Determinants of Neointimal Proliferation and Stent Coverage After Intracoronary Therapy With Drug-Eluting Devices in Stable Coronary Artery Disease: Role of Endothelial Progenitor Cells and Interleukin-1 Family Cytokines

Abstract: Background. Endothelial progenitor cells (EPCs) and cytokines seem to play a pivotal role in arterial healing after stent implantation. Using optical coherence tomography (OCT) as a high-resolution imaging technique, we aimed to assess the influence of circulating EPCs and levels of Il-1 cytokines on stent coverage and in-stent proliferation. Methods. Eighty-nine patients were randomly treated with either Xience V drug-eluting stent (DES; n = 48) or bare-metal stent (BMS) postdilated with the SeQuent Please drug-eluting balloon (DEB; n = 41). EPC populations (CD34+/CD133+ and CD34+/CD133+/KDR+EPC) and cytokines (Il-1ra, Il-18, and Il-1α) were measured before percutaneous coronary intervention using flow cytometry or immunoassay. Vessel remodeling was analyzed using coronary angiography and OCT at 6-month follow-up. Results. Indexed neointimal volume and maximal proliferation thickness correlated inversely with EPC levels in the entire study population (r = -0.220; P=.04 and r = -0.253; P=.02) and the BMS + DEB subgroup (r = -0.344; P=.03 and r = -0.374; P=.02). Late lumen loss (LLL) was associated with the proatherogenic Il-18 concentration in the main population (r = 0.342; P=.01) and the BMS + DEB group (r = 0.471; P=.01). In the DES subgroup, associations with proliferation and LLL were lacking. Associations for stent strut coverage were not observed. Conclusions. A high EPC count seems to be a favorable individual patient factor, since it was associated with less in-stent proliferation. Contrarily, high Il-18 levels lead to more LLL, which emphasizes its proatherogenic properties. 

J INVASIVE CARDIOL 2014;26(12):648-653

Key words: restenosis, endothelial progenitor cells, cytokines, PCI, optical coherence tomography 

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In-stent restenosis (ISR) after percutaneous coronary intervention (PCI) is still an unresolved issue despite modern therapeutic options with drug-eluting stents (DESs), drug-eluting balloons (DEBs), and even bioresorbable vascular scaffolds. Besides the chosen type of intracoronary vascular device, related individual biological factors such as endothelial progenitor cells (EPCs) or cytokines seem to play a pivotal role in vascular response and arterial healing in the short- and long-term course following a coronary intervention.^1,2 Cytokines of the interleukin (IL)-1 family and their link to atherosclerotic risk factors, such as obesity or diabetes mellitus, as well as cardiovascular disease progression, have been investigated before.^2-5 EPCs are bone-marrow derived, pluripotent, mononuclear cells that are characterized by certain surface markers (eg, CD34, KDR, and CD133). They are involved in neovascularization and maintenance of endothelial integrity after endothelial damage.^6,7 In previous studies, reduced levels of CD34+/KDR+ EPCs were associated with endothelial dysfunction and in-stent proliferation and predicted major adverse cardiovascular events.^8-11 

With optical coherence tomography (OCT), a light-based imaging technique, quantification of neointimal growth and stent-architecture in vivo is possible.^12,13 OCT is currently the most sophisticated and precise intracoronary imaging modality and allows simultaneous evaluation of stent coverage, “histology-like” assessment of proliferation patterns, neoatherosclerosis, and even plaque morphology. Since differences after PCI with modern stent devices are subtle, a high-resolution imaging technique is mandatory to evaluate vascular healing processes. So far, in-stent proliferation has been approximated by the rather imprecise method of quantitative coronary angiography, and data for stent coverage have not been provided in former EPC studies.^11,14 Furthermore, the possible influence of the implanted intracoronary device on EPCs and cytokines, and vice versa, has not yet been investigated. 

Using OCT, we aimed to assess the influence of circulating EPCs and levels of cytokines of the Il-1 family on neointimal proliferation and stent coverage 6 months after elective PCI. We hypothesized that incomplete stent strut coverage is determined by the number of circulating EPCs. Furthermore, the possible impact of EPCs and cytokines on vessel healing processes and restenosis after different DES procedures was investigated.

Methods

Trial design and study population. The design and results of the OCTOPUS trial have already been described in detail elsewhere.^15,16 Briefly, patients with stable coronary artery disease and indication for elective PCI due to a native coronary lesion suitable for stent placement and OCT imaging were included in the study at the University Hospital of Jena, Germany. Eligible patients were randomly assigned to receive treatment either with the Xience V (Abbott Vascular) everolimus DES or a combination therapy of the Coroflex Blue bare-metal stent (BMS) postdilated with the paclitaxel SeQuent Please DEB (both B. Braun). Only 2.5 mm or 3.0 mm stent diameters with two stent lengths (DES, 18 or 28 mm; DEB, 16 or 25 mm) were allowed in each study arm. To provide a safety margin at the proximal and distal stent edges, and to avoid “candy-edge” restenosis, all BMSs were postdilated with a longer DEB (20 or 30 mm DEB for 16 or 25 mm BMS, respectively). All patients were given aspirin 100 mg daily and clopidogrel-naive patients were treated with a 300 mg loading dose prior to intervention. Patients were discharged on the condition to continue dual-antiplatelet therapy for at least 6 months. After 6 months, invasive follow-up including OCT imaging with the time-domain, occlusive technique (M2 CV system; LightLab Imaging, Inc) was attempted in all patients (Figure 1). The percentage of struts without coverage (Figure 1) and neointimal proliferation of the study stent after 6 months, defined as peak relative neointimal proliferation area and relative neointimal volumetric proliferation within the study stent (Figure 2), were investigated. Quantitative coronary angiography (QCA) was performed at baseline and at 6 months follow-up assessing reference lumen diameter (RLD), minimal lumen diameter (MLD), percent diameter stenosis ([MLD/RLD] x 100), side-branch involvement, late lumen loss (LLL; defined as final MLD at the end of the index procedure – MLD at follow-up) and net luminal gain (NLG; defined as MLD at follow-up – baseline MLD). Patients gave informed written consent prior to any study-related procedures. The study protocol was approved by the local Ethics Committee and conducted according to the principles of the Declaration of Helsinki.

Laboratory analysis. A day prior to PCI, peripheral venous blood samples were drawn using a 5 mL EDTA tube, a 5 mL heparin tube, and 10 mL serum tube (coated with micronized silica particles) for assessment of EPCs and cytokines of the IL-1 family. Blood work and measurements of EPCs and cytokines were performed by one operator, who was blinded to the patients’ treatment. In addition, routine blood samples were obtained at baseline and follow-up at the Department of Clinical Chemistry of the University Hospital of Jena for the following parameters: complete blood count, white blood count differential, total cholesterol, low-density lipoprotein (LDL; mmol/L), high-density lipoprotein (HDL; mmol/L), triglycerides (mmol/L), and creatinine (µmol/L).

Circulating endothelial progenitor cells. EPCs were identified by flow cytometry according to their coexpression of the different antigens: (1) CD34, a marker of hematopoietic progenitor cells; (2) the more immature hematopoietic stem cell marker CD133; and (3) kinase domain receptor (KDR), a marker of endothelial lineage, also known as the endothelial cell receptor “vascular endothelial growth factor receptor-2” (VEGFR-2) as described previously.^17,18 After lysis of red blood cells with lysing solution, cells were incubated with a FcR blocking reagent (MACS Milteny Biotec) to saturate sites for unspecific binding and washed with phosphate-buffered saline (PBS) twice. Then, staining of peripheral blood mononuclear cells with fluorescein-isothiocyanate (FITC)-conjugated anti-human CD34 monoclonal antibody (MACS Milteny Biotec, Clone AC136), R-phycoerythrin (R-PE) conjugated anti-human CD133 mononclonal antibody (MACS Milteny Biotec, Clone 293C3), and allophycocyanin (APC)-conjugated anti-human VEGFR-2 monoclonal antibody (R&D Systems, Clone 89106) was conducted with an incubation time of 15 minutes at 4 °C. Mouse IgG2a FITC-conjugated antibodies and IgG2b PE-conjugated antibodies were used as isotype control (MACS Milteny Biotec, Clone S43.10 and Clone IS6-11E5.11). Different EPC populations (CD34+/CD133+ and CD34+/CD133+/KDR+) were measured by 3-color flow cytometry (FACSCalibur; BD Biosciences) and analyzed with CellQuestPro software version 4.0.2 (BD Biosciences) as described previously.¹⁷

IL-1 family cytokines. Frozen serum (-70 °C) was used to quantify Il-1ra, Il-18, and Il-1α by a quantitative sandwich enzyme immunoassay technique according to the manufacturers’ instructions (R&D Systems, Inc and MBU). Microplates were precoated with a capture antibody. After the samples were pipetted into the wells, any present antigen binded to the capture antibody. A conjugated anti-human monoclonal antibody was added and incubated. After another washing to remove unbound antibody-enzyme reagent, a substrate solution was added and the optical density of the fluorescently-labeled antibody was measured at 450 nm using a microplate reader. Corresponding IL concentrations were calibrated from a dose-response curve, which was based on reference standards.

Statistical analysis. All patient-related parameters, as well as laboratory and OCT parameters, were archived in a custom-made database (Microsoft Access; Microsoft, Inc). Categorical variables were presented as percentages and analyzed with the Pearson χ² test. Continuous parameters were expressed as mean ± standard deviation (SD), and in case of normally distributed variables, analyzed with the student’s t-test or with the Mann-Whitney test for non-normally distributed variables. Bivariate analysis with the Pearson correlation coefficient was conducted to determine the strength of the linear relationship between EPC or cytokine levels with clinical, proliferation, and coverage parameters. Statistical significance was assumed for P-values <.05 and tested two-tailed with regression analysis. All calculations were done with SPSS for Windows, version 19.0.

Results

Study population. Six-month follow-up with OCT and blood samples for EPC were available for 89 patients (48 patients with DES and 41 patients with BMS + DEB). Blood samples for cytokine levels were available for 60 patients (28 DES patients and 32 BMS + DEB patients). The baseline clinical characteristics as well as angiographic and procedural parameters of the entire study population (n = 89) are summarized in Table 1. The two study groups were well balanced, and except for LDL levels, no significant differences were observed. Table 2 shows the baseline levels of circulating EPCs and cytokines, with no differences of cytokines between the device groups. EPC counts were significantly higher in the DES subgroup (P<.05). Cytokine measurements were available for 55 patients (Il-1ra) and 60 patients (Il-18). Baseline leukocyte and lymphocyte numbers, which could bias EPC levels, did not differ between the groups and ranged within standard values.

Relationships between biological markers, proliferation and strut coverage. Quantitative coronary angiography showed a slightly greater LLL in the BMS + DEB group compared to the DES group (0.16 ± 0.15 mm vs 0.24 ± 0.21 mm, respectively), but failed to reach significance (P=.06). With OCT, significantly more in-stent neointimal proliferation after BMS + DEB therapy was found compared to DES implantation at 6-month follow-up (15.7 ± 7.8 mm³ vs 11.0 ± 5.2 mm³ proliferation volume per cm stent length; P=.01). The proportion of uncovered struts did not differ between the DES group (5.0 ± 9.4%) and the BMS + DEB group (5.6 ± 9.6%; P=.34). 

Table 3 gives an overview of correlations between clinical parameters, as well as in-stent proliferation and stent strut coverage with the measured biological markers.

In the entire study population, indexed neointimal volume and maximal proliferation thickness correlated inversely with CD34+/KDR+ EPC (r = -0.220; P=.04 and r = -0.253; P=.02) and CD34/CD133+/KDR+ EPC counts (r = -0.230; P=.03 and r = -0.251; P=.02). Also, HDL levels showed a positive association with CD34+/KDR+ EPCs (r = 0.253; P=.02) and CD34/CD133+/KDR+ EPCs (r = 0.263; P=.02). LLL positively correlated with Il-18 levels (r = 0.342; P=.01), a proatherogenic cytokine. 

In the further analysis regarding the chosen stent procedure, age (r = 0.451; P=.02) and LDL (r = 0.402; P=.047) correlated positively with the anti-inflammatory receptor antagonist Il-1ra, whereas glomerular filtration rate (r = -0.435; P=.04) showed an inverse relationship with Il-1ra in the DES group. Correlations for proliferation were absent in the DES group.

Corresponding to the found correlations in the main study population (Table 3), similar observations for proliferation assessed with QCA and OCT were found in the DEB stent procedure group (indexed neointimal volume and CD34+/CD133+/KDR+ EPCs: r = -0.344 and P=.03; maximal proliferation thickness and CD34+/CD133+/KDR+ EPCs: r = -0.374 and P=.02; LLL and Il-18: r = 0.471 and P=.01).

Associations for stent strut coverage were lacking in the entire study group, as well as in the subgroup analysis. 

Discussion

In the present study proliferation patterns as well as stent coverage were evaluated simultaneously by using the high-resolution intravascular imaging technique OCT and compared to circulating EPC levels for the first time. 

We observed that lower EPC counts are associated with more in-stent proliferation 6 months after implantation. Accordingly, higher HDL levels were associated with higher numbers of circulating EPC populations (CD34+/KDR+ and CD34+/CD133+/KDR+). Our data support the hypothesis that EPC might exert protective properties in coronary atherosclerosis. On the other hand, Peliccia et al found a positive correlation for higher EPC levels and ISR after elective PCI.¹⁴ However, whether EPCs elicit antiproliferative effects or not seems to be determined by their subpopulations, since the investigated EPC population in that study was different (CD34+/KDR+/CD45+) compared to ours and other studies.^8,9,19-22 Moreover, the ability of EPC recruitment, and functional properties of EPC such as migration and adhesiveness, might be of crucial importance to their protective effects.^11,23 A previous small study raised the question of whether EPCs might influence proliferation patterns since EPC exhaustion correlated with diffuse neointimal hyperplasia, but no association with focal stenosis was found.¹¹ In the current study, using a very precise OCT algorithm, we found an inverse correlation between EPC numbers with both diffuse (indexed neointimal volume/cm stent length describing neointimal growth of the entire stent) and focal (maximal proliferation thickness) in-stent proliferation. 

Interestingly, we found no association between circulating EPCs or cytokines and strut coverage. Since we assessed neointimal growth with the same method, naked stent struts do not seem to stimulate accelerated neointimal proliferation, contrary to a former hypothesis.¹¹ This observation can be emphasized by the well-known vessel healing response after BMS implantation, ie, BMS implantation allows quick restoration of endothelial integrity, but in-stent proliferation compared to DES is also clearly enhanced and rates of ISR are significantly higher.^24,25

Also, other pathways like mobilization of smooth muscle cells (SMCs) and their secretion of extracellular matrix proteins play a role in neointimal tissue formation.^20,26 Yet, it cannot be determined if depletion of circulating EPCs is a mere association or a causal factor for neointimal proliferation. The link between EPC counts and function, SMC activation, and subsequent neointimal proliferation might rely on mutual stimuli excited from proatherogenic cytokines after an inflammatory trigger (eg, endothelial denudation) or due to continuous inflammation.²⁷ In line with this assumption, we also observed a correlation between LLL and the concentration of Il-18. Il-18 has known proatherogenic effects, and has been associated with atherosclerosis and plaque destabilization.²⁸ Cytokines like Il-18 may down-regulate circulating EPC populations directly or indirectly via other induced cytokines. Thus, neointimal proliferation is possibly a result of different pathways that are activated by the same regulators.

In the DES subgroup, we did not find any correlations between proliferation (in OCT or QCA) and circulating EPCs or IL-18 levels. This finding might be due to less in-stent proliferation following the DES procedure and a low number of cases. However, the EPC count in the DES subgroup was also significantly higher compared to the BMS + DEB group, which underlines their influence to suppress excessive neointimal growth. Furthermore, everolimus has entirely different pharmacological effects compared to the cytotoxic mitotic inhibitor paclitaxel.^24,29,30 It can only be speculated that the cytostatic mToR-inhibitor everolimus might implement vessel effects more independently from individual biological factors. Previous studies did not state or differ between stent types.^11,14 Therefore, this vague hypothesis can not be emphasized with additional data. 

The receptor antagonist of Il-1 (Il-1ra) was compensatorily elevated with higher LDL levels and age in the DEB group, which goes along with previous findings and supports its protective features in atherosclerosis.^31,32 However, whether or not Il-1ra plays a role in neointimal proliferation cannot be assessed since we were unable to find any associations with OCT or QCA parameters.

Clinical implications. The causal biological mechanism needs to be further evaluated in order to effectively address the issue of ISR. Circulating EPCs and cytokines could possibly serve as an individual risk stratification and might help identify patients more prone to restenosis after PCI. 

Study limitations. The sample size of the present study is relatively small, and clinically relevant binary restenosis was not observed. We did not assess functional characteristics of EPCs, which could have improved our comprehension of neointimal tissue formation. Also, we cannot provide a stent-naive control group. 

Conclusion

Higher levels of circulating EPCs are associated with less in-stent proliferation, whereas the proatherogenic IL-18 correlates with LLL. Incomplete stent coverage does not trigger in-stent proliferation. Hence, neointimal growth seems to be rather stimulated by processes of the inner media. 

Acknowledgments. We thank the cath lab personnel and Annett Schmidt from the research laboratories of the Cardiology Division at the University of Jena for assisting the study team. 

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From the 1st Clinic of Medicine, Division of Cardiology, University Hospital of Jena, Germany.

Disclosure: The authors have completed and returned the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Goebel reports grants from Abbott Vascular, B. Braun, and Boston Scientific; personal fees from Actelion and Boston Scientific. Dr Poerner reports research grants from Abbott Vascular, B. Braun, and Boston Scientific in addition to local institutional funding; personal fees from Daiichi Sankyo Europe GmbH, MSD Sharp & Dohme GmbH, Bayer Vital GmbH, Boehringer Ingelheim GmbH, Biotronik GmbH, Pfizer GmbH, Boston Scientific Medizintechnik GmbH, and Nicolai Medizintechnik GmbH. Dr Otto reports grants from Abbott Vascular, B. Braun, and Boston Scientific; personal fees from Daiichi Sankyo Europe GmbH, MSD Sharp & Dohme GmbH; other compensation from Bayer Vital GmbH. Dr Jung reports grants from Abbott Vascular, B. Braun, Boston Scientific, and Novo Nordisk; grants and personal fees from Actelion, Bayer Healthcare; personal fees from Boehringer Ingelheim, Vifor Pharma, Pfizer, Abbott Vascular, Boston Scientific, Zoll Lifebridge, and Novartis; personal fees from Terumo, grants from Swedish Heart Lung Foundation and the Swedish Research Council, outside the submitted work. Dr Figulla is a member of the European Advisory Board from Boston Scientific, and reports grants from Abbott Vascular, B. Braun, and Boston Scientific.

Manuscript submitted February 4, 2014, provisional acceptance given February 14, 2014, final version accepted February 25, 2014.

Address for correspondence:  Dr med Sylvia Otto, 1st Clinic of  Medicine, Division of Cardiology, University Hospital of Jena, Germany,  Erlanger Allee 101, 07747 Jena,  Germany. Email: sylvia.otto@med.uni-jena.de