Faculty Bibliography

The Akt signaling pathway controls several cellular functions in the cardiovascular system; however, its role in atherogenesis is unknown. Here, we show that the genetic ablation of Akt1 on an apolipoprotein E knockout background (ApoE(-/-)Akt1(-/-)) increases aortic lesion expansion and promotes coronary atherosclerosis. Mechanistically, lesion formation is due to the enhanced expression of proinflammatory genes and endothelial cell and macrophage apoptosis. Bone marrow transfer experiments showing that macrophages from ApoE(-/-)Akt1(-/-) donors were not sufficient to worsen atherogenesis when transferred to ApoE(-/-) recipients suggest that lesion expansion in the ApoE(-/-)Akt1(-/-) strain might be of vascular origin. In the vessel wall, the loss of Akt1 increases inflammatory mediators and reduces eNOS phosphorylation, suggesting that Akt1 exerts vascular protection against atherogenesis. The presence of coronary lesions in ApoE(-/-)Akt1(-/-) mice provides a new model for studying the mechanisms of acute coronary syndrome in humans

Hypercholesterolemia is associated with increased risk of premature cardiovascular disease in adults, while early atherosclerotic lesions (reversible fatty streaks and non reversible fibrous plaques) are also associated with cardiovascular risk factors including low density lipoprotein-cholesterol (LDL-C). Although LDL-C is a risk factor that should be addressed in high risk children such as those with familial hypercholesterolemia, it is unclear, at present, whether there is a certain plasma LDL-C level that would call for an intervention regardless of the etiology of elevated LDL-C. Therefore, at present, screening the entire population to identify subjects with hypercholesterolemia is not justified. The aims of this review are to familiarize the reader with inherited diseases that are associated with elevated LDL-C and discuss the management of children with elevated LDL-C

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit micro1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in approximately 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.

BACKGROUND: Distraction osteogenesis is a valuable clinical tool; however the molecular mechanisms governing successful distraction remain unknown. We have used a uniaxial in vitro strain device to simulate the uniaxial mechanical environment of the interfragmentary distraction gap. MATERIALS AND METHODS: Using the Flexcell system, normal human osteoblasts were subjected to different levels of cyclical uniaxial mechanical strain. Cellular morphology, proliferation, migration, and the expression of angiogenic (vascular endothelial growth factor [VEGF] and fibroblast growth factor-2 [FGF-2]) and osteogenic (osteonectin, osteopontin, and osteocalcin) proteins and extracellular matrix molecules (collagen IalphaII) were analyzed in response to uniaxial cyclic strain. RESULTS: Osteoblasts exposed to strain assumed a fusiform spindle-shaped morphology aligning parallel to the axis of uniaxial strain and osteoblasts exposed to strain or conditioned media had a 3-fold increase in proliferation. Osteoblast migration was maximal (5-fold) in response to 9% strain. Angiogenic cytokine, VEGF, and FGF-2, increased 32-fold and 2.6-fold (P < 0.05), respectively. Osteoblasts expressed greater amounts of osteonectin, osteopontin, and osteocalcin (2.1-fold, 1.8-fold, 1.5-fold respectively, P < 0.01) at lower levels of strain (3%). Bone morphogenic protein-2 production increased maximally at 9% strain (1.6-fold, P < 0.01). Collagen I expression increased 13-, 66-, and 153-fold in response to 3, 6, and 9% strain, respectively. CONCLUSIONS: Uniaxial cyclic strain using the Flexcell device under appropriate strain parameters provides a novel in vitro model that induces osteoblast cellular and molecular expression patterns that simulate patterns observed in the in vivo distraction gap

OBJECTIVE: The mechanism of neovascularization during the proliferative phase of infantile hemangioma is poorly understood. It is known that circulating bone marrow-derived endothelial progenitor cells (EPCs) form new blood vessels in ischemic tissues using mediators regulated by the transcription factor, HIF-1alpha. Mobilization of EPCs is enhanced by VEGF-A, matrix metalloproteinase (MMP)-9, and estrogen, whereas homing is secondary to localized expression of stromal cell-derived factor-1alpha (SDF-1alpha). We examined whether these mediators of EPC trafficking are upregulated during the proliferation of infantile hemangioma. METHODS AND RESULTS: Surgical specimens and blood samples were obtained from children with proliferating hemangioma and age-matched controls (n=10, each group). VEGF-A and MMP-9 levels were measured in blood, and tissue sections were analyzed for SDF-1alpha, MMP-9, VEGF-A, and HIF-1alpha. The role of estrogen as a modulator of hemangioma endothelial cell growth was also investigated. We found that all these mediators of EPC trafficking are elevated in blood and specimens from children with proliferating infantile hemangioma. In vitro, the combination of hypoxia and estrogen demonstrated a synergistic effect on hemangioma endothelial cell proliferation. CONCLUSIONS: These findings demonstrate that proliferating hemangiomas express known mediators of vasculogenesis and suggest that this process may play a role in the initiation or progression of this disease

OA1 (GPR143) is a pigment cell-specific intracellular glycoprotein consisting of 404 amino acid residues that is mutated in patients with ocular albinism type 1, the most common form of ocular albinism. While its cellular localization is suggested to be endolysosomal and melanosomal, the physiological function of OA1 is currently unclear. Recent reports predicted that OA1 functions as a G protein coupled receptor (GPCR) based on its weak amino acid sequence similarity to known GPCRs, and on demonstration of GPCR activity in OA1 mislocalized to the plasma membrane. Because mislocalization of proteins is often caused by or induces defects in their proper folding/assembly, the significance of these studies remains unclear. A characteristic feature of GPCRs is a seven transmembrane domain structure. We analyzed the membrane topology of OA1 properly localized to intracellular lysosomal organelles in COS-1 cells. To accomplish this analysis, we established experimental conditions that allowed selective permeabilization of the plasma membrane while leaving endolysosomal membranes intact. Domains were mapped by the insertion of a hemagglutinin (HA) tag into the predicted cytosolic/luminal regions of OA1 molecule and the accessibility of tag to HA antibody was determined by immunofluorescence. HA-tagged lysosome associated membrane protein 1 (LAMP1), a type I membrane protein, was employed as a reporter for selective permeabilization of the plasma membrane. Our results show experimentally that the C-terminus of OA1 is directed to the cytoplasm and that the protein spans the intracellular membrane 7 times. Thus, OA1, properly localized intracellularly, is a 7 transmembrane domain integral membrane protein consistent with its putative role as an intracellular GPCR

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism