Son of a Lesser God: The Case of Cell Therapy for Refractory Angina.

In the last decades, various non-pharmacological solutions have been tested on top of medical therapy for the treatment of patients affected by refractory angina (RA). Among these therapeutics, neuromodulation, external counter-pulsation and coronary sinus constriction have been recently introduced in the guidelines for the management of RA in United States and Europe. Notably and paradoxically, although a consistent body of evidence has proposed cell-based therapies (CT) as safe and salutary for RA outcome, CT has not been conversely incorporated into current international guidelines yet. As a matter of fact, published randomized controlled trials (RCT) and meta-analyses (MTA) cumulatively indicated that CT can effectively increase perfusion, physical function and well-being, thus reducing angina symptoms and drug assumption in RA patients. In this review, we (i) provide an updated overview of novel non-pharmacological therapeutics included in current guidelines for the management of patients with RA, (ii) discuss the Level of Evidence stemmed from available clinical trials for each recommended treatment, and (iii) focus on evidence-based CT application for the management of RA.

When Good Guys Turn Bad: Bone Marrow's and Hematopoietic Stem Cells' Role in the Pathobiology of Diabetic Complications.

Diabetes strongly contributes to the development of cardiovascular disease, the leading cause of mortality and morbidity in these patients. It is widely accepted that hyperglycemia impairs hematopoietic stem/progenitor cell (HSPC) mobilization from the bone marrow (BM) by inducing stem cell niche dysfunction. Moreover, a recent study demonstrated that type 2 diabetic patients are characterized by significant depletion of circulating provascular progenitor cells and increased frequency of inflammatory cells. This unbalance, potentially responsible for the reduction of intrinsic vascular homeostatic capacity and for the establishment of a low-grade inflammatory status, suggests that bone BM-derived HSPCs are not only victims but also active perpetrators in diabetic complications. In this review, we will discuss the most recent literature on the molecular mechanisms underpinning hyperglycemia-mediated BM dysfunction and differentiation abnormality of HSPCs. Moreover, a section will be dedicated to the new glucose-lowering therapies that by specifically targeting the culprits may prevent or treat diabetic complications.

New Strategies to Enhance Myocardial Regeneration: Expectations and Challenges from Preclinical Evidence.

Nowadays, cardiac regeneration is an emerging topic in the cardiovascular field because of the compelling need for effective therapies for repairing or replacing cardiac tissue damaged by pathological or physiological conditions. Indeed, irreversible myocardial remodeling which follows acute myocardial infarction represents a serious burden of this century. In this context, a great improvement in pharmacological and interventional techniques is accompanied by a big challenge of cardiac regenerative medicine. In the last 20 years, several clinical trials tried to investigate the role of different types of stem cells in promoting cardiac repair. However, the promising results obtained in the preclinical trials have not yet been reproduced in patients. Thus, the development of novel strategies to improve stem cell efficiency became imperative. Here, an overview of the more recent cell types proposed for cardiac regeneration is presented, together with the most interesting approaches to enhance cell regenerative potential as well as cell-free approaches.

G-CSF for Extensive STEMI.

RATIONALE: In the exploratory Phase II STEM-AMI (Stem Cells Mobilization in Acute Myocardial Infarction) trial, we reported that early administration of G-CSF (granulocyte colony-stimulating factor), in patients with anterior ST-segment-elevation myocardial infarction and left ventricular (LV) dysfunction after successful percutaneous coronary intervention, had the potential to significantly attenuate LV adverse remodeling in the long-term. OBJECTIVE: The STEM-AMI OUTCOME CMR (Stem Cells Mobilization in Acute Myocardial Infarction Outcome Cardiac Magnetic Resonance) Substudy was adequately powered to evaluate, in a population showing LV ejection fraction ≤45% after percutaneous coronary intervention for extensive ST-segment-elevation myocardial infarction, the effects of early administration of G-CSF in terms of LV remodeling and function, infarct size assessed by late gadolinium enhancement, and myocardial strain. METHODS AND RESULTS: Within the Italian, multicenter, prospective, randomized, Phase III STEM-AMI OUTCOME trial, 161 ST-segment-elevation myocardial infarction patients were enrolled in the CMR Substudy and assigned to standard of care (SOC) plus G-CSF or SOC alone. In 119 patients (61 G-CSF and 58 SOC, respectively), CMR was available at baseline and 6-month follow-up. Paired imaging data were independently analyzed by 2 blinded experts in a core CMR lab. The 2 groups were similar for clinical characteristics, cardiovascular risk factors, and pharmacological treatment, except for a trend towards a larger infarct size and longer symptom-to-balloon time in G-CSF patients. ANCOVA showed that the improvement of LV ejection fraction from baseline to 6 months was 5.1% higher in G-CSF patients versus SOC (P=0.01); concurrently, there was a significant between-group difference of 6.7 mL/m2 in the change of indexed LV end-systolic volume in favor of G-CSF group (P=0.02). Indexed late gadolinium enhancement significantly decreased in G-CSF group only (P=0.04). Moreover, over time improvement of global longitudinal strain was 2.4% higher in G-CSF patients versus SOC (P=0.04). Global circumferential strain significantly improved in G-CSF group only (P=0.006). CONCLUSIONS: Early administration of G-CSF exerted a beneficial effect on top of SOC in patients with LV dysfunction after extensive ST-segment-elevation myocardial infarction in terms of global systolic function, adverse remodeling, scar size, and myocardial strain. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01969890.

Abnormal DNA Methylation Induced by Hyperglycemia Reduces CXCR 4 Gene Expression in CD 34+ Stem Cells.

Background CD 34+ stem/progenitor cells are involved in vascular homeostasis and in neovascularization of ischemic tissues. The number of circulating CD 34+ stem cells is a predictive biomarker of adverse cardiovascular outcomes in diabetic patients. Here, we provide evidence that hyperglycemia can be "memorized" by the stem cells through epigenetic changes that contribute to onset and maintenance of their dysfunction in diabetes mellitus. Methods and Results Cord-blood-derived CD 34+ stem cells exposed to high glucose displayed increased reactive oxygen species production, overexpression of p66shc gene, and downregulation of antioxidant genes catalase and manganese superoxide dismutase when compared with normoglycemic cells. This altered oxidative state was associated with impaired migration ability toward stromal-cell-derived factor 1 alpha and reduced protein and mRNA expression of the C-X-C chemokine receptor type 4 ( CXCR 4) receptor. The methylation analysis by bisulfite Sanger sequencing of the CXCR 4 promoter revealed a significant increase in DNA methylation density in high-glucose CD 34+ stem cells that negatively correlated with mRNA expression (Pearson r=-0.76; P=0.004). Consistently, we found, by chromatin immunoprecipitation assay, a more transcriptionally inactive chromatin conformation and reduced RNA polymerase II engagement on the CXCR 4 promoter. Notably, alteration of CXCR 4 DNA methylation, as well as transcriptional and functional defects, persisted in high-glucose CD 34+ stem cells despite recovery in normoglycemic conditions. Importantly, such an epigenetic modification was thoroughly confirmed in bone marrow CD 34+ stem cells isolated from sternal biopsies of diabetic patients undergoing coronary bypass surgery. Conclusions CD 34+ stem cells "memorize" the hyperglycemic environment in the form of epigenetic modifications that collude to alter CXCR 4 receptor expression and migration.

Linking cell function with perfusion: insights from the transcatheter delivery of bone marrow-derived CD133+ cells in ischemic refractory cardiomyopathy trial (RECARDIO).

BACKGROUND: Cell therapy with bone marrow (BM)-derived progenitors has emerged as a promising therapeutic for refractory angina (RA) patients. In the present study, we evaluated the safety and preliminary efficacy of transcatheter delivery of autologous BM-derived advanced therapy medicinal product CD133+ cells (ATMP-CD133) in RA patients, correlating perfusion outcome with cell function. METHODS: In the phase I "Endocavitary Injection of Bone Marrow Derived CD133+ Cells in Ischemic Refractory Cardiomyopathy" (RECARDIO) trial, a total of 10 patients with left ventricular (LV) dysfunction (ejection fraction ≤ 45%) and evidence of reversible ischemia, as assessed by single-photon emission computed tomography (SPECT), underwent BM aspiration and fluoroscopy-based percutaneous endomyocardial delivery of ATMP-CD133. Patients were evaluated at 6 and 12 months for safety and preliminary efficacy endpoints. ATMP-CD133 samples were used for in vitro correlations. RESULTS: Patients were treated safely with a mean number of 6.57 ± 3.45 ×  106 ATMP-CD133. At 6-month follow-up, myocardial perfusion at SPECT was significantly ameliorated in terms of changes in summed stress (from 18.2 ± 8.6 to 13.8 ± 7.8, p = 0.05) and difference scores (from 12.0 ± 5.3 to 6.1 ± 4.0, p = 0.02) and number of segments with inducible ischemia (from 7.3 ± 2.2 to 4.0 ± 2.7, p = 0.003). Similarly, Canadian Cardiovascular Society and New York Heart Association classes significantly improved at follow-up vs baseline (p ≤ 0.001 and p = 0.007, respectively). Changes in summed stress score changes positively correlated with ATMP-CD133 release of proangiogenic cytokines HGF and PDGF-bb (r = 0.80, p = 0.009 and r = 0.77, p = 0.01, respectively) and negatively with the proinflammatory cytokines RANTES (r = - 0.79, p = 0.01) and IL-6 (r = - 0.76, p = 0.02). CONCLUSION: Results of the RECARDIO trial suggested safety and efficacy in terms of clinical and perfusion outcomes in patients with RA and LV dysfunction. The observed link between myocardial perfusion improvements and ATMP-CD133 secretome may represent a proof of concept for further mechanistic investigations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02059681 . Registered 11 February 2014.

Cell therapy for heart disease after 15 years: Unmet expectations.

Over the past two decades cardiac cell therapy (CCT) has emerged as a promising new strategy to cure heart diseases at high unmet need. Thousands of patients have entered clinical trials for acute or chronic heart conditions testing different cell types, including autologous or allogeneic bone marrow (BM)-derived mononuclear or selected cells, BM- or adipose tissue-derived mesenchymal cells, or cardiac resident progenitors based on their potential ability to regenerate scarred or dysfunctional myocardium. Nowadays, the original enthusiasm surrounding the regenerative medicine field has been cushioned by a cumulative body of evidence indicating an inefficient or modest efficacy of CCT in improving cardiac function, along with the continued lack of indisputable proof for long-term prognostic benefit. In this review, we have firstly comprehensively outlined the positive and negative results of cell therapy studies in patients with acute myocardial infarction, refractory angina and chronic heart failure. Next, we have discussed cell therapy- and patient-related variables (e.g. cell intrinsic and extrinsic characteristics as well as criteria of patient selection and proposed methodologies) that might have dampened the efficacy of past cell therapy trials. Finally, we have addressed critical factors to be considered before embarking on further clinical trials.

Preferential myofibroblast differentiation of cardiac mesenchymal progenitor cells in the presence of atrial fibrillation.

Atrial fibrillation (AF) is characterized by electrical, contractile, and structural remodeling mediated by interstitial fibrosis. It has been shown that human cardiac mesenchymal progenitor cells (CMPCs) can be differentiated into endothelial, smooth muscle, and fibroblast cells. Here, we have investigated, for the first time, the contribution of CMPCs in the fibrotic process occurring in AF. As expected, right auricolae samples displayed significantly higher fibrosis in AF vs control (CTR) patients. In tissue samples of AF patients only, double staining for c-kit and the myofibroblast marker α-smooth muscle actin (α-SMA) was detected. The number of c-kit-positive CMPC was higher in atrial subepicardial regions of CTR than AF cells. AF-derived CMPC (AF-CMPC) and CTR-derived CMPC (Ctr-CMPC) were phenotypically similar, except for CD90 and c-kit, which were significantly more present in AF and CTR cells, respectively. Moreover, AF showed a lower rate of population doubling and fold enrichment vs Ctr-CMPC. When exogenously challenged with the profibrotic transforming growth factor-ß1 (TGF-ß1), AF-CMPC showed a significantly higher nuclear translocation of SMAD2 than Ctr-CMPC. In addition, TGF-ß1 treatment induced the upregulation of COL1A1 and COL1A2 in AF-CMPC only. Further, both a marked production of soluble collagen and α-SMA upregulation have been observed in AF-CMPC only. Finally, electrophysiological studies showed that the inwardly rectifying potassium current (IK1) was evenly present in AF- and Ctr-CMPC in basal conditions and similarly disappeared after TGF-ß1 exposure. All together, these data suggest that AF steers the resident atrial CMPC compartment toward an electrically inert profibrotic phenotype.

Cell Therapy for Refractory Angina: A Reappraisal.

Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. Although positive results in terms of surrogate endpoints have been suggested by early and phase II clinical studies as well as by meta-analyses, the more recent reports lacked the provision of definitive response in terms of hard clinical endpoints. Regrettably, pivotal trials designed to conclusively determine the efficacy of cell-based therapeutics in such a challenging clinical condition are therefore still missing. Considering this, a comprehensive reappraisal of cardiac cell-based therapy role in RA seems warranted and timely, since a number of crucial cell- and patient-related aspects need to be systematically analysed. As an example, the large variability in efficacy endpoint selection appears to be a limiting factor for the advancement of cardiac cell-based therapy in the field. This review will provide an overview of the key elements that may have influenced the results of cell-based trials in the context of RA, focusing in particular on the understanding at which the extent of angina-related endpoints may predict cell-based therapeutic efficacy.

BM ageing: Implication for cell therapy with EPCs.

The bone marrow (BM) is a well-recognized source of stem/progenitor cells for cell therapy in cardiovascular diseases (CVDs). Preclinical and clinical studies suggest that endothelial progenitor cells (EPCs) contribute to reparative process of vascular endothelium and participate in angiogenesis. As for all organs and cells across the lifespan, BM and EPCs are negatively impacted by ageing due to microenvironment modifications and EPC progressive dysfunctions. The encouraging results in terms of neovascularization observed in young animals after EPC administration were mitigated in aged patients treated for ischemic CVDs. The limited efficacy of EPC-based therapy in clinical setting might be ascribed at least partly to ageing. In this review, we comprehensively discussed the age-related changes of BM and EPCs and their implication for cardiovascular cell-therapies. Finally, we examined alternative approaches under investigation to enhance EPC potency.

Full GMP-compliant validation of bone marrow-derived human CD133(+) cells as advanced therapy medicinal product for refractory ischemic cardiomyopathy.

According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133(+) cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 10(6) of CD133(+) cells (range 2.85 × 10(6)-30.84 × 10(6)), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133(+) cells of 90,60% (range 81,40%-96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 10(6) cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).

Novel Application of 3-Dimensional Real-Time Cardiac Imaging to Guide Stem Cell-Based Therapy.

Stem cell-based therapy is an emerging treatment for refractory ischemic cardiomyopathy. The transendocardial approach represents the most attractive method that allows direct percutaneous injections of the cell product into the ischemic territories. This clinical case shows a novel strategy designed to optimize cell endocardial delivery, based on the implementation of the 3-dimensional electroanatomical map with the intracardiac-echocardiographic reconstruction of the left ventricle, using acquired multiple slice recordings. Combined imaging was efficacious to detail the anatomical and functional characteristics of the target areas and to guide cell delivery supported by direct real-time visualization of the needle to improve procedural effectiveness and safety.

Characterization of the Pall Celeris system as a point-of-care device for therapeutic angiogenesis.

BACKGROUND AIMS: The Pall Celeris system is a filtration-based point-of-care device designed to obtain a high concentrate of peripheral blood total nucleated cells (PB-TNCs). We have characterized the Pall Celeris-derived TNCs for their in vitro and in vivo angiogenic potency. METHODS: PB-TNCs isolated from healthy donors were characterized through the use of flow cytometry and functional assays, aiming to assess migratory capacity, ability to form capillary-like structures, endothelial trans-differentiation and paracrine factor secretion. In a hind limb ischemia mouse model, we evaluated perfusion immediately and 7 days after surgery, along with capillary, arteriole and regenerative fiber density and local bio-distribution. RESULTS: Human PB-TNCs isolated by use of the Pall Celeris filtration system were shown to secrete a panel of angiogenic factors and migrate in response to vascular endothelial growth factor and stromal-derived factor-1 stimuli. Moreover, after injection in a mouse model of hind limb ischemia, PB-TNCs induced neovascularization by increasing capillary, arteriole and regenerative fiber numbers, with human cells detected in murine tissue up to 7 days after ischemia. CONCLUSIONS: The Pall Celeris system may represent a novel, effective and reliable point-of-care device to obtain a PB-derived cell product with adequate potency for therapeutic angiogenesis.

The CD133+ cell as advanced medicinal product for myocardial and limb ischemia.

Ischemic diseases are the major cause of death and morbidity in Western countries. In the last decade, cell therapy has been suggested to be a promising treatment both in acute/chronic myocardial and peripheral ischemia. Different cell lineages have been tested, including endothelial progenitor cells. A subpopulation of bone marrow-derived immature ECPs, expressing the highly conserved stem cell glycoprotein antigen prominin-1 or CD133 marker, was shown to possess pro-angiogenic and antiapoptotic effects on ischemic tissues. The mechanisms implicated in CD133+ cells ability to contribute to neovascularization processes have been attributed to their ability to directly differentiate into newly forming vessels and to indirectly activate pro-angiogenic signaling by paracrine mechanisms. A large body of in vivo experimental evidences has demonstrated the potential of CD133+ cells to reverse ischemia. Moreover, several clinical trials have reported promising beneficial effects after infusion of autologous CD133+ into ischemic heart and limbs exploiting various delivery strategies. These trials have contributed to characterize the CD133+ manufacturing process as an advanced cell product (AMP). The aim of this review is to summarize available experimental and clinical data on CD133+ cells in the context of myocardial and peripheral ischemia, and to focus on the development of the CD133+ cell as an anti-ischemic AMP.

[Role of bone marrow-derived CD133+ stem cells in cardiac regeneration: from experimental to clinical trials]. / Ruolo delle cellule staminali di origine midollare CD133+ nella rigenerazione miocardica: dalle evidenze sperimentali ai trial clinici sull'uomo.

Recent advances in coronary revascularization techniques have improved the outcomes of ischemic heart disease in both acute and chronic settings. As a drawback, an increase in patients with an advanced stage of ischemic cardiomyopathy refractory to optimal medical treatment has been observed. Among the therapeutic alternatives under investigation, cell therapy showed considerable anti-ischemic potential. Although several types of cells have been used, bone marrow-derived endothelial progenitor cells are among the most appealing therapeutic agents due to their angiogenic properties. In particular, endothelial progenitors expressing the transmembrane protein CD133 have been in vitro and in vivo extensively characterized and clinically tested. The aim of this paper is to discuss the translational process that allowed the clinical application of CD133+ endothelial progenitor cells in the context of ischemic cardiomyopathy.

Emerging treatment options for refractory angina pectoris: ranolazine, shock wave treatment, and cell-based therapies.

A challenge of modern cardiovascular medicine is to find new, effective treatments for patients with refractory angina pectoris, a clinical condition characterized by severe angina despite optimal medical therapy. These patients are not candidates for surgical or percutaneous revascularization. Herein we review the most up-to-date information regarding the modern approach to the patient with refractory angina pectoris, from conventional medical management to new medications and shock wave therapy, focusing on the use of endothelial precursor cells (EPCs) in the treatment of this condition. Clinical limitations of the efficiency of conventional approaches justify the search for new therapeutic options. Regenerative medicine is considered the next step in the evolution of organ replacement therapy. It is driven largely by the same health needs as transplantation and replacement therapies, but it aims further than traditional approaches, such as cell-based therapy. Increasing knowledge of the role of circulating cells derived from bone marrow (EPCs) on cardiovascular homeostasis in physiologic and pathologic conditions has prompted the clinical use of these cells to relieve ischemia. The current state of therapeutic angiogenesis still leaves many questions unanswered. It is of paramount importance that the treatment is delivered safely. Direct intramyocardial and intracoronary administration has demonstrated acceptable safety profiles in early trials, and may represent a major advance over surgical thoracotomy. The combined efforts of bench and clinical researchers will ultimately answer the question of whether cell therapy is a suitable strategy for treatment of patients with refractory angina.