Diagnostic Imaging Europe
September 2000

Intervention:
New strategies prevent platelet aggregation

By Herve Rousseau, M.D., Eric Allaire, M.D., Ph.D., and Philippe Otal, M.D.

Repeat procedures to combat restenosis continue to be the Achilles’ heel of angioplasty, and therapies that may prevent restenosis have been the subject of continuing investigation. Despite initially promising animal model and in vitro results, most clinical trials with pharmaceutical agents have been disappointing. The implantation of stents is currently the only technique proven to decrease restenosis after percutaneous transluminal angioplasty (PTA), but the risk of intrastent restenosis remains.

Subacute coronary stent thrombosis usually occurs within seven to 10 days after stent deployment. It is a rare (less than 3%) complication that may occur up to four weeks after the procedure and results in occlusion of the stented vessel with a platelet-rich thrombus. To lower the risk of this complication, the earlier use of aspirin, heparin, dipyridamole, and dextran was subsequently enhanced by the addition of warfarin. This precaution achieved a stent thrombosis rate of about 3.5% in clinical trials.^1,2

During the past three years, the use of warfarin has been abandoned, as a result of recognition that antiplatelet, rather than anticoagulant, treatment is the key to reducing the risk of subacute stent thrombosis. The synergistic combination of aspirin and ticlopidine hydrochloride is associated with a stent occlusion rate of only 0.8%, compared with 5.4% using anticoagulation treatment in coronary trials.³ Neutropenia (severe in 0.8% of patients), rash, and diarrhea are side effects associated with ticlopidine hydrochloride, and these require regular monitoring of the white cell count. It is likely that ticlopidine hydrochloride will be replaced by clopidogrel (Plavix), a less expensive but structurally similar drug reported to have fewer side effects. Oral ticlopidine hydrochloride or clopidogrel is usually discontinued one month after subacute thrombosis.

Abciximab (Rheopro) is a fragment of monoclonal antibody that binds to the platelet glycoprotein IIb/IIIa receptor involved in the final common pathway of platelet aggregation and inhibits this process. It is as expensive as a stent and its cost-effectiveness needs to be assessed carefully, as long-term follow-up data become available.

Restenosis after stenting is thought to result from the dual mechanism of recoil and constrictive geometric vessel remodeling. The processes involved are rather complex and may be disturbed at various levels. Thrombus formation or intimal hyperplasia can result. A proliferate response to injury results in an intimal hyperplasia, which in turn is caused by smooth muscle cell migration and matrix production. After stenting, the endothelium is inevitably damaged, and the vessel may be completely stripped from its covering layer, exposing subendothelial tissues to the bloodstream. Because these tissues are thrombogenic, their exposure triggers activation of the coagulation system, platelet aggregation and degranulation, and concurrent thrombus formation. In an attempt to repair the damage, a new intima develops and becomes covered by an endothelium. Finally, the stent is covered by this new lining and so has no contact with the blood (Figures 1 through 3).

In contrast to PTA alone, the scaffolding effect of stent deployment may diminish recoil and remodeling, although it is thought to increase neointimal hyperplasia. The impact of stents on restenosis, however, is considered to be purely mechanical. Stent implantation expands the vessel lumen farther than balloon angioplasty by itself, and this larger lumen creates more space for intimal proliferation. Stents do not diminish the cellular response to injury; they increase it. However, endoprostheses do decrease the restenosis rate by increasing the artery’s capacity to tolerate intimal proliferation.

Factors that affect thrombogenicity and intrastent restenosis are surface texture, surface electrostatic charge, free surface energy, and stent expansion and rigidity.

• Surface texture. The smoother the stent surface, the less thrombogenic it is.

• Surface electrostatic charge. Blood cells and proteins are kept away from the endothelium by repelling forces, owing to their similar electronegative charge. Most metals used for stents have a positive surface charge. When exposed to blood, they attract proteins and cells. Within minutes, a thin film appears at the stent surface, consisting mainly of proteins and especially of fibrinogen, which passivates the stent surface. Unlike most metals, tantalum has an electronegative surface charge, induced by thorough cleaning during the manufacturing process. But the material becomes electropositive within a few hours following exposure to electrolyte solutions such as blood.

• Free surface energy. This quality is related to “unsatisfied” intermolecular bonds at the material surface and largely determines how well a fluid is spread over the material (i.e., “wettability”). It can be measured as a critical surface tension (in dyn/cm), and for most stents the material is thrombogenic.

• Stent expansion. Ideally, the metal struts of a stent should be impressed in the vessel wall, although depressions in the vascular wall result. Tissue mounds protrude toward the vessel lumen between the struts, and depressions and stents become filled with thrombus. Multicenter reendothelialization then occurs from the spaces not covered by the stent. To accelerate the endothelialization process in a stented vessel, the surface area covered by metal should be kept to a minimum, while the free luminal surface should be maximized.

  Stents with the least metal show reduced restenosis and intimal hyperplasia during follow-up. When there is too much expansion, tissue mounds protrude into the bloodstream (Figure 4). However, more extensive thrombus deposition is observed if stent expansion is insufficient. About 1.2 times the vessel diameter is recommended to ensure satisfactory embedding of the struts into the vessel wall. If the stent diameter is oversized, damage to the vessel wall and spasms are observed more frequently. Stent lengths should cover the whole lesion, without deliberately extending to neighboring healthy areas.

• Rigidity. Debate continues on whether rigid or flexible stents cause more intimal hyperplasia and a higher frequency of restenosis. The transition between stented and unstented segments should be smooth and abrupt changes in diameter or direction avoided. Transition zones must be modeled to maintain a uniform vessel diameter without abrupt changes in flow direction.

The stiffness of metallic stents generally influences the mechanical properties of the vessel at the implantation site.⁴ Vascular compliance is reduced after stent implantation: An increase in transmural pressure causes less increase in vessel diameter than normal. In other words, the pulsatile diameter change in stented vessels becomes markedly reduced, and loss of pulsatile energy increases at the stented segment compared with a normal vessel. Impedance to local flow increases, resulting in greater pressure wave reflections and pulsatile mechanical stress at the interface between stented and nonstented vessel segments.

At a microscopic level, the increased wave reflections and mechanical stress lead to locally increased velocity gradients and areas of turbulence, with vibratory weakening of the arterial wall. This in turn causes greater local stress. The final consequences of the compliance mismatch are endothelial damage and pronounced neointima formation.⁵

Hemodynamic factors also contribute to thrombus formation following stent implantation. With laminar flow, where the velocity increases toward the center of a vessel, blood cells are preferably located in the middle. This corresponds to the parabolic flow profile over a vascular cross section. When flow velocity decreases, or when laminar flow becomes turbulent, axial accumulation of blood cells is less pronounced and finally disappears. If the arterial wall is damaged, contact with the thrombogenic subendothelial tissues intensifies. Slower flow and localized areas of turbulence mean that coagulation activation is increased and activated coagulation factors are not washed out from the site of activation as fast as usual. The quantity of more stable thrombi increases with decreasing shear stress and turbulence. Because the patency of stents is likely to depend on vessel outflow and the diameter of endoprostheses, stents of 5 mm or smaller tend to occlude. However, for larger arteries like the aortoiliac, the risk of thrombosis is lower.

Despite extensive clinical testing, no mechanical or pharmaceutical therapy to date has been shown to inhibit the proliferative component of restenosis. The large coronary trials have found that antiplatelet treatment reduces the neointimal proliferation, and consequently, platelet aggregation inhibitors should be started the day before stent implantation. Heparin should be administered during the procedure and also for the first 24 to 36 hours after stent placement to minimize the amount of thrombus formation at the stented site and the risk of thrombotic stent occlusion.

Because platelets stimulate proliferation through platelet-derived growth factors, minimizing platelet accumulation may also have a direct effect on the amount of intimal hyperplasia. Anticoagulation therapy should be given as long as a risk of thrombotic stent occlusion remains (i.e., as long as the stent is not completely insulated from the flowing blood by endothelialization, which happens in four to six weeks). Aspirin should be given for at least six months and ticlopidin hydrochloride for two months.

Attempts to reduce stent restenosis include evaluation of intracoronary radiotherapy using catheter-based radiation, radioactive stents, the delivery of a growth factor with a balloon catheter to speed endothelialization of a stent, gene therapy, and local drug delivery. Experimentally, and in clinical studies, a decrease in intimal hyperplasia by low-level radioactivity or antibodies against growth factors has also been tested with some effect.

Brachytherapy

Sources used for endovascular brachytherapy emit gamma or beta radiation. Gamma radiation was tested during the earliest experiments, which included human clinical trials. It has several drawbacks: Tissue penetration is high, protection of cardiovascular interventionists is difficult, and the time necessary to deliver the desired dose is long. However, no major adverse effects have been observed in treated patients. By contrast, beta-radiation does not penetrate tissues easily, and doses delivered beyond 1 cm of the source are low. The dose can be given rapidly, in a time frame compatible with the presence of a catheter in a lumen. Overall, beta radiation appears to be easier to use and may give results comparable to gamma radiation in terms of restenosis prevention.

The effects of radiation on the arterial wall depend on the dose and the condition of the artery, whether it is diseased or has been previously injured. The very high doses used at the beginning of cancer treatments have occasionally induced aneurysmal changes.⁶

The most common lesions are stenoses or occlusions that occur several years after irradiation. Arterial injuries are observed following doses of 20 to 80 Gy. Because the strength of radiation used for endovascular brachytherapy ranges from 5 to 25 Gy, the possibility of delayed ad- verse local effects after endovascular brachytherapy cannot be ruled out.

In vitro, radiation kills dividing cells in a dose-dependent manner. Smooth muscle cells and endothelial cells are equally sensitive to irradiation.⁷ In vivo, vessels display hypocellular and sclerotic media 32 days after irradiation. Sometimes an intrawall hemorrhage is observed, especially for high doses (25 Gy). A mild—but significant—intimal hyperplasia is observed in previously noninjured vessels. When applied after mechanical injury, intracoronary irradiation decreases intimal hyperplasia.⁸ These data provide the rationale for endovascular brachytherapy.

Mechanisms of late gain in luminal volume can be investigated using intravascular ultrasound with three-dimensional reconstruction. One human study suggests that an increase in luminal volume is limited by the external elastic lamina, despite a simultaneous increase in plaque volume.⁹ It is not known whether the effect of brachytherapy is due solely to its effect on the intima and media or if the constrictive process could also be modulated by radiation. This determines the optimal depth at which radiation should be delivered.

Doses are calculated based on emission at 2 mm from the source (e.g., in a 4-mm cylinder) and are ultimately chosen according to the size of the vessel. Doses prescribed in coronary arteries vary from 7 to 18 Gy. Catheters have been designed to maintain the source in the center of the artery, delivering radiation uniformly around the circumference. These centered catheters have not been used in many published clinical trials.

The problem of dose calculation and delivery with noncentered catheters was addressed using large catheters (5-French catheter in coronaries) and by intravascular ultrasound measurements of the distance between the source and the artery wall. In the Beta Energy Restenosis Trial (BERT), the vessel was exposed to the source for between two minutes 20 seconds and three minutes 44 seconds. Three-channel delivery catheters, with a handle in which the source is stored under a quartz shield, are typical. A hydraulic system transfers the source from the handle to the tip of the coronary catheter.

Most clinical trials have been designed in coronaries. The technique appears to be safe and feasible, especially with beta-emitting sources. In the BERT study, the restenosis rate was 15% on coronary asrteriography at six months. In Teirstein et al’s randomized study, restenosis rates at three years were 15.4% after irradiation, and 48.3% in the placebo group, for patients treated for restenosis.¹⁰ However, 6% of late sudden thrombotic closures have been observed up to 15 months after brachytherapy.¹¹

Stents may be another way to deliver radioactivity homogeneously to the vessel wall. Recent data suggest that human coronary radioactive stenting from Albiero et al suggest that although stenosis is prevented within the stent, voluminous hyperplasia de-velops at both edges of the stent.¹² The mechanism of this “candy wrapper” effect is unknown. One explanation could be that radioactivity decreases in areas distant from the stent, leading to increased hyperplasia.

Access is a problem in the treatment of peripheral arteries or arteriovenous dialysis because vessel diameters range from 3 to 14 mm, and currently available beta-emitting sources deliver high doses within 2 to 3 mm. The vessels treated are longer than coronaries. One study of irradiation after dilatation plus stenting of restenotic lesions of femoral arteries suggests good results with follow-ups ranging from several months to six years.¹³ Future directions might include the use of a balloon stent filled with beta-emitting sources, where leakage presents real danger, or radioactive stents.

Brachytherapy is a promising technique. Further experimental and clinical studies are needed, as well as long-term follow-up to rule out the possibility of adverse effects on arterial-wall remodeling.

Gene Therapy

Because naked DNA has a short half-life and does not penetrate cells easily, vectors have been constructed. These vectors are viruses with genomic modifications chosen to attenuate or suppress their pathogenicity. They retain the ability to infect cells, and the cells in turn express proteins encoded by the viral genetic material.¹⁴ Current vectors are derived from adenoviruses or retroviruses, and safety remains a crucial concern because their toxicity is not zero. Vectors can also induce pathologic changes in arteries, such as inflammation and intimal hyperplasia.¹⁵

Genetic material can also be included in liposomes. Alternatively, an electric field, ultrasound, or radiation can be applied to the tissue to increase DNA penetration into the cells. Human gene therapy trials generally rely on injections of high doses of naked DNA.¹⁶ This strategy avoids most of the potential hazards to patients and maintains environmental safety. Because the duration of expression does not exceed two weeks, the short presence of the foreign gene in vivo limits the risk of uncontrolled pathologies from gene recombination, or long-term expression.

Genes controlling restenosis are numerous and cannot be easily identified. Although prevention of constrictive remodeling shows promise, the genes tested at the moment are chosen for their involvement in intimal hyperplasia formation. Many of these genes control smooth muscle cell death, division, or migration.¹⁴ Other strategies, such as the use of antisense technology to interfere with messenger RNA (mRNA) specific to oncogenes like c-myc or c-myb, are designed to block cell division.

Various factors trigger cell division after angioplasty, often functioning in parallel. Alternative procedures aim to control the cell cycle upstream. A 70% decrease in smooth muscle cell proliferation can be achieved by blocking one single factor, E2F, which controls several aspects of cell cycle progression.¹⁷

One difficulty specific to the prevention of restenosis is that large particles such as viruses have difficulty crossing the thick and dense extracellular matrix of the artery wall.¹⁸ The transduction of cells beyond the endothelium necessitates special conditions. Multichannel balloons have been designed to facilitate penetration of the genetic material by convection or diffusion. Multiple curved needles that penetrate the artery wall to inject the genetic material have also been developed. Assessing the efficiency of gene transfer to vessels during the procedure is also difficult. MRI can detect a marker and complex particle composed of the gene of interest.

Several clinical trials in angiogenesis have started, but gene therapy for restenosis remains limited to experimental studies. A better understanding of the biological mechanisms of restenosis is necessary. One key question is whether a control of smooth muscle cell proliferation limited in time is sufficient to keep the artery patent in the long term.

In trials for angiogenesis in peripheral artery diseases, DNA is injected into muscles percutaneously several times at weekly intervals. These iterative injections are difficult and costly. For restenosis, long-term expression and the use of vectors might be necessary, and these requirements may explain the delay of clinical trials in restenosis when compared with angiogenesis. Restenosis is not usually a life-threatening condition, and aggressive development of a new technique is harder to justify.

References

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