Shinohara Masakazu, Kawashima Seinosuke, Yamashita Tomoya, Takaya Tomofumi, Toh Ryuji, Ishida Tatsuro, Ueyama Tomomi, Inoue Nobutaka, Hirata Ken-ichi, Yokoyama Mitsuhiro
Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Chuo-ku, Japan.
Cardiovasc Res. 2005 Nov 1;68(2):249-58. doi: 10.1016/j.cardiores.2005.06.016. Epub 2005 Jul 21.
Intimal hyperplasia plays an important role in a variety of types of vascular remodeling, particularly luminal narrowing after vascular injury. The vascular smooth muscle cells (VSMCs) in the neointimal area are a synthetic phenotype and have different epitopes from VSMCs in the normal media. The synthetic VSMCs in the neointima contain various possible antigens that can be targeted by the immune system. In this study, we tried to develop a new immunotherapy, which targets the synthetic VSMCs, for prevention of neointimal formation after angioplasty.
Rabbits were repeatedly immunized with fixed xenogenic rat cultured VSMCs suspended in adjuvant as immunogens or injected with adjuvant and phosphate-buffered saline (PBS) or rat hepatocytes as controls every 2 weeks for 3 times. One week after the last immunization/injection, balloon injury of the left common carotid artery was performed. Four weeks after the injury, rabbits were euthanized and the neointimal lesion formation was assessed. The mean neointimal area of the PBS-injected, non-immunized group and the rat hepatocyte-immunized, control group was not statistically different (0.339 +/- 0.036 and 0.350 +/- 0.041 mm(2), P = NS). However, immunization with rat VSMCs significantly reduced the intimal lesion area (0.219 +/- 0.0286 mm(2); P < 0.05 vs. PBS-injected, non-immunized group and rat hepatocyte-immunized group.) PCNA-immunopositive proliferating VSMCs in the neointima were suppressed by the rat VSMC immunization (1.34 +/- 0.49% vs. 5.78 +/- 0.47%; P < 0.05 vs. PBS-injected, non-immunized group). Rat VSMC immunization induced antibodies which had strong cross-reactivity against rabbit synthetic VSMCs. In experiments in vitro, proliferation and migration of rabbit VSMCs that were stimulated by serum, angiotensin (AT) II, platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF), and the phorbol ester PMA were significantly suppressed by treatment with immunoglobulin extracted from the VSMC-immunized rabbit plasma, implying that the immunoglobulin had some global effects on VSMCs. The rat VSMC-immunized rabbit immunoglobulin bound the rabbit AT1a receptor protein, which was expressed in COS7 cells by transfection of rabbit AT1a receptor pcDNA3. This binding to AT1a receptor may be one of mechanisms of the effects of VSMC-immunized immunoglobulin.
Xenogenic, synthetic rat VSMC immunization in rabbits induced auto-antibodies against synthetic rabbit VSMCs in a cross-reaction. The induced auto-antibodies against synthetic VSMCs may provide a possibility of new immunotherapy for vascular remodeling that forms neointimal lesions.
内膜增生在多种类型的血管重塑中起重要作用，尤其是血管损伤后的管腔狭窄。新生内膜区域的血管平滑肌细胞（VSMC）呈合成表型，与正常中膜的VSMC具有不同的表位。新生内膜中的合成型VSMC含有各种可能被免疫系统靶向的抗原。在本研究中，我们试图开发一种针对合成型VSMC的新免疫疗法，以预防血管成形术后新生内膜的形成。
将悬浮于佐剂中的固定异种大鼠培养VSMC作为免疫原反复免疫家兔，或每2周注射佐剂和磷酸盐缓冲盐水（PBS）或大鼠肝细胞作为对照，共进行3次。最后一次免疫/注射后1周，对左颈总动脉进行球囊损伤。损伤后4周，对家兔实施安乐死并评估新生内膜病变形成情况。注射PBS的未免疫组和注射大鼠肝细胞的对照组的平均新生内膜面积无统计学差异（分别为0.339±0.036和0.350±0.041mm²，P=无显著差异）。然而，用大鼠VSMC免疫可显著减少内膜病变面积（0.219±0.0286mm²；与注射PBS的未免疫组和注射大鼠肝细胞的免疫组相比，P<0.05）。大鼠VSMC免疫可抑制新生内膜中PCNA免疫阳性的增殖VSMC（分别为1.34±0.49%和5.78±0.47%；与注射PBS的未免疫组相比，P<0.05）。大鼠VSMC免疫诱导产生的抗体与兔合成型VSMC具有强交叉反应性。在体外实验中，用从VSMC免疫的兔血浆中提取的免疫球蛋白处理后，血清、血管紧张素（AT）II、血小板衍生生长因子（PDGF）-BB、成纤维细胞生长因子（FGF）和佛波酯PMA刺激的兔VSMC的增殖和迁移均受到显著抑制，这表明该免疫球蛋白对VSMC具有一些整体作用。大鼠VSMC免疫的兔免疫球蛋白与兔AT1a受体蛋白结合，该蛋白通过转染兔AT1a受体pcDNA3在COS7细胞中表达。与AT1a受体的这种结合可能是VSMC免疫的免疫球蛋白发挥作用的机制之一。
家兔异种合成型大鼠VSMC免疫诱导产生针对合成型兔VSMC的自身抗体，呈交叉反应。诱导产生的针对合成型VSMC的自身抗体可能为形成新生内膜病变的血管重塑提供新的免疫治疗可能性。
