Autologous CD133 + Cells and Laser Revascularization in patients with severe Ischemic Cardiomyopathy

We tested the hypothesis that targeted TMLR combined with intramyocardial injection of autologous CD 133+ progenitor cells is safe and feasible in patients with chronic ischemic cardiomyopathy (ICM) and no revascularization options. Eight male patients (age 62 ± 2.4 years) with multivessel severe ischemic heart disease and no revascularization options were enrolled. Autologous CD 133 + endothelial progenitor cells were derived and purified from the bone marrow on the day of surgery using the clinical-grade closed CliniMACS system. Using a lateral thoracotomy approach, TMLR was performed, followed by transmyocardial transplantation of purified CD133 + cells (mean number of transplanted cells: 12.5 × 106) in the region surrounding the TMLR sites. These sites were selected based on ischemia on pre-procedure perfusion imaging. We performed clinical and myocardial perfusion imaging pre-procedure and then at 6- and 12-month follow-up. No major complications or death occurred during the procedure or during the peri-operative hospital stay. One patient died of cardiac cause 6 months post-procedure. There was a reported short-term improvement in anginal and heart failure symptoms and a modest reduction in the ischemic score as assessed by perfusion imaging. Our phase 1 clinical study examining the combination therapy of targeted transmyocardial laser revascularization therapy and autologous CD133 + endothelial progenitor cells in patients with chronic ICM and no revascularization options demonstrates the feasibility and short-term safety of this combined approach and warrants future larger phase 2 randomized clinical studies.


Introduction
Due to limited organ availability for heart transplantation, alternative therapeutic strategies in patients with chronic ischemic cardiomyopathy (ICM) and no revascularization options have emerged. Stem-cell therapy for ICM has been promising in potentiating myocardial functional recovery [1]. Bone-marrow (BM)-derived cells are the most frequent cell type used, due to ease of procurement and minimal exvivo processing [2]. CD133 + BM-derived hematopoietic stem cells are an established cell source shown to be beneficial when administered in ICM patients [1]. Transmyocardial laser revascularization (TMLR) is another innovative concept shown to potentially induce angiogenesis, primarily through paracrine effects on angiogenic stem cells and maintaining cell retention after transplantation [3][4][5]. The combination of TMLR and subsequent cell therapy was first implemented in a small feasibility study; however, long-term safety and outcomes have not been explored [6].
Herein we report the application of TMLR, and simultaneous intramyocardial infusion of bone marrow-derived autologous CD133 + selected cell product in 8 patients, with severe ICM and no percutaneous or surgical revascularization options, utilizing a lateral thoracotomy approach. The primary outcome of this study is the safety and feasibility of this approach.

Methods
Between July 2016 and October 2019, 8 patients (all males) with end-stage ICM were enrolled in our prospective phase I single-center clinical trial. Patient characteristics and comorbidities are summarized in Table 1. All procedural steps were logistically feasible, and all patients received the cell therapy within 12 h of BM aspiration and less than 1 h after CD133 + stem cell purification, as per the study protocol.
The inclusion and exclusion criteria for the study cohort are outlined in Supplemental Table 1. All patients underwent a baseline evaluation of their functional status and cardiac function using the Kansas City Cardiomyopathy Questionnaire (KCCQ)-12, Seattle Angina Questionnaire (SAQ), and the New York Heart Association heart failure symptoms score. Cardiac function was assessed using a transthoracic echocardiogram. Myocardial ischemia was confirmed at baseline using myocardial single-photon emission computerized tomography (SPECT) imaging. Follow-up evaluations for clinical and functional status, echocardiography, and myocardial perfusion were performed at 6-and 12-months post-procedure. Statistical analyses: Throughout the manuscript, data are expressed as mean ± standard error of the means (SEM). Differences were analyzed using the unpaired Student t-test or analysis of variance (one-way ANOVA) as appropriate. A value of P < 0.05 was considered significant. Statistical analyses were performed using the Prism 9 package (GraphPad, La Jolla, CA) and SPSS version 25 (SPSS, IBM Corp, Armonk, NY).

Technique
On the day of the procedure, patients underwent bone marrow aspiration and CD133 + cells were processed and separated using the clinical-grade closed CliniMACS system (Miltenyi Biotech, Germany) [7]. TMLR was performed using a Heart Laser CO2 Transmyocardial Revascularization System (Novadaq Technologies Inc., BC, Canada) through a lateral thoracotomy followed by transmyocardial transplantation of purified CD133 + cells (mean number of transplanted cells 12.5 × 10 6 cells). Cell viability was greater than 85% and cell purity was greater than 75%. The number of TMLR channels and the territories of the heart where the TMLR and CD 133 + injections were performed are summarized in Table 2. These territories were selected based on the presence of ischemia and viable myocardium on the pre-procedural nuclear stress test. CD133 + cells were injected in the immediate proximity of the TMLR channels using a 25G needle. We obtained serial cardiac enzymes and a 48-h echocardiogram during the postoperative period.  The primary endpoint was procedural feasibility and safety defined as peri-procedural myocardial infarction, infection, arrhythmia, or cardiogenic shock. Secondary exploratory endpoints included 6-and 12-month changes in left ventricular function and myocardial ischemia. Secondary clinical endpoints included major adverse cardiac or cerebrovascular events, or hospitalization for adverse cardiac events at 6-and 12-months. The study protocol was approved by the Institutional Review Board at the University of Kentucky and was performed under an investigational new drug (IND) approval from the FDA. The study was registered in clinicaltrials.gov under the number: NCT03043742. All patients gave written and informed consent to participate in the trial.

Results
No inpatient procedure-related complications were observed. We observed an elevation in troponin T (0.38 ± 0.07 ng/ml) and creatine kinase (mean 1008.8 ± 270.1 mcg/L) peaking 8 h post-procedure, consistent with cardiac surgery and TMLR. Enzymes trended down and were within the normal range at 1-week follow-up. During short-term follow-up, one patient suffered from heart failure-related hospitalization 1-week following the procedure and was managed according to the standard of care, and discharged home (summarized in Table 3). On long-term follow-up, three patients had severe adverse events classified as secondary endpoint events. One patient died from ventricular fibrillation arrest at 6 months. Two patients had HF-related hospitalizations at 6and 12-months, respectively. A short-term improvement in heart failure symptoms was observed (mean NYHA 2.3 ± 0.1 pre-infusion to 1.8 ± 0.1 at 3-month post-infusion, P < 0.05 for baseline vs. 3 months); however, this observed benefit appeared to fade by 12-months of follow-up (2.3 ± 0.1 at 12 months of follow up, P = 0.92). Similarly, the Seattle Angina Questionnaire (pre-infusion score of 19.5 ± 2.1 to 24.7 ± 2.5 at 6-month and 22.9 ± 2.8 at 12-month followup, P = 0.54) (Fig. 1). However, these trends did not reach statistical significance. An improvement in the quality of life was observed during follow-up with an increase in mean KCCQ-12 score (pre-infusion score of 42 ± 4.8 to 47 ± 4.3 at 12 months after surgery, P = 0.82). The mean left ventricular ejection fraction was 42% pre-infusion and 46% at 12 months after surgery (P = 0.7). The assessment of scar size and amount of ischemia using nuclear stress imaging did not show a significant difference (scar: pre: 23.7 ± 5.9 and post: 24.2 ± 5.3, P = 0.95; ischemia: pre: 3.5 ± 1.9 and post: 5.5 ± 2.6, P = 0.55) between baseline and follow-up studies (Fig. 1). These data suggest that the infusion of CD133 proangiogenic cells at the time of TMLR is clinically feasible, safe, and may improve heart failure symptoms although the improvement is transient. It is important to note that due to the fact that our study is primarily a safety and feasibility study, we could not separate the effects of TMLR for those of cell therapy, and future studies focused on the efficacy of these components are warranted.

Discussion
Our single-center phase I study demonstrates the safety and feasibility of the combined stem cell/TMLR approach in patients with severe ischemic heart disease. The rationale for this combination is that TMLR elicits a paracrine effect resulting in the release of homing cytokines, thus enhancing the retention of transplanted cells and their therapeutic benefit [8]. Our data suggest that this approach is clinically feasible and can be safely performed in humans. This prospective study was primarily designed as a safety and feasibility trial without a control group and therefore cannot reach conclusions about the efficacy of this approach.
The exact mechanism behind cardiac recovery after stem cell therapy is still unknown. Proposed mechanisms include modulation of inflammation and reducing adverse cardiac remodeling through the effect of stem cells on extracellular matrix deposition and remodeling. Among these proposed mechanisms, angiogenesis is the most clinically relevant. Studies examining the role of endothelial progenitor cells in patients with end-stage ischemic heart disease showed clinically relevant improvement in symptoms and quality of life [9]. Our study could provide a step further in this therapeutic pathway. By combining TMLR and CD133 cells, signals for cell homing and retention are enhanced, together with pro-angiogenic cytokine release from injured cardiac Heart failure related hospitalization (6 months) Ventricular fibrillation and death (6 months) Heart failure-related hospitalization (12 months) tissue. Combined, these factors could provide important signals for retention and vessel formation for the transplanted CD133 + stem cells [10]. Therefore, our safety findings are significant as they pave the way for future clinical randomized studies exploring this therapeutic approach in a larger patient population.
Our study was not powered to detect significant changes in clinical outcomes, however, we noted trends towards improvement in heart failure in the study population. These changes were paralleled with modest improvement in ejection fraction. Interestingly, the improvement was transient in some of the outcomes measured which is in agreement with what has been observed in human studies of cell therapy and calls for future studies to enhance the long-term effects of cardiac cell therapy.

Conclusion
In this single-center phase I trial, we established a safe and clinically feasible protocol of combining TMLR and CD133 + cardiac cell therapy in patients with ischemic heart failure and no revascularization options. This novel approach resulted in a transient relief of heart failure symptoms and improvement in quality of life. Future randomized controlled studies examining this approach in large cohorts and different clinical scenarios are warranted.