A Novel Drug-Eluting Stent Coated With an Integrin-Binding Cyclic Arg-Gly-Asp Peptide Inhibits Neointimal Hyperplasia by Recruiting Endothelial Progenitor Cells
Rüdiger Blindt, MD*,*,
Felix Vogt, MD*, ,
Irina Astafieva, PhD ,
Christian Fach, MD*, ,
Mihail Hristov, MD,,
Nicole Krott, MSc*,,
Berthold Seitz, MS*,
Aphrodite Kapurniotu, PhD||,
Connie Kwok, PhD,
Manfred Dewor, MSc||,
Anja-Katrin Bosserhoff, PhD¶,
Jürgen Bernhagen, PhD¶,
Peter Hanrath, MD*,
Rainer Hoffmann, MD* and
Christian Weber, MD*,,
* Department of Cardiology, University Hospital Aachen, Aachen, Germany
Interdisciplinary Center for Clinical Research in Biomaterials and Tissue-Material Interaction in Implants BIOMAT, University Hospital Aachen, Aachen, Germany
Guidant Corporation, Santa Clara, California
Department of Molecular Cardiovascular Research, University Hospital Aachen, Aachen, Germany
|| Institute of Biochemistry, University Hospital Aachen, Aachen, Germany
¶ Institute of Pathology, University of Regensburg, Regensburg, Germany
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Figure 1 Characterization and outgrowth of isolated porcine endothelial progenitor cells (EPCs). (A) Mononuclear cells were plated on culture dishes in the presence (1 µg/ml or 100 µg/ml, respectively) or absence of integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) coating. Coating with cRGD provoked a significant dose-dependent increase of spindle-shaped cells compared with uncoated control experiments. Representative images from four isolations are shown, magnification 100x. (B) Flow cytometry analysis of adherent cells at day 7 of culture. Unstained cells served as a control condition. Analysis was performed by manual gating and fluorescence channel (FL)-1/FL-2 dot plot quadrant statistic. 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled Ac-low-density lipoprotein/lectin double-positive cells in the upper right region were judged as EPCs. FITC = fluoresceinisothiocyanat.
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Figure 2 Recruitment of isolated endothelial progenitor cells (EPCs) on integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) matrix. (A) After 7 days in culture, adherent cells were counted in multiple microscopic fields. Coating with cRGD (1 µg/ml or 100 µg/ml) resulted in a significant increase in EPC outgrowth compared with uncoated control experiments. The fibronectin coating served as a positive control condition (n = 4, *p < 0.05). (B) Coating with cRGD (1 µg/ml or 100 µg/ml) resulted in a significant increase in EPC arrest under flow conditions compared with uncoated control experiments. The fibronectin coating served as a positive control condition (n = 4, *p < 0.05). FN = fibronectin.
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Figure 3 Effect of the integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) on endothelial progenitor cell (EPC) and smooth muscle cell (SMC) invasion. (A) cRGD (100 µg/ml) stimulated significantly the invasive potential of EPCs compared with control cells, whereas SMC invasion against different concentrations of cRGD was not altered (B). DMEM = Dulbecco's modified eagle medium.
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Figure 4 Stent design, mechanical properties, and in vitro integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) release from the stent. (A) Scanning electron microscopy photographs showing smooth surfaces of the polymer stent coating after complete expansion of the stent and after simulated use (magnification in the left panel 10x, in the right panel 100x). (B) The cRGD-coated stent release kinetic profile over 72 h.
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Figure 5 In vivo recovery of integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) from vascular tissues surrounding cRGD-loaded stents and quantification of cRGD vascular tissue levels. (A) The high-performance liquid chromatography (HPLC) chromatogram obtained from a vascular tissue lysate surrounding an unloaded polymer stent implanted for 4 weeks and compared with a tissue lysate that was spiked with cRGD after removal from the stent. The spectrum from the cRGD-containing and cRGD-free tissue lysates are essentially superimposable. In the preparation from the cRGD-spiked tissue lysate (blue), a peak is visible at an elution time around 16.2 min that precisely co-elutes with the standard cRGD peak (black). As an HPLC control, pure cRGD peptide alone underwent chromatography (red). Only the relevant part of the spectrum is shown, but overall, only a limited number of substances with a relative molecular weight (Mr) < 3,000 Da was obtained (latter not shown). (B) Recovery of cRGD from the surrounding vascular tissue 4 weeks after stent implantation. The HPLC chromatogram obtained from a vascular tissue lysate surrounding the cRGD-loaded stent implanted for 4 weeks (green). After separation of the tissue from the stent, the tissue was lysed and spiked with additional cRGD (blue). Pure cRGD underwent chromatography as an HPLC standard (red). The two lysates show a peak that elutes at the same time (at 16 min) as the standard cRGD. Spiking the tissue with cRGD leads to a higher peak and shows the chromatographic identity of the spiked cRGD species with that loaded onto the stent. The cRGD peaks are indicated by arrows. (C) Identification of the recovered implanted cRGD by electrospray ionization mass spectrometry analysis. The cRGD from tissue lysate surrounding the cRGD-loaded 4-week implanted stent was detected by HPLC (see B), the assumed cRGD-containing HPLC peak at 16 min (green spectrum in B) was isolated and lyophilized, and the cRGD identity was verified by MS analysis. The M+2 peak of cRGD was clearly detectable as indicated. (D) Confirmation of stent-derived cRGD identification by ESI MS of the spiked tissue lysate. The tissue lysate was spiked with additional cRGD before work-up (which was identical to that as described in panel C) and HPLC. The M+2 peak appears at a markedly enhanced intensity but at the same Mr as in (C).
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Figure 6 Histomorphometric analysis. (A) Representative photomicrographs of coronary tissue sections that were used to determine stenosis rates after implantation of unloaded polymer, integrin-binding cyclic Arg-Gly-Asp peptide (cRGD)-loaded, or bare-metal stents after 4 and 12 weeks (magnification 40x). L = lumen; NI = neointima; arrows indicate neointimal area. (B) Quantification of infiltration with chronic inflammatory cells (CIC) around the stent struts.
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Figure 7 Effects on endothelialization. (A) Representative photomicrographs of endothelialization, analyzed by CD34 staining, of unloaded polymer, integrin-binding cyclic Arg-Gly-Asp peptide (cRGD)-loaded, or bare-metal peri-stent tissue sections after 4 and 12 weeks (magnification 400x). (B) Quantification of endothelialization of unloaded polymer, cRGD-loaded, or bare-metal persistent tissue sections. (C) Electron microscopy of cRGD stent surfaces at 4 weeks showed uniform and complete endothelial coverage (left), whereas analysis of control stents showed incomplete endothelial coverage in the interstrut region. (D) The cRGD coating (left) significantly increased endothelial progenitor cell (EPC) recruitment as compared with control stents (right, magnification 200x). (E) Quantification of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-labeled EPCs by fluorescent image analysis (top) or cell counting (bottom).
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