Citations

AbstractThis unit provides a set of protocols for introducing recombinant genes into normal, injured, and atherosclerotic arteries. The protocols include animal preparation, surgical techniques, and delivery systems. Protocols describe gene delivery to normal, injured, and stented porcine iliofemoral arteries, employing a double balloon infusion catheter to deliver the vector. Another basic protocol describes gene delivery to atherosclerotic arteries using a hyperlipidemic double‐injury rabbit model, and requires surgical exposure of the artery and instillation of the gene vector via a catheter. Additional protocols describe gene delivery to normal and injured murine carotid and femoral arteries. An describes a percutaneous method for arterial gene delivery. These protocols may be adapted to deliver genes to either injured or noninjured atherosclerotic arteries.

The expression of the apolipoprotein A-I (apo A-I) gene was investigated in the myelinating sciatic nerve. Hybridization analysis with an apo A-I cDNA probe obtained from a cDNA library of mRNA isolated from rapidly myelinating chick sciatic nerve indicated that apo A-I coding transcripts increase during development in the chick sciatic nerve in parallel with the increase of myelin lamellae. Substantial apo A-I-like immunoreactivity in chick sciatic nerve homogenates was detected by Western blotting. The amount of antigen increased from the 15-d embryonic stage to 1 d posthatch and then decreased. Two subcellular fractions corresponding to the cytoplasmic compartments were particularly enriched in apo A-I. apo A-I immunoreactivity was also found in highly purified myelin preparations. Immunohistochemical staining provided further evidence for the presence of apo A-I in the endoneurial compartment of the sciatic nerve. Electron microscopic examination of these fractions after negative staining showed the presence of spherical and disc-shaped particles resembling high density lipoproteins. The presence of apo A-I, cholesterol esters, phospholipids, and triacylglycerols in ultracentrifugal fractions corresponding to serum lipoproteins and the behavior of apo A-I on nondenaturing gradient gels implied that apo A-I was associated with lipid. Studies with short-term organ cultures of sciatic nerves from 1-d chicks strengthened the evidence for local synthesis and secretion of apo A-I and apo A-I-containing lipoproteins by this tissue. These results establish that the apo A-I gene is actively expressed in developing sciatic nerve during the period of rapid myelination. These findings support the hypothesis that apo A-I synthesized within the nerve participates in the local transport of lipids used in myelin biosynthesis.

Rupture of an intracranial aneurysm (subarachnoid hemorrhage) is a potentially devastating condition frequently complicated by delayed cerebral ischemia from sustained contraction of intracranial arteries (cerebral vasospasm). There is mounting evidence linking the formation of intracranial aneurysms and the pathogenesis of post-subarachnoid hemorrhage vasospasm to aberrant bioavailability and action of the vasodilator molecule nitric oxide generated by isoforms of nitric oxide synthase. In humans, the gene encoding the endothelial isoform of nitric oxide synthase (eNOS) is known to be polymorphic, with certain polymorphisms associated with increased cardiovascular disease susceptibility. In this prospective clinical study involving 141 participants, we used gene microarray technology to demonstrate that the eNOS gene intron-4 27-base pair variable number tandem repeat polymorphism (eNOS 27 VNTR) predicts susceptibility to intracranial aneurysm rupture, while the eNOS gene promoter T-786C single nucleotide polymorphism (eNOS T-786C SNP) predicts susceptibility to post-subarachnoid hemorrhage vasospasm. We believe that genetic information such as this, which can be obtained expeditiously at the time of diagnosis, may be used as a helpful adjunct to other clinical information aimed at predicting and favorably modifying the clinical course of persons with intracranial aneurysms.

Although all three Vav family members are expressed in T lymphocytes, the role that Vav3 plays in T cell activation is poorly defined. Here we show that, like Vav1, Vav3 undergoes rapid tyrosine phosphorylation after T cell receptor (TCR) cross-linkage and interacts with the adaptor molecules SLP76 and 3BP2 in a SH2-dependent manner. However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity. In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element–mediated gene transcription. These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.

Fluorodeoxyglucose positron emission tomography (PET) studies have found that patients with Alzheimer's dementia (AD) have abnormally low rates of cerebral glucose metabolism in posterior cingulate, parietal, temporal, and prefrontal cortex. We previously found that cognitively normal, late-middle-aged carriers of the apolipoprotein E ε4 allele, a common susceptibility gene for late-onset Alzheimer's dementia, have abnormally low rates of glucose metabolism in the same brain regions as patients with probable AD. We now consider whether ε4 carriers have these regional brain abnormalities as relatively young adults. Apolipoprotein E genotypes were established in normal volunteers 20–39 years of age. Clinical ratings, neuropsychological tests, magnetic resonance imaging, and PET were performed in 12 ε4 heterozygotes, all with the ε3/ε4 genotype, and 15 noncarriers of the ε4 allele, 12 of whom were individually matched for sex, age, and educational level. An automated algorithm was used to generate an aggregate surface-projection map that compared regional PET measurements in the two groups. The young adult ε4 carriers and noncarriers did not differ significantly in their sex, age, educational level, clinical ratings, or neuropsychological test scores. Like previously studied patients with probable AD and late-middle-aged ε4 carriers, the young ε4 carriers had abnormally low rates of glucose metabolism bilaterally in the posterior cingulate, parietal, temporal, and prefrontal cortex. Carriers of a common Alzheimer's susceptibility gene have functional brain abnormalities in young adulthood, several decades before the possible onset of dementia.

Gene transfer involves the use of an engineered biologic vehicle known as a vector to introduce a gene encoding a protein of interest into a particular tissue. In diseases with known defects at a genetic level, gene transfer offers a potential means of restoring a normal molecular environment via vector-mediated entry (transduction) and expression of genes encoding potentially therapeutic proteins selectively in diseased tissues. The technology of gene transfer therefore underlies the concept of gene therapy and falls under the umbrella of the current genomics revolution. Particularly since 1995, numerous attempts have been made to introduce genes into intracranial blood vessels to demonstrate and characterize viable transduction. More recently, in attempting to translate cerebrovascular gene transfer technology closer to the clinical arena, successful transductions of normal human cerebral arteries ex vivo and diseased animal cerebral arteries in vivo have been reported using vasomodulatory vectors. Considering the emerging importance of gene-based strategies for the treatment of the spectrum of human disease, the goals of the present report are to overview the fundamentals of gene transfer and review experimental studies germane to the clinical translation of a technology that can facilitate genetic modification of cerebral blood vessels.

The current study was designed to determine the effect of recombinant heme oxygenase-1 (HO-1) gene expression on endothelial function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector (10 10 PFU/mL, total volume 300 μL) encoding either the HO-1 gene (AdCMVHO-1) or the β-galactosidase (β-Gal) reporter gene (AdCMVβ-Gal). Twenty-four hours after transduction, arterial rings were suspended in organ chamber for isometric force recording. Endothelium-dependent relaxations were obtained in response to bradykinin (10 −10 to 10 −6 mol/L) during contraction to uridine-5′-triphosphate (UTP; 3 × 10 −6 to 3 × 10 −5 mol/L). Certain rings were incubated with oxyhemoglobin (OxyHb; 10 −5 mol/L) overnight (16 to 18 hours of 24 hours). Expression and localization of recombinant protein were shown by Western blot analysis and immunohistochemistry. Endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to forskolin (10 −9 to 10 −5 mol/L) and DEA-NONOate (10 −10 to 10 −5 mol/L) were identical in β-Gal– and HO-1–transduced arteries. Exposure to OxyHb caused impairment of endothelium-dependent relaxation to bradykinin ( P < 0.01). In contrast, OxyHb did not affect endothelium-dependent relaxation in arteries expressing recombinant HO-1 ( P > 0.05). This protective effect of HO-1 was reversed by coincubation with tin protoporphyrin (SnPP9; 10 −5 mol/L), a selective inhibitor of HO-1 ( P < 0.01). Basal levels of 3′,5′-cyclic monophosphate (cGMP) in HO-1–transduced vessels were not significantly different from those in β-Gal–transduced vessels. Pretreatment with OxyHb significantly reduced cGMP level in β-Gal–transduced rings ( P < 0.01), whereas it had no effect in HO-1–transduced rings. These results demonstrate that HO-1 gene transfer does not affect endothelial and smooth muscle function of normal arteries, and that expression of recombinant HO-1 in cerebral arteries protects vasomotor function against OxyHb-induced injury.