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Endothelial Progenitor Cell Function in Patients With Coronary Chronic Total Occlusion and its Relationship With Collateral Circulation
Abstract
Aim. To evaluate the relationship between endothelial progenitor cell (EPC) count and function and collateral circulation in coronary chronic total occlusions (CTOs). Methods. A total of 20 consecutive patients with successfully treated CTO lesions were included during a period of 12 months. EPC count and function were evaluated by flow cytometry and colony-forming unit (CFU) analysis at baseline (before percutaneous coronary intervention) and at 1-year follow-up. Patients were classified, according to Rentrop classification at the baseline angiography, as group 1 (Rentrop 3; n = 7) and group 2 (Rentrop <3; n = 13). Differences in EPC count and function were compared between groups. Results. The EPC count did not differ between the 2 groups, either at baseline or at follow-up. CFU was significantly lower at follow-up compared with baseline in the overall population (16.6 106/mL (IQR, 10.2-29.4 106/mL) vs 7.1 106/mL (IQR, 5.3-25.0 106/mL); P=.046). Group 1 had both higher basal and follow-up CFU values compared with group 2 (35.4 106/mL (IQR, 21.5-41.8 106/mL) vs 13.3 106/mL (IQR, 6.9-17.5 106/mL) and 32.1 106/mL (IQR, 13.9-40.5 106/mL) vs 5.9 106/mL (IQR, 4.4-9.8 106/mL), respectively; P=.01 for both). By linear regression analysis, Rentrop grade 3 flow was an independent predictor of both basal and follow-up CFU levels (odds ratio, 3.66; 95% confidence interval, 6.41-29.69; P<.01; and odds ratio, 5.24; 95% confidence interval, 9.78-25.85; P<.01, respectively). Conclusion. Patients with Rentrop grade 3 collateral circulation exhibited higher EPC activity at baseline and at 1-year follow-up compared with those who had reduced collateral circulation. The role of this higher EPC activity in determining clinical endpoint should be investigated in a larger study.
Key words: collateral circulation, endothelial progenitor cell count
Introduction
The development of collateral circulation is of great clinical importance in coronary chronic total occlusions (CTOs) given that it decreases ischemic burden, improves systolic function, and avoids cardiac injury.1-3 Endothelial progenitor cells (EPCs) can play important roles in this setting, favoring the generation of collateral vessels.3 These cells comprise a heterogeneous population of immature circulating blood cells, initially mobilized and released from the bone marrow,4 and though rare (representing approximately 0.01%-0.0001% of the mononuclear fraction of peripheral blood), they are important for vascular homeostasis, regeneration of injured endothelium, and neovascularization of ischemic tissue. They are also important for cardiovascular health starting from early-stage atherosclerosis and continuing until recovery from CTO. Healthy people at low risk for atherosclerosis have higher levels of circulating EPCs than those at high risk. Moreover, the EPCs in high-risk individuals become senescent more rapidly than those in low-risk individuals.5
We aimed to determine the number of EPC colony-forming units (CFUs) in patients with CTOs with various degrees of collateral vascularization prior to and 1 year following successful recanalization by percutaneous coronary intervention (PCI) and its relationship with the baseline clinical and angiographic characteristics of the patient.
Methods
Patient selection and follow-up. In this single-center, observational, prospective substudy of a larger study, all consecutive patients who underwent an attempted recanalization of a CTO by PCI were screened. Subjects with successful intervention were prospectively included according to inclusion/exclusion criteria. Successful CTO-PCI was defined as the recanalization of the lesion with residual stenosis of <30% and Thrombolysis in Myocardial Infarction (TIMI) flow grade 3.2 CTO-PCI was performed using the following CTO crossing strategies when appropriate: antegrade wire escalation; antegrade dissection/re-entry; and retrograde wire escalation.
Occlusion duration was evaluated considering previous coronary angiographies, date of a prior myocardial infarction, or onset of symptoms.6-8 All patients were symptomatic for angina or had documented ischemia and viability in myocardial regions supplied by the CTO vessel. Patients with contraindications to antiplatelet therapy, known or suspected infection, inflammatory, autoimmune disease, and connective tissue disorders, were excluded.9 For the CTO-PCI, the second-generation zotarolimus-eluting Resolute Integrity stent (Medtronic) was used in all patients. Following PCI, all patients were prescribed clopidogrel for at least 6 months and lifelong aspirin.
The follow-up protocol included a 12-month postprocedure visit and a coronary angiography in order to confirm the persistence of a proper CTO-PCI result after hospital discharge. The study was approved by our institutional ethics committee and complied with the principles of the Declaration of Helsinki. All patients signed informed consent for the procedure(s) and for the research use of their anonymized data.
Angiographic assessment of collaterals. Views with the least foreshortening of the collateral connection were selected for the angiographic diagnostic analysis at a centralized core laboratory. Collateral filling of the recipient artery was evaluated according to the Rentrop classification, which consists of 4 classes: 0 = no filling of any collateral; 1 = filling of sidebranches of the epicardial artery; 2 = partial filling of the epicardial vessel; and 3 = complete filling of recipient artery by collaterals.10 For the purpose of this report, we divided our population into two groups; those with Rentrop grade 3 (group 1) and those with Rentrop 1 or 2 (group 2).
Two independent observers, blinded to clinical characteristics, assessed the Rentrop grade. In case of disagreement, a consensus was reached by a third independent observer.
EPC flow-cytometry. For flow-cytometric determination of circulating endothelial progenitor cells, 50 mL of whole blood was labeled for 20-30 minutes at room temperature using manufacturer-recommended concentrations with antihuman- VE-Cadherin-PE (Becton Dickinson) and antihuman-AC133-APC (Miltenyi Biotec). Blood was drawn immediately before the CTO-PCI procedure and at the 1-year follow-up in all patients. Immunofluoroscent cell staining was performed by incubating peripheral blood mononuclear cells for 15 minutes with the following: the hematopoietic progenitor cell marker fluoresceinisothiocyanate-CD34 (R&D Systems); the immature hematopoietic cell marker phycoerythrin-cyanine5 conjugate-CD133 (Miltenyi Biotec); and the endothelial cell receptor phycoerythrin-VEGFR-2 (R&D Systems), also known as kinase domain receptor (KDR). After staining, erythrocyte lysis, and fixation, cell fluorescence was measured by flow-cytometry. CD34+, CD133+ and CD34+CD133+ were considered progenitor precursor cells, while CD34+KDR+, CD133+KDR+, and CD34+CD133+KDR+ were considered endothelial progenitor cells.11-15
EPC function evaluation. EPC function was evaluated using CFU analysis. Peripheral blood mononuclear cells were isolated by Ficoll density-gradient centrifugation with Histopaque-1077 (Sigma Chemical Company) and washed 3 times in Dulbecco’s phosphate-buffered saline (GIBCO BRL Life Technologies). Recovered cells were resuspended in endothelial cell basal medium-2 (EBM-2) (Clonetivs), and placed in fibronectin-coated tissue culture flasks at 37 ºC. After 4 days, adherent cells were detached and 1 million of them were subsequently cultured for 3 days on fibronectin-coated plates with 1 mL of EBM-2. Plates were studied under phase-contrast microscopy.15-18 Colonies were counted by two independent investigators and expressed as the relative number of colonies per well (number of CFUs per well/number of cells per well). The CFU evaluation was performed prior to CTO-PCI (B-CFU) and at 1-year follow-up (F-CFU).
ΔCFU was computed as difference between B-CFU and F-CFU. ΔCFU% was defined as (ΔCFU/ B-CFU) x 100. The interobserver variability in CFU counts was 4.2 ± 1.4%.
Statistical analysis. Continuous data are expressed as mean ± standard deviation or median (interquartile range [IQR]), according to their distribution as assessed by the Kolmogorov-Smirnov test. Categorical data are presented as counts and percentages. Differences in continuous variables were evaluated by the Mann-Whitney U-test. The Chi-square test was used for comparisons of categorical variables. Changes in EPC parameters between baseline and follow-up were evaluated by paired test for repeated measurements.
A multivariable model was used to assess independent predictors of B-CFU and F-CFU by using as dependent variables age, hypertension, diabetes, and Rentrop class (3 vs others). Our approach was to include in the multivariate model all those variables that could be clinically related with the number of CFUs, such as age, occlusion time, diabetes mellitus, hypertension, and Rentrop grade. A 2-sided P-value <.05 indicated statistical significance in the multivariable analysis. Statistical analyses were performed by SPSS software, version 25.0 (SPSS).
Results
Baseline characteristics. During a 1-year period, 80 patients were screened, and 20 met the criteria for this study (Figure 1), Study participants were divided based on Rentrop grade, a method to rate the collateral filling of the recipient artery (Supplemental Figure S1). Those with Rentrop grade 3 (n = 7) comprised group 1 and all others (n = 13) comprised group 2 (7 participants [53.8%] with Rentrop grade 2 and 6 patients [46.2%] with Rentrop grade 1). Table 1 shows the baseline clinical characteristics. The 2 groups did not differ in any way except in major triglyceride levels, where the median was 2.2 mmol/L (IQR, 1.8-2.3 mmol/L) in group 1 vs 1.0 mmol/L (IQR, 0.5-1.6 mmol/L) for group 2 (P=.01). Although not statistically significant, group 1 had a higher proportion of participants with severe clinical angina or silent ischemia compared with group 2. The medical therapies also did not differ between the 2 groups except that group 2 had a higher proportion of participants treated with oral nitrates (46.2% in group 2 vs 0.0% in group 1; P=.05).
In subgroup analysis, those with Rentrop grade 1 did not differ from those with Rentrop grade 2 in baseline characteristics or medical therapies (Supplemental Table S1).
Angiographic and procedural variables. Across the study population, the CTO lesions were more frequently located at the right coronary artery, but the 2 groups did not differ in CTO coronary location, occlusion duration, or multivessel disease. CTO occurred simultaneously with Rentrop grade 3 in 2 patients; both were successfully treated with PCI. No significant difference in the rate of multivessel disease was observed between Rentrop 3 vs Rentrop <3. Two patients had 2 CTOs with Rentrop grade 3 in each lesion; both lesions were successfully treated with PCI in 1 patient while the other patient had concomitant moderate coronary disease and conservative management was adopted. In the Rentrop <3 group, 4 patients had concomitant intermediate multivessel coronary artery disease not requiring PCI. The mean number of stents implanted was 1.8 ± 1.0. Overlapping stents were implanted in 50% of the study population (n = 10). Size, length, and number of stents, procedural time, fluoroscopy time, and contrast volume did not differ between groups (Table 2). CTO-PCI was successfully performed by antegrade wire escalation technique in all patients with Rentrop < 3. In the Rentrop 3 group, the retrograde wire escalation technique was successfully accomplished in 2 patients and antegrade wire escalation was used in the remaining patients. In subgroup analysis, those with Rentrop grade 1 did not differ from those with Rentrop grade 2 in these angiographic variables (Supplemental Table S2).
Flow-cytometry and function evaluation of EPCs. Across the study population, the median number of CFUs was significantly lower at the 1-year follow-up vs baseline (7.1 x 106/mL [IQR, 5.3-25.0 x 106/mL] vs 16.6 x 106/mL [IQR, 10.2-29.4 x 106/mL]; P=.046).
Neither the number of progenitor precursor cells nor the number of EPCs differed between groups 1 and 2 at baseline and at follow-up (Table 3).
Both B-CFU and F-CFU values were greater in group 1 vs group 2 (Figure 2). Median B-CFU values were 35.4 x 106/mL (IQR, 21.5-41.8 x 106/mL) vs 13.3 x 106/mL (IQR, 6.9-17.5) x 106/mL, respectively (P=.01), whereas F-CFU values were 32.1 x 106/mL (IQR, 13.9-40.5 x 106/mL) vs 5.9 x 106/mL (IQR, 4.4-9.8 x 106/mL), respectively (P=.01). In subgroup analyses, both values were greater in those with Rentrop grade 2 vs those with Rentrop grade 1 (Supplemental Table S3). Neither ΔCFU nor ΔCFU% differed statistically between the 2 groups (Table 3).
Predictors of EPC function. Rentrop grade 3 emerged as an independent predictor of both basal and follow-up CFU levels (odds ratio, 3.66; 95% confidence interval, 6.41-29.69; P<.01 and odds ratio, 5.24; 95% confidence interval, 9.78-25.85; P<.01, respectively) (Table 4 and Table 5). Arterial hypertension predicted a greater F-CFU value (odds ratio, 11.88; 95% confidence interval, 3.43-20.33; P=.01).
Discussion
The major findings of our study are: (1) the number of EPCs does not differ between those with Rentrop grade 3 and those with any lower grade; and (2) an association was observed between Rentrop grade 3 collaterals and CFUs at both baseline and at 1-year follow-up.
This study shows that the number of EPCs does not differ based on Rentrop grade (however, CFU values were greater when Rentrop grade 3 was evidenced) and that grade 3 is an independent predictor of greater B-CFU and F-CFU values. Evidence revealing the relationship between EPC-mediated angiogenesis and coronary collateral development is scarce. Our findings support the results reported by Matsuo et al,19 who studied the effects of EPCs on collateral circulation in patients with single-vessel CTO, demonstrating that those with greater collateral circulation had greater EPC-CFU levels, reduced senescent cells, and higher levels of basic fibroblast growth factor.
Greater EPC function could be a factor contributing to greater collateral filling in group I. High EPC levels are known to indicate vascular regenerative abilities, and they correlate with the coronary collateral flow index, which is a measure of collateral support in coronary circulation.20-24 In the presence of CTO, collateral vessels could be native pre-existing collateral vessels or neocollateral vessels. Stimulated by hypoxia and ischemia, collateral growth occurs when native pre-existing small collateral vessels remodel under the pressure gradient generated by vessel narrowing or occlusion or by the growth of new capillary vessels (angiogenesis), the result of upregulation of various growth factors and their receptors. It is therefore not surprising that patients with higher Rentrop grades also have higher CFU levels.23 Higher rates of clinical angina or ischemia demonstrated by imaging could at least partly explain the differences observed in this study. Unfortunately, during the follow-up, we did not perform any non-invasive test to evaluate residual ischemia after CTO-PCI, which may help explain the difference observed in our group.
In this study population, significant differences in EPC levels were not found between the 2 groups, and we believe that this is related to the small sample size. Higher B-CFU and F-CFU values were found when collateral circulation was broader, and the number of CFUs was significantly lower at the 1-year follow-up than at baseline across the entire study population. These results suggest that the amelioration of myocardial perfusion could provide negative feedback to EPC function and balance endothelium homeostasis.
Additionally, changes in the number of CFUs and in CFU% did not differ between groups, but were associated with other clinical variables. The literature provides scarce data describing the correlation between plasma lipid and lipoproteins and the number of circulating EPCs.25-29 Lipid metabolism has been shown to impact EPC function, notably when cholesterol metabolism has been altered.15,30–32 Oxidized low-density lipoprotein seems to alter the number of CFUs and EPC differentiation by telomerase inactivation and the dephosphorylation of Akt (a serine-threonine protein kinase implied in EPC differentiation), respectively.33,34 Group 1 showed higher plasmatic levels of triglycerides compared with group 2, a finding that corroborates work by Pellegatta et al, who demonstrated that plasmic levels of high-density lipoprotein and triglycerides and of the ratio between total cholesterol and high-density lipoprotein cholesterol positively correlate with the number of CFUs.35 We found this as well; however, we did not find differences in low-density lipoprotein levels between the 2 groups. We note that it is difficult to find differences in a small population in which the majority are treated with potent statins.
It is interesting to note the difference in nitrate use between the 2 groups. No individual in group 1 was treated with nitrates, yet 6 of the individuals in group 2 (46.2%) were treated with an oral nitro derivate (P=.05). Many factors, such as the practice of the treating physician, the activity level of the individual, and other characteristics, could play a role in this scenario.
Growing evidence underlines the clinical relevance of EPCs as therapeutic agents and biomarkers of cardiovascular function, risk, and prognosis. Despite these promising data, EPC clinical applications, such as exogenous or autologous cell therapies, remain unclear. Data describing CTOs are scarce. One study showed that after successful recanalization of the occluded vessel and subsequent randomization to either stem cell therapy or a control therapy, hibernating myocardial segments were reduced by 31% in the treatment group but unchanged in controls. In spite of this, more evidence is required. With the hope that various EPC problems can be overcome early, real clinical applications such as biomarkers and regenerative cell agents are being developed.36,37
Study limitations. The primary limitation of this study is its small sample size. Our approach was to include every plausibly important variable to select variables to include in a multivariate model. Because the P-value is affected by sample size, our relatively small sample could make some variables appear to have substantive importance even when they are not significant. Therefore, we selected every variable that could be related to the number of CFUs (ie, age, occlusion time, diabetes mellitus, hypertension, and Rentrop grade). Our findings are hypothesis generating and should be confirmed using larger studies. Moreover, future studies should aim to include information on the extent of atherosclerotic disease in other arterial regions, serial angiograms to more precisely define the time of occlusion, as well as healthy controls. Each of these was beyond the scope of this study.
Conclusion
Patients with Rentrop grade 3 collateral circulation present major EPC activity at baseline and at 1-year follow-up compared with those who demonstrate poorer filling. Moreover, Rentrop grade 3 is an independent predictor of B-CFU and F-CFU values. Finally, EPC activity could be a key point for collateral vessel formation, which is a promising area of research that could improve quality of life for those with CTOs.
Affiliations and Disclosures
From the 1Cardiology Department, Clinic Cardiovascular Institute, Hospital Clínic, IDIBAPS, Barcelona, Spain; and the 2Cardiovascular Science Institute – ICCC, IIB-Sant Pau, Hospital de Sant Pau, Barcelona, Spain.
Disclosure: The authors have completed and returned the ICMJE Form for Disclosure of Potential Conflicts of Interest. The authors report no conflicts of interest regarding the content herein.
Manuscript accepted November 24, 2020.
Address for correspondence: Salvatore Brugaletta, MD, PhD, Villaroel Street 170, Barcelona, 08036, Spain. Email: sabrugaletta@gmail.com
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