Observations were made on thoracic aortas and carotid arteries from male rabbits that were normal or experimental (those infused with epinephrine, norepinephrine, angiotensis II, endotoxin and cholesterol-fed animals).
Procedures were in accordance with the general methods for electron microscopy.
I. Fine structures of no-treatment animals (normal state).
II. Characteristics of normal fine structures and physiologically changed fine structures.
A. Connection between endothelial and smooth muscle cells
1. Attachment plates connected by foot processes grown from endothelial cells and smooth muscle cells.
2. Three types of connections were observed through the fenestrations of internal elastic laminae.
a. Type 1: Processes of medial smooth muscle cells in the endothelial lines.
b. Type 2: Long processes of endothelial cells in smooth muscle cells.
c. Type 3: Connections made apart from both cells.
3. The nature of connection was mainly simple apposition, but the so-called gap junctions were observed in some cases.
B. Initial contraction-relaxation-response appeared immediately after an infusion of angiotensin II, catecholamine, endotoxin or one-shot treatment of cholesterol. Local edema surrounding the connection site was then observed.
The myo-endothelial connections may have an important role to play as sensors of stimuli. In structural terms, the same myofilaments exist in the endothelial cells near the connecting site. These findings suggest that they (=myofilaments) are probably involved in the function of contraction and/or relaxation of endothelial cells and medial smooth muscle cells. Through this structure, endothelial cells and smooth muscle cells form a syncytium in the inner part of aortic wall.
C. Endothelial cell-leucotyte interaction.
1. Adhesion of leucocytes, especially monocytes, to the endothelial cells was observed in cases of angiotensin II infusion, endotoxin infusion and one-shot cholesterol treatment. Leucotytes successively enter the subendothelial space by diapedesis.
2. Leucotytes (monocytes) were often observed in the endothelial cell lines. Structurally. these cells were differentiated with nuclei and a number of marginal folds. Functionally, these cells demonstrated strong phagocytotic activity against carbon particles, but not in the neighboring endothelial cells.
These findings suggest that the interaction was probably induced by some humoral mediator or cytokines.
D. Endothelial cell-platelet interaction.
Completely normal endothelial cells block platelet adhesion on their cell surface. Following damage to the endothelial cell or formation of the giant endothelial cell, platelets adhere to endothelial cell surface. This finding, which seems to signify induction of mitogens into the gaint cell, seems to play an important role in the course of the interaction between endothelial cells and platelets.
III. Prepathological or pathological changes.
A. Diffuse edematous arterial reaction in subendothelial space. Repeats of contraction-relaxation response and increased permeability brought about this change from the local edema. The first step of change showed a homogeneous, low-density extracellular space, and the next step gave rise to high-density extracellular spaces containing small lipid droplets.
B. At the same time, subendothelial smooth muscle cells migrated from the inner layer of media and attached themselves to the endothelial cells by small foot processes.
IV. Observation of the process of protecting damaged endothelial cells. (Replacement)
Replacement for damaged endothelial cells was observed in an early stage of pathological change.
A. The first type of protection was observed in the prolongation of the marginal folds of neighboring endothelial cells or neighboring giant endothelial cells.
B. The second type of protection occurred in cases showing loss of continu

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We investigated reparative response at the site of angioplasty injury both in human coronary arteries and in subclavian arteries of the Watanabe heritable hyperlipidemic (WHHL) rabbit, using conventional and immunocytochemical techniques.
I. At the target site of PTCA in patients with unstable angina and acute myocardial infarction, fissure of atheroma is the most common angioplasty injury. In patients with stable angina, however, the PTCA injury is much more variable.
II. At the site of medial injury induced by PTCA, de-differentiation of medial smooth muscle cells occurs at an early stage. During the evolution of the proliferative response, re-differentiation of smooth muscle cells gradually occurs, revealed by changes in the expression of cytoskeletal proteins.
III. Angioplasty injury to a lipid-rich plaque in human coronary arteries causes a different type of tissue reaction, composed of abundant macrophages, smooth muscle cells and occasional neovascularization.
IV. Injury and response after angioplasty in the WHHL rabbit are almost identical to that seen in humans after PTCA.
These findings suggest that the WHHL rabbit may serve as a good model for further studies on mechanisms of the response to injury after PTCA in humans.

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To develop an effective strategy to prevent neointimal formation after angioplasty injury, we have identified cell cycle regulatory proteins as targets for inhibition using antisense oligonucleotides (ODN). In addition, we have developed a novel intraluminal molecular delivery method that employs the protein coat of the Hemagglutinating virus of Japan (HVJ) complexed with liposomes, to enhance the efficiency of cellular uptake and the stability of antisense oligonucleotides while minimizing non-specific toxicity. Phosphorothioate ODN are complexed with liposomes and the protein coat of the inactivated HVJ. This method results in a more rapid cellular uptake and a ten-fold higher transfection efficiency of plasmids or ODN than lipofection or passive uptake methods. HVJ-liposome complex containing ODN was incubated within the rat carotid artery for 10 minutes immediately after balloon injury. First, we examined the effect of antisense cdc (cell division cycle) 2 kinase, proliferating cell nuclear antigen (PCNA), cdk (cyclin dependent kinase) 2 kinase and cyclin B₁ ODN on neointimal formation after balloon injury in the rat carotid artery model. Reverse transcription PCR in antisense ODN (PCNA and cdc 2 kinase) transfected vessels showed a marked decrease in cdc 2 and PCNA gene expression as compared to that in sense control ODN transfected vessels. The measurement of DNA synthesis as assessed by bromodeoxyuridine index and DNA content revealed a decrease in antisense PCNA and cdc 2 kinase ODN treated vessels on day 4 after injury as compared to sense ODN treated vessels. Transfection with either antisense cdc 2, cdk 2 kinase and cyclin B₁ ODN alone resulted in a partial inhibition of neointimal formation. We then evaluated the strategy of combined antisense ODN to achieve complete inhibition. The combination of antisense ODN directed against both cdc 2 kinase and PCNA inhibited completely neointimal formation at 2 weeks after angioplasty. Moreover, the inhibitory effect of this antisense ODN combination on neointimal formation persisted up to 8 weeks after a single transfection. The combination of antisense cdc 2 kinase and cyclin B₁ ODN, or antisense cdc 2 and cdk 2 kinase ODN, also resulted in near complete inhibition of neointimal formation. The presistent inhibition of neointimal formation after balloon injury was also observed by the combination of antisense cdc 2 kinase and cyclin B₁ ODN. The present study documents that a single intraluminal molecular in vivo delivery of antisense cell cycle regulatory genes ODN results in a inhibition of neointimal formation in the rat carotid balloon injury model as a model of gene therapy. Interestingly, our results reveled that multiple blockade of cell cycle regulatory genes by antisense ODN are necessary to achieve the complete inhibition of neointimal formation after angioplasty.

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Hyperplasia of smooth muscle cells (SMC) in the artery wall is an essential component of atherosclerosis, restenosis following angioplasty and allograft rejection after heart transplantation. The inhibition of SMC migration, along with proliferation, has long been investigated as a potential strategy for the treatment of cardiovascular disease. In vivo transfer of genetic material into the arterial wall offers the devolopment of a medical intervention to alter the genetic program of SMC for therapeutic purposes. Given that local vascular diseases such as atherosclerosis and restenosis are caused by abnormalities of gene expression, it is logical to attempt to treat these diseases through the gene replacement, the antisense inactivation of gene expressiton or the augmentation of expression of genes that are capable of playing a therapeutic role. To date, however, no therapeutic genes for preventing intimal accumulation of SMC in humans have yet been reported. We summarize recent gene therapy strategies for restenosis with special reference to the biological effects of transfer of the human calponin gene, encoding an actin-binding protein which has sequence similarity with the Vav Ras/Rho nucleotide exchange factor and a yeast gene CDC24, into the arteries of live animals. Our results suggest that overexpression of calponin, through its ability to suppress SMC growth and migration may inhibit arterial myointimal hyperplasia in vivo. Direct gene transfer of calponin into the arterial wall may thus be of potential therapeutic value in cardiovascular disease.

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We previously reported a clinical study in which (1) Probucol prevented restenosis after coronary angioplasty and (2) Pravastatin reduced the risk of restenosis associated with plasma cholesterol concentration. To examine these mechanism, we analyzed the effect of probucol and pravastatin on the intimal proliferation, the cellular makeup of lesion and the expression of PDGF after balloon injury in rabbits. Probucol study: Probucol was given orally 1.3 g/day from 2 weeks prior to carotid balloon injury to the time of killing (2 or 4 weeks after balloon injury). Probucol remarkably decreased intimal area by 70%, the number of Smooth Muscle Cell (SMC) and Proliferating Cell Nuclear Antigen (PCNA)-labeled cells in the intima. The expression of PDGF-A mRNA was markedly suppressed with probucol treatment. However, probucol did not suppress SMemb expression. SMemb is a good molecular marker for de-differentiated SMC. Probucol is effective in preventing SMC proliferation, which is possibly due to a decrease in the expression of PDGF.
Pravastatin study: Pravastatin was received orally 20mg/day with 0.5% cholestrol diet 2 weeks before iliac balloon injury. After 2 or 4 weeks, the rabbits were killed. Pravastatin reduced plasma cholesterol concentration, neointimal macrophage content and intimal area. The neointimal area was related to macrophage content. Moreover, macrophage content was correlated to plasma cholesterol level. Pravastatin effectively inhibits myointimal proliferation, which may be partly explained by its hypocholesterolemic activity.

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