Local Delivery of Imatinib Mesylate (STI571)-Incorporated Nanoparticle Ex Vivo Suppresses Vein Graft Neointima Formation
Background— Clinical outcome of surgical revascularization using autologous vein graft is limited by vein graft failure attributable to neointima formation. Platelet-derived growth factor (PDGF) plays a central role in the pathogenesis of vein graft failure. Therefore, we hypothesized that nanoparticle (NP)-mediated drug delivery system of PDGF-receptor (PDGF-R) tyrosine kinase inhibitor (imatinib mesylate: STI571) could be an innovative therapeutic strategy.
Methods and Results— Uptake of STI571-NP normalized PDGF-induced cell proliferation and migration. Excised rabbit jugular vein was treated ex vivo with PBS, STI571 only, FITC-NP, or STI571-NP, then interposed back into the carotid artery position. NP was detected in many cells in the neointima and media at 7 and 28 days after grafting. Significant neointima was formed 28 days after grafting in the PBS group; this neointima formation was suppressed in the STI571-NP group. STI571-NP treatment inhibited cell proliferation and phosphorylation of the PDGF-R-β but did not affect inflammation and endothelial regeneration.
Conclusions— STI571-NP-induced suppression of vein graft neointima formation holds promise as a strategy for preventing vein graft failure.
Clinical outcome of surgical revascularization using autologous vein graft is limited by vein graft failure resulting from accelerated neointima formation; 30% to 50% of vein grafts fail within 10 years.1 Because platelet-derived growth factor (PDGF), expressed by proliferating vascular smooth muscle cells (VSMCs) and infiltrating monocytes, plays a central role in the pathogenesis of vein graft failure,2 targeted molecular blockade of PDGF signaling is a potential strategy for preventing vein graft failure. Imatinib mesylate (STI571),3 a potent inhibitor of the c-Abl tyrosine kinase (TK), the c-Kit receptor kinase, and the PDGF-R TK, is approved for the treatment of patients with chronic myeloid leukemia. It has been shown that c-Kit-positive progenitor cells can differentiate into α-actin-positive VSMCs and may contribute to neointima formation after vascular injury.4 It has also been reported that c-Abl TK is involved in angiotensin II-induced VSMC hypertrophy.5 In contrast, STI571 has been shown to have little antiproliferative effects on endothelial cells.6 These data suggest that STI571 appropriately inhibits neointima formation without negative effects on endothelial regeneration/vascular healing, and thus provide a rationale for the use of STI571 as a VSMC-selective molecular targeting drug in the prevention of neointima formation associated with vein graft failure.
STI571 has been reported to inhibit balloon injury-induced neointima formation in rats7 when dosages beyond the clinical norm were used (50 mg/kg per day). In contrast, STI571 had no effect on neointima formation in rabbits when administered in a clinically relevant dosage (10 mg/kg per day).8 Recent clinical studies in humans have detected no beneficial effects of oral administration of STI571 (600 mg/d for 10 days)9 on in-stent restenosis. These data suggest that systemic administration of STI571 at clinical dosages may not be sufficient to antagonize PDGF-induced vascular responses at the site of vascular injury. It has been suggested that STI571 administered at standard dosages (400≈800 mg/d) may not reach sufficient serum concentrations (maximum concentration: <10 μmol/L) to function as an inhibitor of PDGF-R signaling.10 Furthermore, long-term administration of STI571 causes cardiac mitochondrial dysfunction that results in cardiotoxicity and ventricular dysfunction.11
Therefore, preventing vein graft failure via STI571-mediated PDGF-R signaling blockade requires an efficient local drug delivery system. Ex vivo local delivery of drugs or genes to the vein has been used as a clinically relevant approach. We have recently developed bio-absorbable polymeric nanoparticles (NP) formulated from the polymer poly-ethylene-glycol (PEG)-modified poly(DL-lactide-co-glycolide) (PLGA).12,13 PEG-PLGA NP offers the advantages of safety, efficient intracellular delivery of different classes of therapeutic agents, and the capacity for sustained intracytoplasmic release.14,15 Therefore, we hypothesized that STI571-incorporated NP could be an innovative therapeutic strategy for preventing vein graft failure. We investigated whether our NP-based drug delivery system worked as an intracellular ex vivo delivery system to the excised vein, and whether blockade of PDGF-R TK by STI571-incorporated NP suppressed vein graft neointima formation in vivo.
Materials and Methods
Preparation of PEG-PLGA NP
PEG-PLGA NP encapsulated with fluorescence marker or STI571 was prepared using an emulsion solvent diffusion method, as previously reported.12,13 Additional details can be found in the online Data Supplement.
Cellular Uptake and Intracellular Distribution of NP In Vitro
We cultured rat aortic smooth muscle cells (SMCs) and evaluated the cellular uptake of PEG-PLGA NP by fluorescence microscopy. Additional details can be found in the online Data Supplement.
Measurement of In Vitro FITC Release Kinetics From NP
To measure FITC release kinetics, FITC-NP (n=8) was immersed in Tris-EDTA buffer, and the released FITC from NP was measured.
Cell Proliferation, Migration, Cytotoxicity, and TUNEL Assay
We cultured human coronary artery SMCs and evaluated proliferation, migration,16 cytotoxicity, and apoptosis. Additional details can be found in the online Data Supplement.
Experimental Animal Models
Male Japanese white rabbits (KBT Oriental, Tokyo, Japan) weighing 2.5 to 3.0 kg were fed a high-cholesterol diet for 2 weeks before the operation. Animals were anesthetized, a midline neck incision was made, and an approximately 3-cm segment of the jugular vein was dissected free; all side branches were ligated. The vein segments were gently flushed, and placed in a buffer alone (n=11) or in a solution containing either FITC-encapsulated PEG-PLGA NP at 0.5 mg/mL (n=11), STI571- encapsulated PEG-PLGA NP at 0.5 mg/mL containing STI571 at 100 μmol/L (n=11), or STI571 alone at 100 μmol/L (n=11) for 30 minutes at room temperature. The treated vein segments were interposed into ipsilateral carotid arteries in an end-to-side fashion. The animals were maintained on the same high-cholesterol diet throughout experimental period.
All animals received aspirin at 20 mg/d from 3 days before the graft procedure until euthanasia. After venous blood samples were taken, animals were killed with a lethal dose of anesthesia on days 7 (n=5 each) and 28 (n=6 each). The vein grafts were harvested, flushed with saline, and used for histopathologic, immunohistochemical, and biochemical studies.
Ex Vivo NP Delivery in Human Vein
Segments of internal thoracic vein were obtained from patients undergoing coronary arterial bypass surgery. Additional details can be found in the online Data Supplement.
Histopathologic and Immunohistochemical Analysis
Tissue sections from the grafts were harvested and prepared for analysis. Additional details can be found in the online Data Supplement.
Western Blot Analysis
Protein was extracted from cultured VSMCs or frozen vein graft tissues. Western blot analysis was performed with antibodies against human PDGF-R-β, phospho-PDGF-R-β, phosphotyrosine, phosho-p44/42 MAPK (ERK 1/2), ERK 1/2, c-Abl TK, or actin (additional details can be found in the online Data Supplement).
Data are expressed as the mean±standard error of the mean (SEM). Statistical analysis of differences was performed by ANOVA and Bonferroni’s multiple comparison tests. Statistical significance was set at P<0.05.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
In Vitro Cell Uptake and Intracellular Distribution of NP and NP Release Kinetics In Vitro
When incubated with rat aortic and human coronary artery SMCs, the fluorescence-encapsulated NP showed excellent capacity of intracellular distribution (Figure 1A). In contrast, no fluorescence was detected when the SMCs were incubated with blank NP or fluorescent molecules only. A large fraction (>90%) of the NP rapidly entered into the cells, and this incorporation rate sustained until 24 hours (Figure 1B). An endocytosis inhibitor (chlorpromazine hydrochloride) attenuated the NP-mediated intracellular incorporation of fluorescence (supplemental Figure I). Fluorescence confocal microscopic study revealed the intracellular retention of NP (Figure 1C). Transmission electron microscopy of the cellular cross-sections revealed the intracellular localization of NP at 1 hour of incubation (Figure 1D).
An analysis of the in vitro FITC release kinetics from FITC-NP showed an early burst of FITC release such that approximately 40% of the total amount ultimately released was present on day 1, followed by sustained release of the remaining FITC over the next 28 days (Figure 1E).
In Vitro Effects of STI571-NP on PDGF-Induced Proliferation and Migration of VSMCs and on Receptor Phosphorylation
As reported by other investigators,6,17 non-NP-STI571 attenuated PDGF-induced proliferation and migration in a dose-dependent manner (Figure 2A and B). STI571 at a concentration of 10 μmol/L prevented the PDGF-induced cell proliferation and migration. The PEG-PLGA NP-containing STI571 at 10 μmol/L also prevented the PDGF-induced responses of VSMCs (Figure 2A and B). Results of a TUNEL assay showed no detectable increase in STI571-induced increase in apoptotic cells (data not shown). Results of a cytotoxicity assay showed that human coronary artery SMCs incubated with blank PEG-PLGA NP at a concentration of 1 mg/mL remained 100% viable relative to control (data not shown).
Western blot analysis showed that in human coronary artery SMCs, PDGF-induced phosphorylation of PDGF-R-β was suppressed by STI571 at 10 μmol/L as well as by STI571-NP (Figure 2C).
Efficacy of NP-Mediated Drug Delivery System to Vein Grafts
Ex vivo incubation of NP with excised rabbit jugular vein or human internal thoracic vein for 30 minutes resulted in high SMC uptake in the media and in some cells in the adventitia (supplemental Figure IIA and IIB). After 7 days of grafting in rabbits when a thin neointima was formed, FITC-positive cells were detected in the neointima and media (% positive area: 51±9%). After 28 days, many FITC-positive cells were still noted in the neointima and media (% positive area: 12±5%). In contrast, no FITC immunoreactivity was noted in veins incubated with PBS.
Effects of STI571-NP on Vein Graft Failure in Rabbits
As we previously reported,18 significant neointima developed 28 days after grafting in animals interposed with control PBS-treated vein grafts. Ex vivo treatment with STI571-NP, but not with STI571 only or FITC-NP, markedly attenuated neointima formation at day 28 (Figure 3).
Increased monocyte infiltration and PCNA-positive proliferating cells were observed in the intima-media and adventitia at 7 and 28 days after grafting (supplemental Figure IIIA). No effects on inflammatory changes were noted in STI571-NP-treated vein graft (supplemental Figure IIIB). In contrast, treatment with STI571-NP, but not with FITC-NP or STI571 only, markedly attenuated the number of PCNA-positive cells observed on day 7 (supplemental Figure IIIC). There were no significant differences in endothelial cell linings among the 4 groups at 7 and 28 days after grafting (supplemental Figure IIID). There were no significant differences in serum cholesterol levels after 4 weeks among the 4 groups (data not shown).
Effects of STI571-NP on PDGF, PDGF-R, PDGF-R Phosphorylation, and MAPK Pathway
Immunohistochemical studies showed that no PDGF was detected in normal veins. In contrast, intense immunohistochemical staining for PDGF was noted in vein graft tissues 7 days after grafting. There were no significant differences in the degrees of the positive staining area among the 4 groups (supplemental Figure IV). Western blot analysis showed that ex vivo treatment with STI571-NP, but not with STI571 only or FITC-NP, markedly attenuated expression of PDGF-R protein and phosphorylation of PDGF-R kinase, phosphorylation of ERK1/2, and c-Abl TK, at 7 days after grafting (Figure 4A through 4C).
We demonstrated for the first time that PEG-PLGA NP is an excellent system for intracellular delivery of molecular targeting drugs in excised veins. This NP system is bio-absorbable polymer with a long history of safe use in medical applications. Therefore, this system may represent a novel NP-mediated drug delivery system to prevent vein graft failure.
We showed that the NP was endocytosed rapidly by VSMCs and was retained stably in the intracellular space. Impressive ex vivo delivery of NP into human veins suggests that this NP-mediated drug delivery system can be applied to clinical settings for humans. An important finding was that long-term retention of NP in neointima and medial cells of vein grafts was detected until day 28. After cellular uptake of NP, NP slowly releases encapsulated drugs or genes into the cytoplasm as PLGA is hydrolyzed, resulting in an intracellular drug delivery. The bio-absorption time of PLGA in the body can be controlled by changing material make-up of PLGA, thus the function of the intracellular drug delivery system can be modified. Therefore, (1) this NP-mediated drug delivery system works as an excellent ex vivo delivery system for the excised vein; and (2) this system provides an effective means of delivering drugs or genes that target intracellular proteins involved in the pathogenesis of vein graft neointima formation.
We selected STI571 because this compound is known to target the PDGF-R TK (see Introduction). NP-incorporated with STI571 attenuated the proliferation of human VSMCs in vitro and the formation of vein graft neointima formation in vivo, both of which are known to be associated with the inhibition of the target molecules of STI571 (PDGF-R TK, c-Abl TK) and downstream signal of PDGF-R (ERK). NP-incorporated with STI571 did not affect endothelial regeneration process after vein grafting. In preliminary experiments, tissue concentrations of STI571 were measured immediately after and 6 hours after incubation of excised veins with STI571-NP by HPLC system, which showed under the limit of detection (1 ng/mL). Although the precise intracellular concentration and distribution of STI571 is unclear, our present data (Figures 2 and 4⇑) provide evidence that (1) STI571-incorporated NP may block PDGF-R signaling possibly via slow release of STI-571 into the cytoplasm as NP is hydrolyzed; and (2) PDGF-R signaling blockade by NP-incorporated with STI571 is a means for treating vein graft neointima formation in vivo.
Inflammatory-proliferative changes have been shown to play a central role in the pathogenesis of vein graft neointima formation. In early stages, the neointima lesion has an inflammatory nature characterized by mononuclear cell infiltration, followed by VSMC proliferation.19 We recently reported that blockade of monocyte chemoattractant protein-1 (MCP-1) by adenovirus-mediated ex vivo transfer of 7ND gene to autologous vein grafts suppressed neointima formation in dogs.18 We also have demonstrated that MCP-1 plays a central role in neointima formation following arterial mechanical injury.20,21,22 In a previous study,18 we showed that blockade of MCP-1 attenuated both inflammation (monocyte infiltration) and proliferation (appearance of proliferating VSMC) in vein grafts. In contrast, data from this study show that NP-mediated delivery of STI571 reduced PDGF-induced proliferation but not inflammation, suggesting that (1) PDGF-mediated proliferative changes might be located downstream of inflammatory changes, or (2) the mechanism of action of STI571-mediated inhibition of proliferation might be distinct from that of anti-MCP-1-mediated attenuation of proliferation and inflammation. If STI571 and anti-MCP-1 treatment exert their effects through different pathways, it would be interesting to examine whether combined blockade of PDGF and MCP-1 would have additive inhibitory effects on vein graft failure.
Expression of PDGF is known to be low in normal blood vessels, but mechanical forces stimulate SMC expression and release of PDGF, and induce PDGF-R phosphorylation (activation).23 We show here that PDGF and the phosphorylation levels of its receptor were up-regulated in vein grafts. STI571-incorporated NP did not affect increased PDGF expression, but it did suppress the protein expression of PDGF-R, PDGF-R kinase, and c-Abl TK in vivo. This could suggest the presence of a positive-feedback loop that, in the absence of STI571, potentiates PDGF-mediated proliferation in vein grafts. It is also possible that reduced PDGF-producing cells (PCNA-positive cells) in the vein graft or blockade of multiple intracellular kinases might have contributed to the beneficial effects of STI571-incorporated NP on vein graft neointima formation in vivo.
One limitation in the present study is that only single dose of STI571-NP was examined. It is practically difficult to obtain the dose-response relationship of this NP system in small animals, because the dose-response relation of STI571 and polymer needs to be examined. For translation of our present findings into clinical medicine, further studies are therefore needed to define a dose-response relation in large animal models.
In conclusion, blockade of PDGF signaling by STI571-incorporated NP-inhibited proliferation of VSMCs in vitro and suppressed vein graft neointima formation in vivo. This NP-mediated drug delivery system provides an innovative and clinically feasible therapeutic strategy for preventing vein graft failure.
Sources of Funding
This study was supported by Grants-in-Aid for Scientific Research (19390216, 19650134) from the Ministry of Education, Science, and Culture, Tokyo, Japan and by Health Science Research Grants (Research on Translational Research and Nano-medicine) from the Ministry of Health Labor and Welfare, Tokyo, Japan.
Dr Egashira holds a patent on the results reported in the present study. The other authors report no conflicts.
Presented at the American Heart Association Scientific Sessions, November 4–7, 2007, Orlando, Fla.
The online Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.740613/DCI.
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