Isolation of murine lung endothelial cells

Several protocols for the isolation of endothelial cells (ECs) from murine lung have been described in the literature. We, however, encountered a number of problems while using these procedures that prevented us from consistently or reliably obtaining pure populations of ECs from the lungs of mice. By incorporating specific elements from previously published protocols, as well as adding some novel features, we developed a new strategy for isolating ECs from murine lung. In this approach, a suspension of lung cells is initially prepared from the lungs of 7- to 14-day-old mouse pups using procedures that prevent intravascular clotting and leukocyte activation, minimize mechanical trauma to the lung tissue, and limit exposure to the digesting enzymes. The resulting cell suspension is cultured for 2–3 days, trypsinized to produce a suspension of single cells, and then subjected to fluorescence-activated cell sorting using an anti-ICAM-2 antibody. The sorted cells are then plated and split 1:2 at each passage to maintain a high density of the cells. Using this approach, we have been able to isolate pure populations of ECs that were sustainable for extended periods in culture without the emergence of fibroblast overgrowth or the development of senescence. We believe the success of this approach will provide opportunities to take advantage of the large and growing number of knockout and transgenic mouse lines to investigate the endothelial-specific roles of targeted molecules in the pulmonary vasculature

Mapping and Initial Analysis of Human Subtelomeric Sequence Assemblies

Physical mapping data were combined with public draft and finished sequences to derive subtelomeric sequence assemblies for each of the 41 genetically distinct human telomere regions. Sequence gaps that remain on the reference telomeres are generally small,well-defined,and for the most part,restricted to regions directly adjacent to the terminal (TTAGGG)n tract. Of the 20.66 Mb of subtelomeric DNA analyzed, 3.01 Mb are subtelomeric repeat sequences (Srpt),and an additional 2.11 Mb are segmental duplications. The subtelomeric sequence assemblies are enriched >25-fold in short,internal (TTAGGG)n-like sequences relative to the rest of the genome; a total of 114 (TTAGGG)n-like islands were found,55 within Srpt regions,35 within one-copy regions,11 at one-copy/Srpt or Srpt/segmental duplication boundaries,and 13 at the telomeric ends of assemblies. Transcripts were annotated in each assembly,noting their mapping coordinates relative to their respective telomere and whether they originate in duplicated DNA or single-copy DNA. A total of 697 transcripts were found in 15.53 Mb of one-copy DNA,76 transcripts in 2.11 Mb of segmentally duplicated DNA,and 168 transcripts in 3.01 Mb of Srpt sequence. This overall transcript density is similar (within ∼10%) to that found genome-wide. Zinc finger-containing genes and olfactory receptor genes are duplicated within and between multiple telomere regions

Intracisternal basic fibroblast growth factor enhances functional recovery and up-regulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction

Focal cerebral infarction (stroke) due to unilateral occlusion of the middle cerebral artery in mature rats produces deficits in sensorimotor function of the contralateral limbs that recover partially over time. We found that biweekly intracisternal injection of basic fibroblast growth factor (bFGF; 0.5 μg/injection), a potent neurotrophic polypeptide, markedly enhanced recovery of sensorimotor function of the contralateral limbs during the first month after stroke without apparent adverse side effects. Immunostaining for growth-associated protein 43 (GAP-43), a molecular marker of axonal sprouting, showed a selective increase in GAP-43 immunoreactivity in the intact sensorimotor cortex contralateral to cerebral infarcts following bFGF treatment. These results show that bFGF treatment can enhance functional recovery after stroke, and that the mechanism may include stimulation of neuronal sprouting in the intact brain

Angiogenesis in Platelet Endothelial Cell Adhesion Molecule-1-Null Mice

Platelet endothelial cell adhesion molecule (PECAM)-1 has been previously implicated in endothelial cell migration; additionally, anti-PECAM-1 antibodies have been shown to inhibit in vivo angiogenesis. Studies were therefore performed with PECAM-1-null mice to further define the involvement of PECAM-1 in blood vessel formation. Vascularization of subcutaneous Matrigel implants as well as tumor angiogenesis were both inhibited in PECAM-1-null mice. Reciprocal bone marrow transplants that involved both wild-type and PECAM-1-deficient mice revealed that the impaired angiogenic response resulted from a loss of endothelial, but not leukocyte, PECAM-1. In vitro wound migration and single-cell motility by PECAM-1-null endothelial cells were also compromised. In addition, filopodia formation, a feature of motile cells, was inhibited in PECAM-1-null endothelial cells as well as in human endothelial cells treated with either anti-PECAM-1 antibody or PECAM-1 siRNA. Furthermore, the expression of PECAM-1 promoted filopodia formation and increased the protein expression levels of Cdc42, a Rho GTPase that is known to promote the formation of filopodia. In the developing retinal vasculature, numerous, long filamentous filopodia, emanating from endothelial cells at the tips of angiogenic sprouts, were observed in wild-type animals, but to a lesser extent in the PECAM-1-null mice. Together, these data further establish the involvement of endothelial PECAM-1 in angiogenesis and suggest that, in vivo, PECAM-1 may stimulate endothelial cell motility by promoting the formation of filopodia