Intracoronary Serum Smooth Muscle Myosin Heavy Chain Levels Following PTCA may Predict Restenosis

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1 Clinical Studies Intracoronary Serum Smooth Muscle Myosin Heavy Chain Levels Following PTCA may Predict Restenosis Yasuhiro TSUCHIO,MD,ShigetoNAITO, MD, Akihiko NOGAMI,MD, Hiroshi HOSHIZAKI, MD, ShigeruOSHIMA, MD, Koichi TANIGUCHI, MD, Hirohisa KATOH, PhD, Toru SUZUKI, 2 MD,MasahikoKURABAYASHI, 3 MD, Akira HASEGAWA, 3 MD, and Ryozo NAGAI, 2 MD SUMMARY Recently a novel biochemical method that uses an immunoassay to quantitate serum smooth muscle myosin heavy chain (SMMHC) levels was developed for diagnosis of aortic dissection. ) The purpose of this study was to determine whether SMMHC released from the coronary arterial wall can be used to predict restenosis after percutaneous transluminal coronary angioplasty (PTCA). Fifty-two consecutive patients undergoing successful PTCA for single vessel disease were examined (40 men, 2 women, 63 ± 8 years). Intracoronary blood samples were obtained distal to the lesion, and from the femoral artery after PTCA. In 0 patients, blood samples were taken immediately after the final balloon inflation, and 0 and 20 minutes after PTCA. SMMHC levels were measured by ELISA using SMMHC-specific monoclonal antibodies. Follow-up coronary angiography was performed 3 months after PTCA. Intracoronary serum SMMHC levels were significantly higher than those obtained from the femoral artery (0.6 ±.5 vs 2. ± 0. ng / ml, p 0.00). Of 40 patients without apparent dissection, the 23 patients who did not develop restenosis in the follow-up study were found to have had higher levels of intracoronary SMMHC levels immediately after PTCA compared to the 7 patients with restenosis (5.2 ± 2.9 vs 7. ±.2 ng / ml, p 0.05). We suggest that elevated intracoronary SMMHC levels after PTCA may reflect the extent of injury to the arterial wall. Intracoronary SMMHC may be a possible biochemical marker for the prediction of restenosis. (Jpn Heart J 2000; 4: 3-40) From the Cardiology Division, Gunma Prefectural Cardiovascular Center, Maebashi, Immunology Laboratory, Diagnostics Division, Yamasa Corporation, Chiba, 2 Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, 3 The Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan. Address for correspondence: Shigeto Naito, MD, Cardiology Division, Gunma Prefectural Cardiovascular Center, 3-2 Kameizumi, Maebashi, Gunma , Japan. Received for publication November, 999. Revised and accepted December 27,

2 32 TSUCHIO, ET AL Jpn Heart J March 2000 Key words: Smooth muscle myosin heavy chain, Percutaneous transluminal coronary angioplasty, Restenosis SMOOTH muscle myosin heavy chain (SMMHC) levels in normal human sera average 0.9 ± 0. ng / ml. Patients with aortic dissection have markedly elevated serum SMMHC levels acutely, which rapidly decrease to normal levels. Measurement of serum SMMHC is a promising technique for detecting other vascular damage. In this study, we used an immunoassay to measure serum SMMHC levels, and examined the relationship between these values and the occurrence of restenosis after percutaneous transluminal coronary angioplasty (PTCA). METHODS Patient registry and criteria: A prospective registry of patients admitted for ischemic heart disease (34 with angina pectoris, 8 with myocardial infarction) was constructed between October 995 and December 997. All patients underwent elective PTCA, and follow-up coronary angiography (CAG) was performed 3 months after PTCA. The objectives and methods of the study were explained to all patients, and informed consent was obtained from each patient. Clinical variables: The following clinical variables were evaluated: age, sex, target lesion, length of lesion, morphology of stenosis by CAG, presence or absence of coronary dissection as detected by CAG, and presence of prior PTCA. We used the criteria of Ambrose 2,3) to define the morphology of each stenosis as one of four types: concentric lesions; type I eccentric lesions; type II eccentric lesions; and multiple irregularities. Concentric lesions were symmetric and usually smooth. Type I eccentric lesions were asymmetric and smooth. Type II eccentric lesions were either smooth with a narrow neck or had irregular borders. Multiple irregularities included vessels with serial lesions or severe diffuse disease. We used the terms concentric lesions and type I eccentric lesions for non- complex lesions, type II eccentric lesions, and multiple irregularities for complex lesions. We defined restenosis as recurrence of a 50 % stenosis of the index lesion. Clinical techniques: Blood samples were obtained from an area of the artery which was distal to the lesion, immediately after final balloon inflation. In 0 patients, blood samples were taken immediately after the final balloon inflation, and 0 and 20 minutes after PTCA. Blood samples were taken from the femoral artery as controls. Serum samples were stored frozen at 20 until testing. Immunoassay of serum SMMHC: A double monoclonal sandwich enzyme immunoassay was used to measure serum SMMHC. The assay uses monoclonal

3 Vol 4 No 2 BIOCHEMICAL MARKER FOR PREDICTING RESTENOSIS 33 antibodies obtained from mice immunized with smooth muscle myosin protein purified from the human uterus. In brief, BALB/c mice were immunized at 2-week intervals. 4) After six immunizations and an additional booster injection, splenocytes obtained from the mouse with the highest titer, as assessed by ELISA, 5) were fused with mouse myeloma cells. 6) The supernatant of the resultant cultured hybridoma cells was screened for anti-smooth muscle myosin antibody production by ELISA, and specific antibody-producing hybridoma cells were cloned by limiting dilution. Monoclonal antibodies were produced in ascitic fluid of BALB/c mice primed with pristane and were purified by protein A affinity chromatography. Western blot analysis confirmed the reactivity of the monoclonal antibodies with SMMHC. The combination of paired antibodies with the highest assay sensitivity was used for the double monoclonal antibody sandwich assay. Measurements could be completed within 5 hours. The assay showed reliable detection of SMMHC in human serum. The lower sensitivity limit of the assay was 0.4 ng / ml. Cross-reactivity with cardiac, skeletal, or platelet myosin was negligible at < 0. %, and the cross-reactivity with aortic myosin protein was 00 %. The analytical recovery showed good agreement with theoretical values, and within and between-run variation showed good reproducibility. Analysis of 75 healthy individuals showed SMMHC levels in normal human sera to average 0.9 ± 0.ng/ml. Statistical analysis: Results are presented as mean ± SEM. Comparison between means of two independent samples was performed by the two-tailed unpaired t test. Statistical significance was defined as p < RESULTS Patient characteristics: Tables I and II summarize the demographic and clinical characteristics of the patients enrolled in the study. A total of 52 patients (40 men and 2 women; mean age, 63 ± 8 years) with ischemic heart disease were enrolled. Of these patients, 34 had angina pectoris and 8 had myocardial infarction (Table I). The coronary target lesion was the left anterior descending coronary artery (LAD) in 24 cases (9 of seg 6, and 5 of seg 7); the left circumflex coronary artery (LCx) in 0 ( of seg, of seg 2, and 8 of seg 3); and the right coronary artery (RCA) in 8 (7 of seg, 5 of seg 2, 5 of seg 3, and of seg 4) (Table II). Underlying diseases included hypertension in 0 patients, diabetes mellitus in 8 patients, and hypercholesteremia in 6 patients (Table I). There were no patients with acute myocardial infarctions, and all procedures were performed as elective PTCA. The indication for PTCA with regard to post myocardial infarction was the presence of ischemia or viability of the myocardial infarction site.

4 34 TSUCHIO, ET AL Jpn Heart J March 2000 Table I. Patient Profile Number of patients Age (years) Male/Female Diagnosis Myocardial infarction Angina pectoris Unstable angina pectoris Underlying disease Hypertesion Diabetes mellitus Hypercholesterolemia Apical hypertrophy WPW syndrome Old cerebral infarction Abdominal aortic aneurysm / Table II. Lesion Characteristics Location Left anterior descending artery segment 6 segment 7 Left circumflex artery segment segment 2 segment 3 Right coronary artery segment segment 2 segment 3 segment 4 Complex lesion (Ambrose type I and II) Non-complex lesion (Ambrose type III and IV) First PTCA Second PTCA Dissection (+) Dissection ( ) Restenosis (+) Restenosis ( ) SMMHC level analysis: Intracoronary serum SMMHC levels after PTCA were significantly higher than those obtained in serum from the femoral artery (0.6 ±.5 vs 2. ± 0. ng / ml, p 0.00) (Figure ). No significant difference was found in intracoronary SMMHC levels between patients with complex lesions and those with non-complex lesions (8. ±.8 vs 3.0 ± 2.3 ng / ml), although SMMHC levels from patients with non-complex lesions tended to be higher (Figure 2). No significant differences were found in intracoronary SMMHC

5 Vol 4 No 2 BIOCHEMICAL MARKER FOR PREDICTING RESTENOSIS 35 Figure. Comparison of smooth muscle myosin heavy chain serum levels obtained from the coronary artery and the femoral artery. Intracoronary serum SMMHC levels after PTCA were significantly higher than those obtained in serum from the femoral artery. Figure 2. Comparison of smooth muscle myosin heavy chain serum levels with complex and non complex lesions. Results are presented as mean ± SEM. No significant difference was found in intracoronary SMMHC levels between patients with complex lesions and those with non complex lesions.

6 36 TSUCHIO, ET AL Jpn Heart J March 2000 levels when patients were grouped based on the target vessel (LAD; 0.6 ±.5 vs LCX; 0.6 ± 5.7 vs RCA; 0.8 ± 2.4 ng / ml). No significant difference was found in intracoronary SMMHC levels when the patients were grouped according to whether this was their first or second PTCA (.3 ± 2.0 vs 8.6 ±.8 ng / ml), however SMMHC levels tended to be higher in those undergoing their first PTCA. No significant difference was found in intracoronary SMMHC levels between patients with and without minor dissection (7.0 ± 2.0 vs.8 ±.8 ng / ml) (Figure 3). We defined minor dissections as intracoronary radiolucencies or parallel tracts during contrast injection which disappeared after dye clearance. In cases of major dissection, we used coronary stenting to avoid acute occlusion. Restenosis after PTCA occurred in 23 of 52 patients (44 %). No significant difference was found in intracoronary SMMHC levels between patients with and without restenosis (7.7 ±.3 vs 3.0 ± 2.4 ng / ml) (Figure 4). Of 40 patients without apparent dissection, the 23 patients who did not demonstrate restenosis at the time of the follow-up study were found to have had higher levels of intracoronary SMMHC levels immediately after PTCA compared to the 7 patients with restenosis (5.2 ± 2.9 vs 7. ±.2 ng / ml, p < 0.05) (Figure 5). The time courses of intracoronary SMMHC levels in 0 patients are shown in Figure 6. Intracoronary SMMHC levels immediately after PTCA were significantly Figure 3. Comparison of smooth muscle myosin heavy chain serum levels with and without coronary dissection. No significant difference was found in intracoronary SMMHC levels between patients with and without coronary dissection.

7 Vol 4 No 2 BIOCHEMICAL MARKER FOR PREDICTING RESTENOSIS 37 Figure 4. Comparison of smooth muscle myosin heavy chain serum levels with and without restenosis. No significant difference was found in intracoronary SMMHC levels between patients with and without restenosis. Figure 5. Comparison of smooth muscle myosin heavy chain serum levels with and without restenosis of 40 patients without apparent dissections. The 23 patients who did not demonstrate restenosis at the time of the follow up study were found to have had higher levels of intracoronary SMMHC levels immediately after PTCA compared to the 7 patients with restenosis.

8 38 TSUCHIO, ET AL Jpn Heart J March 2000 Figure 6. The time course of intracoronary SMMHC levels in 0 patients. Intracoronary SMMHC levels immediately after PTCA were significantly higher than those 0 and 20 minutes after PTCA. higher than those 0 and 20 minutes after PTCA (6.7 ± 6.2 vs 3.6 ± 0.6, 3.7 ±.ng/ml, p < 0.05). DISCUSSION PTCA is an effective and important alternative to medical treatment or bypass surgery in the management of patients with obstructive coronary disease. However, PTCA has three limitations: restenosis, acute coronary closure, and anatomy prohibiting PTCA. The incidence of restenosis following elective PTCA varies from 30 to 40 % within 3 to 6 months after PTCA. 7,8) Mechanical injury signals activate the cellular mechanisms responsible for intimal thickening and incomplete dilatation (as well as thrombus formation, coronary artery spasm, and elastic recoil) contribute to the restenosis process. 9-) Recently, new devices such as coronary stenting and directional coronary atherectomy, have been developed which address some of the limitations of PTCA. 2,3) There are almost no biochemical markers for predicting restenosis, therefore, we investigated in-

9 Vol 4 No 2 BIOCHEMICAL MARKER FOR PREDICTING RESTENOSIS 39 tracoronary SMMHC levels following PTCA. If a balloon catheter injures the coronary intima, smooth muscle cells are easily disrupted, making SMMHC a good candidate as a marker for predicting restenosis. Intracoronary serum SMMHC levels were significantly higher than those in serum from the femoral artery. This suggests that intimal and medial smooth muscles were injured by PTCA. Although not statistically significant, SMMHC levels tended to be higher in patients undergoing their first PTCA than in those with their second PTCA. We hypothesized that intracoronary serum SMMHC levels at a second PTCA would be higher than those at a first PTCA. However, our data do not support this idea. Reasons for this may include outer membranes and new intima constriction in addition to smooth muscle proliferation, or that restenotic lesions could not be adequately dilatated. No significant difference was found in intracoronary SMMHC levels between patients with and without minor dissection. In patients with major dissection, we used coronary stenting. Although dissection has been associated with more severe intimal and medial injury, no large quantities of SMMHC were released with minor dissection, because coronary arteries are of small caliber, and vascular injury did not extend into the media. Although the difference was not statistically significant, intracoronary SMMHC levels in patients with complex lesions tended to be higher than in those with non-complex lesions. The reason for this finding may be that non-complex lesions were more easily dilated. Of 40 patients without apparent dissection, the 23 patients who did not have detectable restenosis at the follow-up study were found to have had higher levels of intracoronary SMMHC levels immediately after PTCA as compared to the 7 patients with restenosis. This suggests that the lesions of patients who did not develop restenosis were more adequately dilated, and that the conditions which may prevent restenosis include sufficient injury of the coronary arterial wall without apparent dissection. Because intracoronary SMMHC levels rapidly decreased after PTCA, if this is to be used as a marker for restenosis, we must examine intracoronary SMMHC levels immediately after PTCA. Recently, intravascular ultrasound imaging has provided new insight into plaque composition and geometric distribution inside the vessel. The absence of plaque fracture, the existence of a major dissection, and greater plaque burden were associated with increased incidence of restenosis. 4) On the other hand, Horie et al have reported that serum lipoprotein (a) levels were significantly higher, and fell significantly after PTCA in the restenosis group. 5) We hope to predict restenosis more accurately using both SMMHC levels and intravascular ultrasound imaging.

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