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Ribonuclease protection assay (RPA) is a sensitive solution hybridization method for quantitation of specific RNAs (1–3). The method is based on the ability of single-strand specific ribonuclease to degrade single-stranded RNA while leaving intact fragments of labeled antisense ...

Phenotypic modulation of arterial smooth muscle cells (SMC) is essential for the evolution of atheromatosis and restenosis after angioplasty (1). During these pathological phenomena, SMC express numerous genes such as those responsible for cell migration and proliferation (2). T ...

Apoptosis is a programmed cell death process in which surplus or damaged cells are eliminated through a highly regulated procedure. The first description of apoptosis relied upon morphological differences among apoptotic, necrotic, and healthy cells (1). Indeed, there are usually a nu ...

A variety of delivery systems, both viral and nonviral, have been employed to genetically modify vascular endothelial cells and smooth muscle cells (SMC) in vitro and by direct in vivo gene transfer into the vessel wall. The most recent addition to gene delivery technology for the vasculature has ...

Adenoviruses are icosahedral viruses 70–90 nm in diameter with a double-stranded, linear DNA genome of approx 36 kb. They are widely utilized in gene transfer protocols owing to their relative ease of genetic manipulation, ability to grow to high titres (109–1012 plaque forming units /mL), and t ...

Cultured endothelial cells have provided a powerful tool for discovery of the molecular regulators of a range of vascular processes from angiogenesis to fibrinolysis (1). Yet, the utility of genetic manipulation of endothelial culture systems to dissect critical intracellular si ...

Since the first report of in vivo direct gene transfer to the vessel wall in 1990 (1) several vectors, such as adenovirus, liposomes, and adeno-associated virus have been employed to introduce foreign genes to the vascular tissue in vivo. Hemagglutinating virus of Japan (HVJ, Sendai virus), a memb ...

The techniques of gene transfer using transfection via electroporation, CaPO4, or cationic lipids rely on selectable markers because of the low efficiency of this approach. Selectable markers range from fluorescent molecules to a variety of cytotoxic compounds, with the most common ...

The monocyte/macrophage (M�) contributes to atherosclerotic lesion initiation and progression through a variety of interactions with cells of the artery wall that depend on the elucidation of a host of cytokines and growth factors by cells residing in the intima. The number and complexity ...

The human saphenous vein is the most commonly used conduit for coronary artery bypass grafting owing to its ready availability, ease of harvesting, and favorable surgical handling (1). However, it suffers from a progressive decline in patency, resulting in a graft failure rate of 50% after 10 yr (2, ...

The formation of new blood vessels from existing blood vessels has been referred to as angiogenesis to distinguish the process from de novo embryonic vessel formation or vasculogenesis (1). This chapter will describe an in vivo assay to measure angiogenesis. There are several important re ...

Methods for gene delivery in vivo vary dramatically in their relative efficiencies. The most efficient to date involve the use of recombinant viruses, most notably adenoviruses, adeno-associated viruses (AAVs), or retroviruses (see Chapters 32 and 34). The creation of these recombina ...

Emerging knowledge of molecular pathology of vascular diseases provides new targets for vascular gene therapy. Sufficient expression of a gene of interest in the vessel wall can be achieved using either extravascular or intravascular gene delivery approaches (1). Plasmid DNA, trans ...

A number of animal models are available to investigators wishing to study the use of gene transfer to prevent neointimal formation after vascular injury. The majority are models of primary vascular injury rather than the human situation, where recurrence of a stenosis occurs in an abnormal bl ...

Antisense oligonucleotides are short segments of synthetic DNA designed to contain sequences of bases complementary to the DNA or RNA of a particular target gene of interest. By binding to the target, the antisense oligonucleotides can prevent translation of the gene into protein via diff ...

Hemostasis is a host defense mechanism that protects the integrity of the vascular system after tissue injury. It works in conjunction with other inflammatory, immune, and repair mechanisms to produce a coordinated response. Hemostatic systems are generally quiescent, but followi ...

Blood samples for most coagulation tests are collected into 3.8% trisodium citrate in a ratio of 1 part anticoagulant to 9 parts blood. Whole-blood samples for DNA isolation can be stored at −50�C and the DNA prepared at a later stage. A more convenient method requiring less freezer space is to store buffy c ...

The polymerase chain reaction (PCR) has revolutionized many areas of medicine, including hemostasis. Although this volume is not devoted to PCR, many of the chapters employ the technique at some point to amplify specific DNA or RNA sequences. This chapter, therefore, provides a brief outline ...

A novel approach to generating high-quality single-stranded DNA involves solid-phase sequencing. A biotin group is incorporated at the 5′-end of one of the amplification primers and as a result becomes incorporated into PCR product during the amplification reaction. The DNA can then be im ...

Silver stains are widely used for the detection of both proteins and nucleic acids in acrylamide gels or on various membranes. They have several advantages over conventional staining methods, including the ability to detect very small amounts of material, while avoiding the hazards of oth ...