction of the extracellular matrix protein collagen type V alpha 1 (col5a1). Furthermore, after high-fat feeding, zebrafish plxnd1 mutant VAT was resistant to expansion, and excess lipid was disproportionately deposited in SAT, leading to an even greater exacerbation of altered body fat distribution. Plxnd1-deficient zebrafish were protected from high-fat-diet-induced insulin resistance, and human VAT PLXND1 mRNA was positively associated with type 2 diabetes, suggesting a conserved role for PLXND1 in insulin sensitivity. Together, our findings identify Plxnd1 as a novel regulator of VAT growth, body fat distribution, and insulin sensitivity in both zebrafish and humans.

PURPOSE: To determine whether transforming growth factor (TGF)-beta inhibition increases the response to radiation therapy in human and mouse non-small-cell lung carcinoma (NSCLC) cells in vitro and in vivo. METHODS AND MATERIALS: TGF-beta-mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-beta type 1 receptor kinase, or with the pan-isoform TGF-beta neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, gammaH2AX foci formation, or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. RESULTS: The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced gammaH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-beta signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-beta neutralizing antibody exhibited fewer gammaH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. CONCLUSIONS: Inhibition of TGF-beta before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-beta inhibition and RT to provide therapeutic benefit in NSCLC.

BACKGROUND AND PURPOSE: To improve oral delivery of AmB by achieving increased oral bioavailability and synergistically enhanced antileishmanial activity using copaiba oil (Cop) through nano-emulsified carrier system (CopNEC). EXPERIMENTAL APPROACH: The AmB encapsulated nano-emulsified carrier (CopNEC-AmB) comprised of Cop, d-alpha Tocopheryl Polyethylene Glycol 1000 Succinate and phosphatidylcholine was prepared by high pressure homogenization method. Stability study of CopNEC-AmB was carried out in simulated gastric fluid and simulated intestinal fluid. The CopNEC-AmB and plain AmB were compared for in vitro antileishmanial activity, their pharmacokinetics, organ distribution and toxicity profiling. RESULTS: The optimized CopNEC-AmB has globule size of 127+/-21 nm, PDI 0.11+/-0.02, and zeta potential (-)38.5+/-2.7 with encapsulation efficiency 91.9+/-1.4 %w/w. The high resolution transmission electron microscopy illustrates spherical particle geometry with homogeny in their sizes. The optimized CopNEC-AmB was found to be stable in gastro-intestinal fluids showing insignificant changes in globule size and encapsulation efficiency. The AUC0-48 value of CopNEC-AmB in rats was significantly improved showing 7.2 folds higher oral bioavailability than free drug. The in vitro antileishmanial activity of CopNEC-AmB was significantly higher (p<0.05) than the free drug because copaiba oil synergistically enhanced chemotherapy of leishmaniasis by causing drastic changes in morphology of Leishmania parasite and rupture of the plasma membrane with loss of their contents. The CopNEC-AmB showed significantly lesser hemolytic toxicity and cell cytotoxicity, and absence of any changes in histopathology of kidney tissues as compared to free drug. CONCLUSION: This prototype CopNEC formulation showed improved bioavailability and nontoxic synergistic antileishmanial chemotherapy of AmB due to copaiba oil against leishmaniasis.

Structural and functional studies of ribosomal subunits require the dissociation of intact ribosomes into individual small and large ribosomal subunits. The dissociation of the prokaryotic 70S ribosomes into the 50S and 30S subunits is achieved by dialysis against a buffer containing a lower Mg(2+) concentration. Eukaryotic 80S ribosomes are dissociated into 60S and 40S subunits by incubation in a buffer containing puromycin and higher KCl and Mg(2+) concentrations.

OBJECTIVE: The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. APPROACH AND RESULTS: Control mice and mice lacking the endothelial glucocorticoid receptor were bred onto an Apoe knockout background and subjected to high-fat diet feeding for 12 weeks. Assessment of body weight and total cholesterol and triglycerides before and after the diet revealed no differences between the 2 groups of mice. However, mice lacking the endothelial glucocorticoid receptor developed more severe atherosclerotic lesions in the aorta, brachiocephalic artery, and aortic sinus, as well as a heightened inflammatory milieu as evidenced by increased macrophage recruitment in the lesions. CONCLUSIONS: These data suggest that the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model.

Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-kappaB signaling and beta-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases.