During angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in an in vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fat in situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent an in vitro population of cells that accurately duplicate the in vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides an in vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in an in vitro system amenable to precise experimental manipulation.

We have used gene amplification in Drosophila follicle cells as a model of metazoan DNA replication to address whether changes in histone modifications are associated with replication origin activation. We observe that replication initiation is associated with distinct histone modifications. Acetylated lysines K5, K8, and K12 on histone H4 and K14 on histone H3 are specifically enriched during replication initiation at the amplification origins. Strikingly, H4 acetylation persists at an amplification origin well after replication forks have progressed significantly outward from the origin, indicating that H4 acetylation is associated with origin regulation and not histone deposition at the replication forks. Origin recognition complex subunit 2 (orc2) mutants with severe amplification defects do not abolish H4 acetylation, whereas the dup/cdt1 mutant delays the appearance of acetylation foci, and mutants in rbf result in temporal persistence. These data indicate that core histone acetylation is associated with origin activity. Furthermore, follicle cells undergoing gene amplification exhibit high levels of histone H1 phosphorylation. The patterns of H1 phosphorylation provide insights into cell cycle states during amplification, as H1 kinase activity in follicle cells is responsive to high Cyclin E activity, and it can be abolished by overexpressing the retinoblastoma homolog, Rbf, that represses Cyclin E. These data suggest that amplification origins are able to initiate when the cells are in a late S-phase, when the genome is normally not licensed for replication.

We molecularly characterized human immunodeficiency virus type 1 (HIV‐1) present in pure populations of astrocytes, macrophages, and multinucleated giant cells isolated using laser capture microdissection from brain tissue of two patients who died with HIV‐associated dementia. The V3 region of the HIV‐1 envelope (env) gene was amplified from the pure‐cell populations, and multiple clones were sequenced. In both patients, the V3 env sequences were distinct in astrocytes compared with neighboring macrophages or multinucleated giant cells and were characteristic of CCR5‐using (R5) HIV‐1. These results demonstrate cell‐specific compartmentalization of distinct R5‐like viral strains in the central nervous system microenvironment. Ann Neurol 2004

No abstract

Aqueous drainage devices for the treatment of glaucoma are subject to the same limitations as most polymeric implants, namely a healing response comprised of chronic inflammation and fibrosis. The most widely used devices are currently made of silicone or polypropylene, materials that exhibit biocompatibility difficulties when they are implanted on the sclera underneath the conjunctiva of the eye. Decreased outflow of aqueous fluid to the conjunctival space caused by the development of a fibrous capsule around the device accounts for at least 20% of aqueous shunts failures. Clearly, the need exists to improve the healing response to aqueous drainage devices, and one approach is to develop new polymers or polymer modifications. Improved devices would elicit a limited fibrotic response while increasing neovascularization around the implant. Previous studies have indicated that denucleation markedly improves the healing characteristics and biocompatibility of expanded polytetrafluoroethylene (ePTFE). We reasoned that altering the design of drainage devices to allow the use of denucleated ePTFE in vivo might minimize fibrosis, thereby improving shunt function. We found that after 8 weeks in vivo, experimental shunt function was equivalent to the Baerveldt shunt, while there was less scarring with increased neovascularizatin. These findings suggest that ePTFE has potential as an improved, long-term alternative material for use in constructing glaucoma shunts.