Edge restenosis: impact of low dose irradiation on cell proliferation and ICAM-1 expression.

BACKGROUND: Low dose irradiation (LDI) of uninjured segments is the consequence of the suggestion of many authors to extend the irradiation area in vascular brachytherapy to minimize the edge effect. Atherosclerosis is a general disease and the uninjured segment close to the intervention area is often atherosclerotic as well, consisting of neointimal smooth muscle cells (SMC) and quiescent monocytes (MC). The current study imitates this complex situation in vitro and investigates the effect of LDI on proliferation of SMC and expression of intercellular adhesion molecule-1 (ICAM-1) in MC. METHODS: Plaque tissue from advanced primary stenosing lesions of human coronary arteries (9 patients, age: 61 +/- 7 years) was extracted by local or extensive thrombendarterectomy. SMC were isolated and identified by positive reaction with smooth muscle alpha-actin. MC were isolated from buffy coat leukocytes using the MACS cell isolation kit. For identification of MC flow-cytometry analysis of FITC-conjugated CD68 and CD14 (FACScan) was applied. SMC and MC were irradiated using megavoltage photon irradiation (CLINAC2300 C/D, VARIAN, USA) of 6 mV at a focus-surface distance of 100 cm and a dose rate of 6 Gy min-1 with single doses of 1 Gy, 4 Gy, and 10 Gy. The effect on proliferation of SMC was analysed at day 10, 15, and 20. Secondly, total RNA of MC was isolated 1 h, 2 h, 3 h, and 4 h after irradiation and 5 microg of RNA was used in standard Northern blot analysis with ICAM-1 cDNA-probes. RESULTS: Both inhibitory and stimulatory effects were detected after irradiation of SMC with a dose of 1 Gy. At day 10 and 15 a significant antiproliferative effect was found; at day 20 after irradiation cell proliferation was significantly stimulated. Irradiation with 4 Gy and 10 Gy caused dose dependent inhibitory effects at day 10, 15, and 20. Expression of ICAM-1 in human MC was neihter inhibited nor stimulated by LDI. CONCLUSION: Thus, the stimulatory effect of LDI on SMC proliferation at day 20 days after irradiation may be the in vitro equivalent of a beginning edge effect. Extending the irradiation area in vascular brachytherapy in vivo may therefore merely postpone and not inhibit the edge effect. The data do not indicate that expression of ICAM-1 in quiescent MC is involved in the process.

Triple-coated stents (Hirudin/Iloprost/Paclitaxel): an in vitro approach for characterizing the antiproliferative potential of each individual compound.

BACKGROUND: Hirudin (H)/iloprost (I)/paclitaxel (P)-coated stents represent a multifactorial approach to reducing the proliferative response caused by ballooning and stenting. The study presented compares the net effect of each individual compound of HIP-coated stents with the summed effect of the compounds in the stent coating. METHODS AND RESULTS: For proliferation prescreening studies, human coronary smooth muscle cells were incubated with H (0.005-500 microg/ml), I (0.00001-1 microg/ml), and P (0.0001-10 microg/ml). After 5 days, cell number was studied in a cell analyzer system. Secondly, 8-mm stents were coated with (1) HI, (2) HIP-10 microg/20 microg/40 microg (HIP5%/10%/20%), (3) P-40 microg (P), (4) IP-40 microg (IP), and (5) HP-40 microg (HP). After 5 days, the effect on cell proliferation and cytoskeletal structures was studied. No antiproliferative effect was found after incubation with H; significant inhibition was seen after incubation with I (p<0.05) or lipophilically dissolved P (p<0.001). After 5 days incubation with HIP5%-, HIP10%-, HIP20%-, P20%-, IP20%-, and HP20%-coated stents, cell proliferation was inhibited by 55.5% (p<0.05), 61% (p<0.05), 57.9% (p<0.05), 59.5% (p<0.001), 59.8% (p<0.001), and 63.3% (p<0.001), respectively. HI- and HIP-coated stents caused a severe destruction of the cytoskeletal structures smooth muscle alpha-actin and alpha-tubulin; despite the destruction, vital cells could be identified with positive FDA staining. CONCLUSIONS: Although both lipophilically dissolved P and hydrophilically dissolved I contributed to the antiproliferative effect, no additive effect of the two compounds was detected. In vivo P can be released more easily from the coating material due to the permanent lipophilic contact of the stent struts with the vessel wall. The current study is the first report on a clear and uncomplicated technique to obtain information on the antiproliferative potential of coated stents before large experimental studies are initiated.

Rapamycin attenuates vascular wall inflammation and progenitor cell promoters after angioplasty.

Rapamycin combines antiproliferative and antiinflammatory properties and reduces neointima formation after angioplasty in patients. Its effect on transcriptional programs governing neointima formation has not yet been investigated. Here, we systematically analyzed the effect of rapamycin on gene expression during neointima formation in a human organ culture model. After angioplasty, renal artery segments were cultured for 21 or 56 days in absence or presence of 100 ng/ml rapamycin. Gene expression analysis of 2312 genes revealed 264 regulated genes with a peak alteration after 21 days. Many of those were associated with recruitment of blood cells and inflammatory reactions of the vessel wall. Likewise, chemokines and cytokines such as M-CSF, IL-1beta, IL-8, beta-thromboglobulin, and EMAP-II were found up-regulated in response to vessel injury. Markers indicative for a facilitated recruitment and stimulation of hematopoetic progenitor cells (HPC), including BST-1 and SDF-1, were also induced. In this setting, rapamycin suppressed the coordinated proadhesive and proinflammatory gene expression pattern next to down-regulation of genes related to metabolism, proliferation, and apoptosis. Our study shows that mechanical injury leads to induction of a proinflammatory, proadhesive gene expression pattern in the vessel wall even in absence of leukocytes. These molecular events could provide a basis for the recruitment of leukocytes and HPC. By inhibiting the expression of such genes, rapamycin may lead to a reduced recruitment of leukocytes and HPC after vascular injury, an effect that may play a decisive role for its effectiveness in reducing restenosis.