Animal model that mimics atherosclerotic plaque rupture.

Atherosclerotic plaque rupture is the main cause of coronary thrombosis and myocardial infarcts. Currently, there is no animal model of plaque disruption. We have developed a rabbit model in which an atherosclerotic plaque can be ruptured at will after an inflatable balloon becomes embedded into the plaque. Furthermore, the pressure needed to inflate the plaque-covered balloon may be an index of overall plaque mechanical strength. The thoracic aorta of hypercholesterolemic rabbits underwent mechanical removal of endothelial cells, and then a specially designed balloon catheter was introduced into the lumen of the thoracic aorta. As early as 1 month after catheter placement, atherosclerotic plaque formed around the indwelling balloon. The plaques were reminiscent of human atherosclerotic lesions, in terms of cellular composition, patterns of lipid accumulation, and growth characteristics. Intraplaque balloons were inflated both ex vivo and in vivo, leading to plaque fissuring. The ex vivo strategy is designed to measure the mechanical strength of the surrounding plaque, while the in vivo scenario permits an analysis of the plaque rupture consequences, eg, thrombosis. In addition, our model allows local delivery of various substances into the plaque. The model can be used to study the pathogenesis of plaque instability and to design plaque stabilization therapy.

Graft permeabilization facilitates gene therapy of transplant arteriosclerosis in a rabbit model.

BACKGROUND: Smooth muscle cell (SMC) replication plays a central role in the pathogenesis of transplant arteriosclerosis. One strategy to eliminate dividing cells is to express a herpesvirus thymidine kinase (tk) gene that phosphorylates the nucleoside analogue ganciclovir into a toxic form leading to cell killing. However, medial SMCs are resistant to gene transfer unless the artery undergoes deendothelialization. We hypothesized that manipulations that increase the "porosity" of the artery can make SMCs prone to gene transfer without denudation. METHODS AND RESULTS: In organ culture of rabbit aorta, longitudinal stretch and supraphysiological pressure applied for 3 hours during incubation with adenoviral vector facilitated gene transfer into medial SMCs without denudation. Of the SMCs, 10.2+/-3.8% expressed a reporter gene of human placental alkaline phosphatase (hpAP), whereas SMCs in control arteries did not express hpAP. To evaluate the feasibility of transgene expression in arterial grafts, we performed such permeabilization-assisted reporter gene transfer into aortas of donor Dutch Belted rabbits and transplanted them into carotid arteries of recipient New Zealand White rabbits. Unstretched transfected grafts were used as a control. SMCs expressed hpAP (7. 3+/-2.4% of cells in 2 days and 4.2+/-1.9% in 2 weeks) in stretched grafts only. In the next series of experiments, we transfected stretched grafts with ADV-tk and combined transplantation with systemic administration of ganciclovir. Stretched ADV-hpAP grafts were used as a control. In 2 weeks, the formation of intimal thickening in tk-expressing grafts was significantly reduced (P<0. 01) because of a decrease in proliferating SMCs. CONCLUSIONS: Manipulations within target tissues can enhance the efficiency of gene transfer into SMCs. Although mechanical permeabilization is clinically problematic, in principle, targeting SMC replication may provide a genetic approach to the treatment of transplant arteriosclerosis.

Gene transfer into normal and atherosclerotic human blood vessels.

Gene transfer to blood vessels is a promising new approach to the treatment of the vascular diseases, but the feasibility of gene transfer to adult human vessels has not been explored. We introduced an adenovirus vector encoding a marker gene human placental alkaline phosphatase into normal and atherosclerotic human vessels in organ culture. In the normal vessels, recombinant gene was expressed preferentially in the endothelial cells (approximately 100%), intimal smooth muscle cells (1.3+/-0.4%, 1.4+/-1.0%, and 3.8+/-0.8% in the internal mammary arteries, saphenous veins, and normal coronary arteries, respectively), and various adventitial cells. Advanced, complicated atherosclerotic plaques demonstrated a similar efficiency of recombinant gene expression (3.1+/-0.5% and 3.8+/-0.3% of nonendothelial intimal cells in the coronary artery and carotid artery plaques, respectively). Of these intimal cells, macrophages and smooth muscle cells expressed a transgene, identifying them as targets for gene transfer. Areas of plaque rupture and thrombus are sites of predilection for expression of recombinant genes. Collagenase and elastase treatment increased the percentage of transgenic alkaline phosphatase-positive cells 7 times (P<0.001), suggesting that the pattern of gene expression was affected by the amount of surrounding extracellular matrix. These studies demonstrate the feasibility of gene transfer to human blood vessels. However, these studies also highlight important barriers to adenoviral gene delivery to the actual normal and atherosclerotic human vessels of clinical interest.

Adenotin and adenotin-like proteins coexist with adenosine receptors in mammalian tissues.

The relationship of adenotin, a low-affinity adenosine-binding protein, to adenosine receptors was examined in two human tissues and two mammalian cultured cell lines. An adenosine A2 receptor exists in the membranes from platelets, PC-12 cells, and JAR cells as shown by a stimulation of adenylate cyclase related to 5'-N-ethylcarboxamidoadenosine (NECA) or a NECA-related increase in intracellular cAMP levels. In contrast, binding studies with tritiated NECA revealed typical adenotin-like low-affinity binding sites on the membranes from the sources studied with agonist potencies as follows: NECA greater than 2-chloroadenosine greater than R-PIA. No evidence was found of coupling to a guanine nucleotide regulatory protein. Solubilization of platelet and placental membranes and precipitation with polyethylene glycol separated adenotin or the adenotin-like protein from a second adenosine binding site in each tissue. The pharmacologic properties of the precipitated binding sites were compatible with an adenosine A2 receptor in platelets and an adenosine A1 receptor in placenta. Our observations indicate that adenotin-like proteins exist outside the placenta. In addition, adenotin and adenotin-like proteins coexist with the adenosine A1 or A2 receptor in a number of cells and tissues and do not couple to a guanine nucleotide regulatory protein and stimulate adenylate cyclase. Therefore, adenotin is pharmacologically distinct from adenosine receptors, and its function remains to be discovered.

Properties of adenotin reconstituted into phospholipid vesicles.

Adenotin is a low affinity adenosine binding protein that has amino terminal homology with mammalian and avian stress proteins. Human placental adenotin was solubilized and reconstituted into phospholipid vesicles with an overall yield of 30%. The properties of adenotin in vesicles were similar to the native membranes as follows: association has a Kobs of 0.61 +/- 0.03 minute-1; equilibrium is reached in approximately 15 minutes; and the first order dissociation constant is 5.0 +/- 0.3 minute-1. Displacement analysis reveals an agonist potency order and Ki values as follows: N-ethylcarboxamidoadenosine, 0.35 microM; 2-chloroadenosine, 1.5 microM; R-phenylisopropyladenosine, greater than 1000 microM. The addition of 100 microM 5'-guanylylimidodiphosphate did not decrease binding of 5'-N-ethylcarboxamidoadenosine (NECA) at 37 degrees C or 4 degrees C but did decrease the IC50 for PC12 and JAR cell membrane agonist binding from 9.9 to 3.3 microM and increase the binding to 150-211% of the control value at 37 degrees C. The latter studies at 37 degrees C showed high variability. Using binding sites reconstituted into vesicles and gel filtration chromatography and agonist related guanine nucleotide release, the authors investigated whether these changes were related to an interaction between adenotin and a guanine nucleotide regulatory protein. No evidence for such an interaction was found. These data suggest that adenotin retains its binding properties when reconstituted into phospholipid vesicles. The function of this low affinity adenosine binding site remains to be discovered. However, the reconstitution of adenotin into phospholipid vesicles provides a method to study its function.

Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein.

The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[3H]ethylcarboxamidoadenosine [( 3H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [3H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

Characteristics of an adenosine A1 binding site in human placental membranes.

Binding sites were solubilized from human placental membrane using 1.5% sodium cholate and were assayed using polyethylene glycol precipitation. These soluble binding sites had properties of an adenosine A1 binding site. 2-[3H]Chloroadenosine and N-[3H]-ethylcarboxamidoadenosine (NECA) binding were time dependent and reversible. Scatchard plots indicate two classes of binding sites with Kd values of 6 and 357 nM for 2-chloro[8-3H]adenosine and 0.1 and 26 nM with [3H]NECA. The specificity of [3H]NECA binding was assessed by the ability of adenosine analogs to complete for binding sites. Using this approach the estimated IC50 values were 60 nM for (R-PIA), 160 nM for S-PIA, 80 nM for NECA, and 20 nM for 2-chloroadenosine. Binding of [3H]NECA to the soluble sites is inhibited to 48% of the control value by 100 microM guanylyl-5'-imidodiphosphate (Gpp(NH)p). The IC50 value for NECA binding to the soluble binding site was increased from 80 nM to 1500 by Gpp(NH)p. There was a shift of binding affinity from a mixture of high and low affinity to only low affinity with 100 microM Gpp(NH)p. Despite these alterations a NECA prelabeled molecular species of 150 kDa did not decrease in molecular weight upon the addition of 100 microM Gpp(NH)p during high-performance liquid chromatography on a Superose 12 column. Other evidence to support the concept of preferential solubilization and assay of a small population of A1 binding sites was obtained. Following solubilization adenosine A2-like binding sites could be detected only in reconstituted vesicles. The existence of small amounts of A1 binding sites in intact human placental membranes was directly demonstrated using the A1 agonist ligand N6-[3H]cyclohexyladenosine and the A1 antagonist ligand 8-[3H]cyclopentyl-1,3-dipropylxanthine. JAR choriocarcinoma cells have "A2-like" membrane binding sites. In contrast to placental membranes, only A2-like binding sites could be solubilized from JAR choriocarcinoma cells. These observations indicate that human placental membranes contain adenosine A1 binding sites in addition to A2-like binding sites. These sites are guanine nucleotide sensitive, but do not shift to a lower molecular weight form upon assumption of a low affinity state.