| Effects of Copper Vapor Laser Irradiation onApoptosis and Proliferation Inhibiting of VascularSmooth Muscle CellsAbstractPercutaneous transluminal coronary angioplasty (PTCA) has been widely adopted as a therapeutic modality for coronary atherosclerosis. However, late restenosis (RS) is developed among 30-50% of patients who undergo PTCA. The main reason causing restenosis is vascular injury, which can be induced by the inflated balloon or similar devices. Restenosis is accompanied by platelet thrombus formation and a change of the phenotype of vascular smooth muscle cells (VSMC). VSMC's change from their resting contractile state to one capable of migratory, proliferative and secretory functions. So, excessive proliferation of VSMC is considered to be the major contributing factor to restenosis. One viable strategy for the provention of restenosis is to inhibit this proliferative response and to induce apoptosis of the proliferative VSMC. Most of the previous studies in this field concentrated on coronary stenting, ionizing radiation, drugs and gene therapy. No effective methods have been found to resolve this problem. Laser therapy is expected to be highly effective for the management of restenosis because of the advantages of laser. Some advantages of laser include accurate direction, exact irradiation dosage, easy manipulation and no systematic toxicity. Till now, the prevention of RS with laser irradiation has not been reported and the mechanism of this alternative treatment is still not understood.The objectives of this study were: 1. Select one or several laser irradiation dosages by which quite high apoptotic rate of VSMC can be induced, and obtain the relation between the laser irradiation dose and the apoptotic rate of VSMC; 2.Observe the morphological and biochemica1 characteristics of aPoptotic VSMC; 3.lnvestigate the factors that influnce the VSMC aPoptotic fate induced by laserirradiation, by analyzing the correlation between VSMC aPoPtotic rate and thecell proliferative statllsi PCNA-positive expression Tate, cell cycIe, c-myc and bc1-2 mRNA leve1s in VSMC; 4. Observe the effects of laser irradiation on theproliferative and matabolic statlls of VSMC, PCNA-positive expression, p53, c-myc and bc1-2 mRNA leve1s in VSMC, so as to propose the mechanism ofinduction aPoptosis and inhibition of the proliferation of VSMC by laserirradiation. This stUdy will provide a foundation for the amer stUdy in vivo andfor designing a nove1 management strategies for local laser irradiation by WhichRS can be prevented.Methods and Results:1. Study the effects of the copper vaPor laser on VSMC aPoPtosis. (l)Morphological characteristics of VSMC were observed under both light andtransmission electron microscope. Morpho1ogical characteristics were observed 4,8, 12, 16, and 24h after irradiated by laser with a PD dose of 300mWcm2 and EDof 200J/cm2. Cell shrinkage, dense chromatin, edging in nuclei, cy'toplasm andnuclei condensation, and aPoptosis bodies fOrmation, which were tyPicalcharacteristic of aPoptosis cells were all found. These changes in VSMC werefound to be time-dependellt. After short time irradiation, early aPoptosisaPpeared While after a long time irradiation, the later stage of aPoptosis occurred.(2) DNA 1addering on agarose demonstrated that a slender small molecule DNAfragment (180~200bp) in VSMC irradiated with low dosage of PDl00 mW/cmand ED 200 mWcm2. More distinCt aPoptotic bands were found When using aPD 200 mW/cm2 and ED 200 mW/cm2 irradiation grouP, and when the laser dosewas PD300 mW/cm', ED 200 mW/cm2, the bands were the most pronounced. (3)The aPoptotic rate of VSMC studied by propidium iodide staining was measuredby flow cytometry (FCM). Measurements were taken 24h after laser irradiationWth a PD300 mWfom', ED 200 mWfom2 dose. The data was analyzed withCellQuest software and revealed that aPoptotic rate of the irradiation grouP was(31.1 l 8.9)%, which had no diffeence Wth the value measu...
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