Background: Coronary microvascular dysfunction results in angina and adverse outcomes in patients with evidence of ischemia and nonobstructive coronary artery disease; however, no specific therapy exists. CD34+ cell therapy increases microvasculature in preclinical models and improves symptoms, exercise tolerance, and mortality in refractory angina patients with obstructive coronary artery disease. The objective of this research was to evaluate the safety, tolerability, and efficacy of intracoronary CD34+ cell therapy in patients with coronary microvascular dysfunction. Methods: We conducted a 2-center, 20-participant trial of autologous CD34+ cell therapy (protocol CLBS16-P01; NCT03508609) in patients with ischemia and nonobstructive coronary artery disease with persistent angina and coronary flow reserve ≤2.5. Efficacy measures included coronary flow reserve, angina frequency, Canadian Cardiovascular Society angina class, Seattle Angina Questionnaire, SF-36, and modified Bruce exercise treadmill test obtained at baseline and 6 months after treatment. Autologous CD34+ cells (CLBS16) were mobilized by administration of granulocyte-colony stimulating factor 5µg/kg/day for 5 days and collected by leukapheresis. Participants received a single intracoronary left anterior descending infusion of isolated CD34+ cells in medium that enhances cell function. Results: Coronary flow reserve improved from 2.08±0.32 at baseline to 2.68±0.79 at 6 months after treatment ( P <0.005). Angina frequency decreased ( P <0.004), Canadian Cardiovascular Society class improved ( P <0.001), and quality of life improved as assessed by the Seattle Angina Questionnaire ( P ≤0.03, all scales) and SF-36 ( P ≤0.04, all scales). There were no cell-related serious adverse events. Conclusions: In this pilot clinical trial of microvascular angina, patients with ischemia and nonobstructive coronary artery disease receiving intracoronary infusion of CD34+ cell therapy had higher coronary flow reserve, less severe angina, and better quality of life at 6 months. The current study supports a potential therapeutic role for CD34+ cells in patients with microvascular angina. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03508609.

Background & Aim No-Option Refractory Disabling Angina (NORDA) is an orphan-sized population with an estimated prevalence between 18,194 and 94,338 persons in the US. CLBS14 has previously been studied for the treatment of NORDA in phase 1 proof of concept, phase 2 dose-defining, and partially completed 3 studies. These studies enrolled up to 1.9% of the prevalent population. The Phase 2 study demonstrated statistically significant improvements in key outcome measures including angina frequency, exercise time, and mortality and is considered pivotal evidence of efficacy. Two independent analyses of combined data from all three studies further support the conclusion of efficacy. Thus, existing evidence may support accelerated or conditional approval in some regions. In the event that additional evidence is required, confirmatory investigations including post-marketing commitments to be potentially completed during the review of a marketing authorization application were designed. Methods, Results & Conclusion We describe a Phase 3 pivotal study to meet regulatory requirements as a confirmatory study in subjects with NORDA, defined as 1) experiencing ongoing angina despite use of maximally tolerated doses of anti-anginal medications, 2) limited by angina after at least 3 minutes but no more than 10 minutes on a modified Bruce-protocol exercise tolerance test, 3) experiencing a minimum of 7 episodes of angina per week during a 2-week lead-in period, 4) with objective evidence of inducible ischemia, 5) not having gained relief from or not being eligible for revascularization procedures, and 6) being assessed by an independent committee as CCS angina class III or IV. Approximately 400 subjects representing up to 2.2% of the prevalent population will be randomized 4:3:1 to receive double-blind intramyocardial CLBS14, double-blind intramyocardial placebo or open-label standard of care (SOC). The primary endpoint will be total exercise time at 6 months, with secondary endpoints of angina frequency and change in CCS angina class. Mortality will be followed for 2 years. SOC subjects will have the option to receive CLBS14 treatment after an initial 6-month observation. A favorable outcome in conjunction with previous data has regulatory support for submission and review of a BLA for CLBS14 for this orphan-sized indication. Considerations around study design and regulatory implications for this first potentially approvable cardiovascular cell therapy, including pathways to conditional approval, will be discussed. No-Option Refractory Disabling Angina (NORDA) is an orphan-sized population with an estimated prevalence between 18,194 and 94,338 persons in the US. CLBS14 has previously been studied for the treatment of NORDA in phase 1 proof of concept, phase 2 dose-defining, and partially completed 3 studies. These studies enrolled up to 1.9% of the prevalent population. The Phase 2 study demonstrated statistically significant improvements in key outcome measures including angina frequency, exercise time, and mortality and is considered pivotal evidence of efficacy. Two independent analyses of combined data from all three studies further support the conclusion of efficacy. Thus, existing evidence may support accelerated or conditional approval in some regions. In the event that additional evidence is required, confirmatory investigations including post-marketing commitments to be potentially completed during the review of a marketing authorization application were designed. We describe a Phase 3 pivotal study to meet regulatory requirements as a confirmatory study in subjects with NORDA, defined as 1) experiencing ongoing angina despite use of maximally tolerated doses of anti-anginal medications, 2) limited by angina after at least 3 minutes but no more than 10 minutes on a modified Bruce-protocol exercise tolerance test, 3) experiencing a minimum of 7 episodes of angina per week during a 2-week lead-in period, 4) with objective evidence of inducible ischemia, 5) not having gained relief from or not being eligible for revascularization procedures, and 6) being assessed by an independent committee as CCS angina class III or IV. Approximately 400 subjects representing up to 2.2% of the prevalent population will be randomized 4:3:1 to receive double-blind intramyocardial CLBS14, double-blind intramyocardial placebo or open-label standard of care (SOC). The primary endpoint will be total exercise time at 6 months, with secondary endpoints of angina frequency and change in CCS angina class. Mortality will be followed for 2 years. SOC subjects will have the option to receive CLBS14 treatment after an initial 6-month observation. A favorable outcome in conjunction with previous data has regulatory support for submission and review of a BLA for CLBS14 for this orphan-sized indication. Considerations around study design and regulatory implications for this first potentially approvable cardiovascular cell therapy, including pathways to conditional approval, will be discussed.

Background & Aim CLBS12 (autologous, mobilized peripheral blood derived selected CD34 cells) has previously been studied for the treatment of No-Option Critical Limb Ischemia (NOCLI) in Phase 1 and 2 studies in Japan and the United States. These studies demonstrated statistically significant improvement in key outcome measures including reduced Rutherford category, improved walking distance, reduction in pain, improvement in blood flow and tissue oxygenation, reduction in size and number of ulcers, and improved amputation-free survival. In one of the Japan studies, more than 80% of subjects treated with a single administration of CD34 cells became CLI-free and maintained this status through 4 years of observation. The promising results of these studies led to discussions with the PMDA regarding the design of a confirmatory study that, with a favorable outcome, could support registration of CLBS12 in Japan for NOCLI under the new regenerative medicine regulations. Methods, Results & Conclusion The study is a prospective, randomized clinical trial in subjects with arteriosclerosis obliterans (ASO; n=30) or Buerger's Disease (BD; n=5). All patients receive standard-of-care treatment (SOC). The study calls for randomization of 30 subjects with ASO to receive either CLBS12 or to continue with SOC alone. Additionally, 5 subjects with BD were to be allocated to a sub-study and receive CLBS12 while continuing SOC treatment. The primary endpoint for the study is ability to achieve a continuous CLI-free state. Other key measures include progression-free, amputation-free, and overall survival; Rutherford category; tissue oxygenation; blood flow; ulcer size, depth, and infection; pain; and quality-of-life. For preparation of CLBS12, subjects are mobilized with GCSF 5 µg/day for 5 days, then undergo apheresis to collect a mononuclear cell fraction. CD34 cells are separated using a magnetic separation method. CLBS12 is formulated as a cell suspension in proprietary media in a 10 mL volume for administration as a series of 20 intramuscular injections in one affected limb. The study was initiated in December 2017 and is expected to be completed in 2020. The BD sub-study has enrolled 6 subjects and half have achieved a continuous CLI-free state. Interim data from the trial will be presented. CLBS12 (autologous, mobilized peripheral blood derived selected CD34 cells) has previously been studied for the treatment of No-Option Critical Limb Ischemia (NOCLI) in Phase 1 and 2 studies in Japan and the United States. These studies demonstrated statistically significant improvement in key outcome measures including reduced Rutherford category, improved walking distance, reduction in pain, improvement in blood flow and tissue oxygenation, reduction in size and number of ulcers, and improved amputation-free survival. In one of the Japan studies, more than 80% of subjects treated with a single administration of CD34 cells became CLI-free and maintained this status through 4 years of observation. The promising results of these studies led to discussions with the PMDA regarding the design of a confirmatory study that, with a favorable outcome, could support registration of CLBS12 in Japan for NOCLI under the new regenerative medicine regulations. The study is a prospective, randomized clinical trial in subjects with arteriosclerosis obliterans (ASO; n=30) or Buerger's Disease (BD; n=5). All patients receive standard-of-care treatment (SOC). The study calls for randomization of 30 subjects with ASO to receive either CLBS12 or to continue with SOC alone. Additionally, 5 subjects with BD were to be allocated to a sub-study and receive CLBS12 while continuing SOC treatment. The primary endpoint for the study is ability to achieve a continuous CLI-free state. Other key measures include progression-free, amputation-free, and overall survival; Rutherford category; tissue oxygenation; blood flow; ulcer size, depth, and infection; pain; and quality-of-life. For preparation of CLBS12, subjects are mobilized with GCSF 5 µg/day for 5 days, then undergo apheresis to collect a mononuclear cell fraction. CD34 cells are separated using a magnetic separation method. CLBS12 is formulated as a cell suspension in proprietary media in a 10 mL volume for administration as a series of 20 intramuscular injections in one affected limb. The study was initiated in December 2017 and is expected to be completed in 2020. The BD sub-study has enrolled 6 subjects and half have achieved a continuous CLI-free state. Interim data from the trial will be presented.

Early evidence from COVID-19 patients and autopsy series indicates that moderate to severe cases are accompanied by damage to the pulmonary endothelium and microcirculation. These data indicate that SARS-CoV-2 infection is particularly avid for lung microvascular endothelium, reflecting endothelial cell infection, inflammation, and focal thrombi occluding the pulmonary circulation leading to progressive fibrosis reminiscent of endothelial-mesenchymal transition. This appears to be a potential mechanism by which COVID-19 causes progressive fibrotic changes, impairment in the pulmonary vasculature, and loss of lung function that persists after acute recovery. Early data from the SARS-CoV-1 epidemic indicated that CD34+ cells in the lung could also be a target of infection and that destruction of lung CD34+ progenitors could account for persistence of pulmonary manifestations. Preclinical studies reveal that restoration of microvasculature in the lung can trigger and sustain regeneration of lung tissue. The microvascular repair function of CD34+ has been documented in preclinical models and multiple human trials in ischemic tissue repair. Accordingly, we designed a clinical trial to evaluate autologous CD34+ cells (CLBS119) for amelioration/repair of COVID-19 lung damage. Subjects must be hospitalized for COVID-19 and have ongoing pulmonary involvement based on room air hypoxia. The study will include approximately 50% of subjects who cannot be liberated from ventilatory support. CD34+ cells will be mobilized with plerixafor and selected from the mononuclear fraction following apheresis. Doses up to 500 x 10 6 cells will be administered IV. Subjects will receive a single administration of cells. Key measures will include assessment of oxygenation, need for supplemental oxygen, pulmonary function assessments, radiographic assessments, and biomarkers. Subjects will be followed for 6 months following administration. Interim findings will be presented.

Patients with refractory angina who are suboptimal candidates for further revascularization have improved exercise time, decreased angina frequency, and reduced major adverse cardiac events with intramyocardial delivery of CD34 + cells. However, the effect of CD34 + cell therapy on health care expenditures before and after treatment is unknown. We determined the effect of CD34 + cell therapy on cardiac-related hospital visits and costs during the 12 months following stem cell injection compared with the 12 months prior to injection. Cardiac-related hospital admissions and procedures were retrospectively tabulated for patients enrolled at one site in one of three double-blinded, placebo-controlled CD34 + trials in the 12 months before and after intramyocardial injections of CD34 + cells vs placebo. Fifty-six patients were randomized to CD34 + cell therapy (n = 37) vs placebo (n = 19). Patients randomized to cell therapy experienced 1.57 ± 1.39 cardiac-related hospital visits 12 months before injection, compared with 0.78 ± 1.90 hospital visits 12 months after injection, which was associated with a 62% cost reduction translating to an average savings of $5500 per cell therapy patient. Patients in the placebo group also demonstrated a reduction in cardiac-related hospital events and costs, although to a lesser degree than the CD34 + group. Through 1 January 2019, 24% of CD34 + subjects died at an average of 6.5 ± 2.4 years after enrollment, whereas 47% of placebo patients died at an average of 3.7 ± 1.9 years after enrollment. In conclusion, CD34 + cell therapy for subjects with refractory angina is associated with improved mortality and a reduction in hospital visits and expenditures for cardiac procedures in the year following treatment.

In 1997, the seminal manuscript by Asahara, Murohara, Isner et al outlined the evidence for the existence of circulating, bone marrow-derived cells capable of stimulating and contributing to the formation of new blood vessels. Consistent with the paradigm shift that this work represented, it triggered much scientific debate and controversy, some of which persists 2 decades later. In contrast, the clinical application of autologous CD34 cell therapy has been marked by a track record of consistent safety and clinical benefit in multiple ischemic conditions. In this review, we summarize the preclinical and clinical evidence from over 700 patients in clinical trials of CD34 cell therapy.

Background: Coronary microvascular dysfunction (CMD) has been shown to result in ischemia and cause angina and is linked to adverse clinical outcomes. Thus, CMD may serve as the underlying mechanis...

Address correspondence to: Dr. Gangjian Qin, Department of Biomedical Engineering, Molecular Cardiology Program, The University of Alabama at Birmingham, 1720 2nd Ave S, Volker Hall G094L, Birmingham, AL 35294, USA, Tel: (205) 934-6690, Fax: (312) 503-0137, gqin@uab.edu. S.X. and J.T. contributed equally to this work DISCLOSURES None. HHS Public Access Author manuscript Circ Res. Author manuscript; available in PMC 2019 March 02. Published in final edited form as: Circ Res. 2018 March 02; 122(5): 701–711. doi:10.1161/CIRCRESAHA.117.311814. A uhor M anscript

RATIONALE:The majority of current cardiovascular cell therapy trials use bone marrow progenitor cells (BM PCs) and achieve only modest efficacy; the limited potential of these cells to differentiate into endothelial-lineage cells is one of the major barriers to the success of this promising therapy. We have previously reported that the E2F transcription factor 1 (E2F1) is a repressor of revascularization after ischemic injury.OBJECTIVE:We sought to define the role of E2F1 in the regulation of BM PC function.METHODS AND RESULTS:Ablation of E2F1 (E2F1 deficient) in mouse BM PCs increases oxidative metabolism and reduces lactate production, resulting in enhanced endothelial differentiation. The metabolic switch in E2F1-deficient BM PCs is mediated by a reduction in the expression of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase kinase 2; overexpression of pyruvate dehydrogenase kinase 4 reverses the enhancement of oxidative metabolism and endothelial differentiation. Deletion of E2F1 in the BM increases the amount of PC-derived endothelial cells in the ischemic myocardium, enhances vascular growth, reduces infarct size, and improves cardiac function after myocardial infarction.CONCLUSION:Our results suggest a novel mechanism by which E2F1 mediates the metabolic control of BM PC differentiation, and strategies that inhibit E2F1 or enhance oxidative metabolism in BM PCs may improve the effectiveness of cell therapy.

Aims:Autologous CD34+ (auto-CD34+) cells represent an attractive option for the treatment of refractory angina. Three double-blinded randomized trials (n = 304) compared intramyocardial (IM) auto-CD34+ cells with IM placebo injections to affect total exercise time (TET), angina frequency (AF), and major adverse cardiac events (MACE). Patient-level data were pooled from the Phase I, Phase II ACT-34, ACT-34 extension, and Phase III RENEW trials to determine the efficacy and safety of auto-CD34+ cells.Methods and results:Treatment effects for TET were analysed using an analysis of covariance mixed-effects model and for AF using Poisson regression in a log linear model with repeated measures. The Kaplan-Meier rate estimates for MACE were compared using the log-rank test. Autologous CD34+ cell therapy improved TET by 46.6 s [3 months, 95% confidence interval (CI) 13.0 s-80.3 s; P = 0.007], 49.5 s (6 months, 95% CI 9.3-89.7; P = 0.016), and 44.7 s (12 months, 95% CI - 2.7 s-92.1 s; P = 0.065). The relative frequency of angina was 0.78 (95% CI 0.63-0.98; P = 0.032), 0.66 (0.48-0.91; P = 0.012), and 0.58 (0.38-0.88; P = 0.011) at 3-, 6- and 12-months in auto-CD34+ compared with placebo patients. Results remained concordant when analysed by treatment received and when confined to the Phase III dose of 1 × 105 cells/kg. Autologous CD34 + cell therapy significantly decreased mortality (12.1% vs. 2.5%; P = 0.0025) and numerically reduced MACE (38.9% vs. 30.0; P = 0.14) at 24 months.Conclusion:Treatment with auto-CD34+ cells resulted in clinically meaningful durable improvements in TET and AF at 3-, 6- and 12-months, as well as a reduction in 24-month mortality in this patient-level meta-analysis.

Although clinical trials of cell-based approaches to cardiovascular disease have yielded some promising results, no cell-based therapy has achieved regulatory approval for a cardiovascular indication. To broadly assess the challenges to regulatory approval and identify strategies to facilitate this goal, the Cardiac Safety Research Consortium sponsored a session during the Texas Heart Institute International Symposium on Cardiovascular Regenerative Medicine in September 2017. This session convened leaders in cardiovascular regenerative medicine, including participants from academia, the pharmaceutical industry, the US Food and Drug Administration, and the Cardiac Safety Research Consortium, with particular focus on treatments closest to regulatory approval. A goal of the session was to identify barriers to regulatory approval and potential pathways to overcome them. Barriers identified include manufacturing and therapeutic complexity, difficulties identifying an optimal comparator group, limited industry capacity for funding pivotal clinical trials, and challenges to demonstrating efficacy on clinical end points required for regulatory decisions. Strategies to overcome these barriers include precompetitive development of a cell therapy registry network to enable dual-purposing of clinical data as part of pragmatic clinical trial design, development of standardized terminology for product activity and end points to facilitate this registry, use of innovative statistical methods and quality of life or functional end points to supplement outcomes such as death or heart failure hospitalization and reduce sample size, involvement of patients in determining the research agenda, and use of the Food and Drug Administration's new Regenerative Medicine Advanced Therapy designation to facilitate early discussion with regulatory authorities when planning development pathways.

Although clinical trials of cell-based approaches to cardiovascular disease have yielded some promising results, no cell-based therapy has achieved regulatory approval for a cardiovascular indication. To broadly assess the challenges to regulatory approval and identify strategies to facilitate this goal, the Cardiac Safety Research Consortium sponsored a session during the Texas Heart Institute International Symposium on Cardiovascular Regenerative Medicine in September 2017. This session convened leaders in cardiovascular regenerative medicine, including participants from academia, the pharmaceutical industry, the US Food and Drug Administration, and the Cardiac Safety Research Consortium, with particular focus on treatments closest to regulatory approval. A goal of the session was to identify barriers to regulatory approval and potential pathways to overcome them. Barriers identified include manufacturing and therapeutic complexity, difficulties identifying an optimal comparator group, limited industry capacity for funding pivotal clinical trials, and challenges to demonstrating efficacy on clinical end points required for regulatory decisions. Strategies to overcome these barriers include precompetitive development of a cell therapy registry network to enable dual-purposing of clinical data as part of pragmatic clinical trial design, development of standardized terminology for product activity and end points to facilitate this registry, use of innovative statistical methods and quality of life or functional end points to supplement outcomes such as death or heart failure hospitalization and reduce sample size, involvement of patients in determining the research agenda, and use of the Food and Drug Administration’s new Regenerative Medicine Advanced Therapy designation to facilitate early discussion with regulatory authorities when planning development pathways.

HomeCirculation ResearchVol. 122, No. 2The (Translational) Road Less Traveled Free AccessArticle CommentaryPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessArticle CommentaryPDF/EPUBThe (Translational) Road Less Traveled Timothy D. Henry, Thomas J. Povsic and Douglas W. Losordo Timothy D. HenryTimothy D. Henry From the Cedars-Sinai Heart Institute, Los Angeles, CA (T.D.H.); Duke Clinical Research Institute, Duke Medicine, Durham, NC (T.J.P.); and Caladrius Biosciences, Inc, Basking Ridge, NJ (D.W.L.). , Thomas J. PovsicThomas J. Povsic From the Cedars-Sinai Heart Institute, Los Angeles, CA (T.D.H.); Duke Clinical Research Institute, Duke Medicine, Durham, NC (T.J.P.); and Caladrius Biosciences, Inc, Basking Ridge, NJ (D.W.L.). and Douglas W. LosordoDouglas W. Losordo From the Cedars-Sinai Heart Institute, Los Angeles, CA (T.D.H.); Duke Clinical Research Institute, Duke Medicine, Durham, NC (T.J.P.); and Caladrius Biosciences, Inc, Basking Ridge, NJ (D.W.L.). Originally published19 Jan 2018https://doi.org/10.1161/CIRCRESAHA.117.311987Circulation Research. 2018;122:207–209An anonymous editorial in Nature Biotechnology asserted that cell therapy for heart disease has been a failure and should not continue. In actuality, the development of CD34+ cells for treatment of refractory angina was a well-informed and well-designed pathway toward Food and Drug Administration (FDA) approval to address an important unmet clinical need. Contrary to what was asserted in the Nature Biotechnology editorial, trials of CD34+ cell therapy for refractory angina not only met their primary end points but were based on a clear mechanism of action.I shall be telling this with a sighSomewhere ages and ages hence:Two roads diverged in a wood, and I—I took the one less traveled by,And that has made all the difference.Robert Frost (1874–1963), "The Road Not Taken"In April 2017, an anonymous editorial was published in Nature Biotechnology entitled "A Futile Cycle in Cell Therapy: Should a Cell Therapy for Heart Disease With Scant Evidence of Efficacy Continue to be Tested in Humans?"1 The authors cite a single, phase 2 study in patients with recent acute myocardial infarction—the PreSERVE-AMI trial—as evidence that "cell therapy for heart disease" has been a failure and that the field should focus more on "bona fide cardiomyocytes or progenitors that may differentiate into heart muscle and defined paracrine factors that may tip the balance from fibrosis toward repair."We take issue with several points in the editorial, the primary being the anonymous author or authors' apparent disregard of basic principles of therapeutic development, the failure to distinguish between types of heart disease, and a poor understanding of the available evidence in the field, particularly as it relates to the cell type under study.The test agent in PreSERVE-AMI was the autologous CD34+ cell.2 To date, there have been at least 700 patients enrolled in clinical trials of CD34+ cell therapy for ischemic tissue repair. The editors of Nature Biotechnology seem to equate the failure of a single, phase 2 clinical trial to show a statistically significant improvement in a clinically ambiguous end point (SPECT [single-photon emission computed tomography] imaging) with a mandate to terminate all therapeutic development efforts for this product, and indeed, more broadly, for all cellular therapeutics near clinical application.In the world of therapeutic development, it is typical for an agent to be tested in multiple clinical indications to make observations of bioactivity and determine whether an overall clinical benefit is evident in specific settings. In phases 1 and 2, an evaluation of the totality of evidence is mandatory, and exclusive focus on a single end point, outside of the setting of a pivotal phase 3 study, is not appropriate.3 Furthermore, when discussing the bioactivity of a therapeutic, it is important to review all of the evidence available to make a determination as to whether further study is warranted. Failure to do so would be highly detrimental to medical progress. Termination of clinical development based on a negative imaging end point in 1 study for a single indication would have deprived the medical community of many of its most effective therapeutics. In the case of CD34+ cell therapy, the weight of evidence is, in fact, positive, and the clinical development of this therapy for refractory angina is worth reviewing.Refractory AnginaThere is a unique and growing patient population with advanced coronary artery disease no longer amenable to surgical or percutaneous revascularization. These challenging, "no option" patients have significant symptoms despite optimal medical management that severely impair their quality of life and create a large economic burden on society.4–6 The only therapies for these patients approved in the United States beyond antianginal and secondary prevention medications are enhanced external counterpulsation (which has not been shown to improve exercise time or mortality) and transcutaneous myocardial laser revascularization (which has been shown to increase mortality in this patient population).4–6 The majority of these patients have preserved left ventricular function, and the source of their problem is myocardial ischemia related to inadequate myocardial perfusion.CD34+ Identification and Preclinical TrialsIn 1997, Asahara et al7 first described a circulating endothelial progenitor cell noting that these CD34+ cells were capable of differentiating into an endothelial lineage. Subsequent studies documented endothelial progenitor cells increased vascularization, perfusion, and function in ischemic cardiac and skeletal muscle providing a mechanistic underpinning for use in treatment of ischemic conditions. Purified CD34+ cells were more potent than nonpurified cell sources supporting further clinical study of this particular cell type.8–11Refractory Angina Clinical Trials With CD34+Extensive preclinical data in small and large animals provided evidence for safety, demonstrated improvements in perfusion, and laid the scientific foundation for progressing to clinical application, notably a phase I/IIa double-blind, randomized, placebo-controlled dose escalation trial in patients with class III/IV angina on optimal medication therapy that began in December 2003.12 Patients received GCSF (granulocyte colony-stimulating factor) 5 μg/kg per day subcutaneously for 5 days followed by leukopheresis and selection of CD34+ cells. All patients then underwent electromechanical mapping followed by intramyocardial injection of CD34+ cells versus placebo into ischemic areas of myocardium using the NOGA catheter system. The trial demonstrated a reduction in angina from 20.5±11.5 to 9.6±13.3 episodes per week from baseline to 6 months in the cell-treated group compared with 21±16 to 27.0±23.8 episodes per week observed in the placebo group.12 Nitroglycerin use was decreased, and exercise tolerance was improved in CD34+-treated patients.A large phase II double-blind placebo-controlled trial, ACT34,11 using basically identical inclusion/exclusion criteria and cell acquisition/processing procedures, randomized patients to 1 of 2 doses of CD34 cells (1×105 or 5×105 cells/kg), vs placebo. At follow up, the cell-treated patients showed a significant improvement in the primary end point of angina frequency at both 6 and 12 months (P=0.02) and a significant improvement in exercise time in cell-treated patients 139±151 versus 69±122 s at 6 months (P=0.014) and 140±171 versus 58±146 s at 12 months (P=0.017).13 For the record, this was the first double-blind, placebo-controlled trial in "no option" patients ever to demonstrate a significant improvement in exercise time!Based on these positive phase I/IIa and phase II trials, an appropriately powered phase III trial the RENEW study (n=440) was designed under a Special Protocol Assessment negotiated with FDA to achieve approval if the primary end point of the study was met.14,15 The inclusion/exclusion criteria were basically identical to the double-blind, placebo-controlled phase 2 trial design and included 2:1 randomization to CD34+ (1×105 cells per kg) versus placebo with an additional 100 patients in an unblinded standard-of-care arm as mandated by regulatory authorities. The only significant trial design difference was that the cell processing was done at a central location and the cell product was shipped to the trial sites. The trial began enrollment in 2012. Unfortunately, on December 4, 2013, the sponsor halted enrollment not because of safety or efficacy concerns (no data analysis was done before study termination) but for financial reasons. The results of the 112 patients enrolled in the trial demonstrated a consistent and significant improvement in the primary end point of exercise time, as well as consistent benefits for angina and major adverse cardiac events.15Most recently, a patient-level meta-analysis of the phase I, phase II, and phase III trials demonstrated a significant improvement in exercise time, a significant reduction in angina, and a significant reduction in mortality after a single administration of autologous CD34+ cells.16 These results, encompassing a total sample size similar to what was planned for RENEW, provide strong evidence that intramyocardial CD34+ is an effective therapy for this high-risk patient population with limited options. Furthermore, multiple meta-analyses in trials of "no option" patients with or without left ventricular dysfunction have documented significant improvements in a variety of end points.17–19To summarize the current status of CD34+ cell therapy for refractory angina, preclinical studies in small and large animals have clearly demonstrated improvement in vascularity and myocardial perfusion, and the cell type, CD34+ cells, has been clearly shown to be important for angiogenesis and vascular repair, thereby providing a mechanism of action. Therefore, contrary to the pronouncements of the anonymous editorial, "sound scientific evidence and a well-characterized mechanism of action" have been established, and "much more preclinical work defining a mechanism of action and demonstrating a strong rationale for exposing humans to these procedures" is not needed. Indeed, the primary end points for both the phase II and III trials were met.13,15The mechanism, cell type, and method of delivery were ideal for this specific patient population—a hypothesis that was born out in subsequent double-blind, placebo-controlled phase I, phase II, and phase III trials demonstrating consistent treatment effects on exercise tolerance, angina, and (although not powered for clinical events) a reduction in major adverse cardiac events, including mortality.Cell Therapy CriticismUnfortunately, much of the criticism of the field of cell therapy seems uninformed and fails to distinguish between different patient populations, cell types, and methods of delivery. In particular, for the refractory angina patient population with impaired blood flow but typically preserved left ventricular function, the overwhelming focus on "myocardial regeneration" is a distraction because these patients need improved myocardial perfusion, not increased muscle mass. In addition, many of the criticisms leveled at the field, such as the failure to show benefit in well-designed placebo-controlled trials, are unfounded. For example, the IxCell-DCM was a double-blind, placebo-controlled trial, which demonstrated a significant reduction in the primary end point using clinical events (death and cardiac hospitalization) in patients with class 3/4 ischemic heart failure.20In contrast to comments in the recent anonymous editorial,1 from our perspective, the scientific pathway toward FDA approval of CD34+ cells for refractory angina was indeed "well-informed, disciplined progress toward the generation of data that satisfy the conditions of the regulatory agency." Indeed, the negotiation of a Special Protocol Assessment with the FDA attests to this fact. The weak link in this process was an industry partner that for financial reasons abrogated their responsibility to complete the phase III trial.In fact, for the patient with refractory angina, CD34+ cell therapy is unique because no other therapy has demonstrated an improvement in exercise time and no other therapy has shown an improvement in mortality heretofore. Perhaps the anonymous editor(s) at Nature Biotechnology would do well to more carefully review available evidence before issuing blanket statements or avoid wading into conversations in areas with which they seem to have little familiarity.DisclosuresT.D. Henry reports that his institution has received research grants from Baxter Healthcare for conduct of the ACT-34 (autologous cell therapy 34) and RENEW studies and was on the Steering Committee for ACT-34 and RENEW. T.J. Povsic reports that his institution has received research grants from Baxter Healthcare for conduct of the ACT-34 and RENEW studies, as well as from Celyad, and reports having received modest consulting fees from Pluristem and Caladrius. D.W. Losordo is employed by Caladrius Biosciences.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Timothy D. Henry, MD, Cedars-Sinai Heart Institute, 127 S San Vicente Blvd, A3100, Los Angeles, CA 90048. E-mail [email protected]References1. A futile cycle in cell therapy.Nat Biotechnol. 2017; 35:291. doi: 10.1038/nbt.3857.CrossrefMedlineGoogle Scholar2. Quyyumi AA, Vasquez A, Kereiakes DJ, et al. PreSERVE-AMI: a randomized, double-blind, placebo-controlled clinical trial of intracoronary administration of autologous CD34+ cells in patients with left ventricular dysfunction post STEMI.Circ Res. 2017; 120:324–331. doi: 10.1161/CIRCRESAHA.115.308165.LinkGoogle Scholar3. Hare JM, Bolli R, Cooke JP, et al; Cardiovascular Cell Therapy Research Network. Phase II clinical research design in cardiology: learning the right lessons too well: observations and recommendations from the Cardiovascular Cell Therapy Research Network (CCTRN).Circulation. 2013; 127:1630–1635. doi: 10.1161/CIRCULATIONAHA.112.000779.LinkGoogle Scholar4. Henry TD, Satran D, Jolicoeur EM. Treatment of refractory angina in patients not suitable for revascularization.Nat Rev Cardiol. 2014; 11:78–95. doi: 10.1038/nrcardio.2013.200.CrossrefMedlineGoogle Scholar5. Henry TD, Satran D, Hodges JS, Johnson RK, Poulose AK, Campbell AR, Garberich RF, Bart BA, Olson RE, Boisjolie CR, Harvey KL, Arndt TL, Traverse JH. Long-term survival in patients with refractory angina.Eur Heart J. 2013; 34:2683–2688. doi: 10.1093/eurheartj/eht165.CrossrefMedlineGoogle Scholar6. Povsic TJ, Broderick S, Anstrom KJ, Shaw LK, Ohman EM, Eisenstein EL, Smith PK, Alexander JH. Predictors of long-term clinical endpoints in patients with refractory angina.J Am Heart Assoc. 2015; 4:e001287. doi: 10.1161/JAHA.114.001287.LinkGoogle Scholar7. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis.Science. 1997; 275:964–967.CrossrefMedlineGoogle Scholar8. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, Kearne M, Magner M, Isner JM. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.Circ Res. 1999; 85:221–228.LinkGoogle Scholar9. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara T. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia.Circulation. 2001; 103:634–637.LinkGoogle Scholar10. Kawamoto A, Tkebuchava T, Yamaguchi J, et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia.Circulation. 2003; 107:461–468.LinkGoogle Scholar11. Kawamoto A, Iwasaki H, Kusano K, Murayama T, Oyamada A, Silver M, Hulbert C, Gavin M, Hanley A, Ma H, Kearney M, Zak V, Asahara T, Losordo DW. CD34-positive cells exhibit increased potency and safety for therapeutic neovascularization after myocardial infarction compared with total mononuclear cells.Circulation. 2006; 114:2163–2169. doi: 10.1161/CIRCULATIONAHA.106.644518.LinkGoogle Scholar12. Losordo DW, Schatz RA, White CJ, et al. Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: a phase I/IIa double-blind, randomized controlled trial.Circulation. 2007; 115:3165–3172. doi: 10.1161/CIRCULATIONAHA.106.687376.LinkGoogle Scholar13. Losordo DW, Henry TD, Davidson C, et al; ACT34-CMI Investigators. Intramyocardial, autologous CD34+ cell therapy for refractory angina.Circ Res. 2011; 109:428–436. doi: 10.1161/CIRCRESAHA.111.245993.LinkGoogle Scholar14. Povsic TJ, Junge C, Nada A, et al. A phase 3, randomized, double-blinded, active-controlled, unblinded standard of care study assessing the efficacy and safety of intramyocardial autologous CD34+ cell administration in patients with refractory angina: design of the RENEW study.Am Heart J. 2013; 165:854–861.e2. doi: 10.1016/j.ahj.2013.03.003.CrossrefMedlineGoogle Scholar15. Povsic TJ, Henry TD, Traverse JH, et al; RENEW Investigators. The RENEW trial: efficacy and safety of intramyocardial autologous CD34(+) cell administration in patients with refractory angina.JACC Cardiovasc Interv. 2016; 9:1576–1585. doi: 10.1016/j.jcin.2016.05.003.CrossrefMedlineGoogle Scholar16. Henry TD, Losordo DW, Traverse JH, Schatz RA, Jolicoeur EM, Schaer GL, Clare R, Chiswell K, White CJ, Fortuin FD, Kereiakes DJ, Zeiher AM, Sherman W, Hunt AS, Povsic TJ. Autologous CD34+ cell therapy improves exercise capacity, angina frequency and reduces mortality in no-option refractory angina: a patient level pooled analysis of randomized double-blinded trials.Eur Heart J. In press. doi: 10.1093/eurheartj/ehx764.Google Scholar17. Kandala J, Upadhyay GA, Pokushalov E, Wu S, Drachman DE, Singh JP. Meta-analysis of stem cell therapy in chronic ischemic cardiomyopathy.Am J Cardiol. 2013; 112:217–225. doi: 10.1016/j.amjcard.2013.03.021.CrossrefMedlineGoogle Scholar18. Fisher SA, Dorée C, Brunskill SJ, Mathur A, Martin-Rendon E. Bone marrow stem cell treatment for ischemic heart disease in patients with no option of revascularization: a systematic review and meta-analysis.PLoS One. 2013; 8:e64669. doi: 10.1371/journal.pone.0064669.CrossrefMedlineGoogle Scholar19. Khan AR, Farid TA, Pathan A, Tripathi A, Ghafghazi S, Wysoczynski M, Bolli R. Impact of cell therapy on myocardial perfusion and cardiovascular outcomes in patients with angina refractory to medical therapy: a systematic review and meta-analysis.Circ Res. 2016; 118:984–993. doi: 10.1161/CIRCRESAHA.115.308056.LinkGoogle Scholar20. Patel AN, Henry TD, Quyyumi AA, Schaer GL, Anderson RD, Toma C, East C, Remmers AE, Goodrich J, Desai AS, Recker D, DeMaria A; ixCELL-DCM Investigators. Ixmyelocel-T for patients with ischaemic heart failure: a prospective randomised double-blind trial.Lancet. 2016; 387:2412–2421. doi: 10.1016/S0140-6736(16)30137-4.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Bolli R and Kahlon A (2020) Time to end the war on cell therapy, European Journal of Heart Failure, 10.1002/ejhf.1767, 22:5, (893-897), Online publication date: 1-May-2020. Bolli R and Hare J (2018) Introduction to a Compendium on Regenerative Cardiology, Circulation Research, 123:2, (129-131), Online publication date: 6-Jul-2018. January 19, 2018Vol 122, Issue 2 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.117.311987PMID: 29348249 Originally publishedJanuary 19, 2018 Keywordsanginacell therapystem cellsbiotechnologyregenerationPDF download Advertisement SubjectsAnginaCell TherapyStem Cells

Sonic Hedgehog (Shh) signaling induces neovascularization and angiogenesis. It is not known whether the hedgehog signaling pathway in endothelial cells is essential to angiogenesis. Smoothened (Smo) transduces hedgehog signaling across the cell membrane. This study assessed whether endothelial Smoothened-dependent Shh signaling is required for Shh-mediated angiogenesis and ischemic tissue repair. Endothelial-specific smoothened knockout mice, eSmo Null were created using Cre-lox recombination system. eSmo Null mice had no observable phenotype at baseline and showed normal cardiac function. Smoothened in CD31+ cells isolated from eSmo Null hearts was significantly reduced compared to CD31+ cells from eSmo WT littermate control hearts. Fluorescence immunostaining of eSmo Null heart sections showed Smo expression in endothelial cells was abolished. The hind-limb ischemia (HLI) model was used to assess the response to ischemic injury. Perfusion ratio, limb motor function, and limb necrosis were not significantly different after HLI between eSmo Null mice and eSmo WT . Capillary densities in the ischemic limb in eSmo Null mice were also similar to eSmo WT at 4 weeks after HLI. Next, response to exogenous Shh was assessed in the corneal angiogenesis model. There was no significant difference in corneal angiogenesis induced by administration of Shh pellets between eSmo WT and eSmo Null mice. Furthermore, in vitro experiments demonstrated that direct Shh had limited effects on endothelial cell proliferation and migration. However, conditioned media from Shh-treated fibroblasts had a more potent effect on endothelial cell proliferation and migration than non-treated conditioned media. Furthermore, Shh treatment of fibroblasts dramatically stimulated angiogenic growth factor expression, including PDGF-B, VEGF-A, HGF and IGF. PDGF-B was the most upregulated and may contribute to the large neo-vessels associated with Shh-induced angiogenesis. Taken together, these data demonstrate that Shh signaling via Smoothened in endothelial cells is not required for angiogenesis and ischemic tissue repair. Shh signaling via stromal cells likely mediates its angiogenic effects.

HomeCirculation ResearchVol. 120, No. 5Reprogrammed Human Endothelial Cells Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBReprogrammed Human Endothelial CellsA Novel Cell Source for Regenerative Vascular Medicine Zhongjian Cheng, Suresh K. Verma, Douglas W. Losordo and Raj Kishore Zhongjian ChengZhongjian Cheng From the Center for Translational Medicine (Z.C., S.K.V., R.K.), Department of Pharmacology (R.K.), Lewis Katz School of Medicine, Temple University, Philadelphia, PA; Caladrius Biosciences, Basking Ridge, NJ (D.W.L.); Division of Cardiology, Department of Medicine, Northwestern University School of Medicine, Chicago, IL (D.W.L.); and Division of Cardiology, Department of Medicine, New York University School of Medicine (D.W.L.). , Suresh K. VermaSuresh K. Verma From the Center for Translational Medicine (Z.C., S.K.V., R.K.), Department of Pharmacology (R.K.), Lewis Katz School of Medicine, Temple University, Philadelphia, PA; Caladrius Biosciences, Basking Ridge, NJ (D.W.L.); Division of Cardiology, Department of Medicine, Northwestern University School of Medicine, Chicago, IL (D.W.L.); and Division of Cardiology, Department of Medicine, New York University School of Medicine (D.W.L.). , Douglas W. LosordoDouglas W. Losordo From the Center for Translational Medicine (Z.C., S.K.V., R.K.), Department of Pharmacology (R.K.), Lewis Katz School of Medicine, Temple University, Philadelphia, PA; Caladrius Biosciences, Basking Ridge, NJ (D.W.L.); Division of Cardiology, Department of Medicine, Northwestern University School of Medicine, Chicago, IL (D.W.L.); and Division of Cardiology, Department of Medicine, New York University School of Medicine (D.W.L.). and Raj KishoreRaj Kishore From the Center for Translational Medicine (Z.C., S.K.V., R.K.), Department of Pharmacology (R.K.), Lewis Katz School of Medicine, Temple University, Philadelphia, PA; Caladrius Biosciences, Basking Ridge, NJ (D.W.L.); Division of Cardiology, Department of Medicine, Northwestern University School of Medicine, Chicago, IL (D.W.L.); and Division of Cardiology, Department of Medicine, New York University School of Medicine (D.W.L.). Originally published3 Mar 2017https://doi.org/10.1161/CIRCRESAHA.117.310573Circulation Research. 2017;120:756–758Nothing in nature is more elegant than the transformation of fertilized oocyte into a unique and complex individual containing some 10 trillion cells with more than 200 specialized functions. Also astonishing, however, is that a cell's career decision is not necessarily permanent, but can be reversed. Committed specialized somatic cells can, in effect, go back and start again from scratch. The generation of induced pluripotent cells, reported first by Takahashi and Yamanaka1 and now generated routinely in laboratories all over the world clearly demonstrates that differentiated somatic cells can be reprogrammed into a cell of desired phenotype. This concept of transcription factor (TF)–based alterations in a cell's identity was further demonstrated by several reports attempting direct reprogramming of a differentiated somatic cells into another differentiated somatic cell without the need for pushing the reprogrammed cell to a pluripotent state, a method now known as "direct reprogramming."2,3 In this issue of Circulation Research, Lee et al4 report their success in reprogramming human dermal fibroblasts (HDFs) into functional and mature endothelial cells (ECs) using a single EC TF ER71/ETV2 (E-26 variant 2). This approach opens a novel era in translational cardiovascular medicine by increasing the availability of autologous ECs to be used in cell-based vascular regenerative therapy.Article, see p 848Derivation of engraftable human ECs could be beneficial to patients with vascular diseases. However, purification and expansion of adult ECs in therapeutically large numbers is technically challenging. Embryonic stem cells and induced pluripotent cells serve as promising alternatives for ECs generation and potentially provide great therapeutic potential; however, several problems limit the clinical applications of derivative cells, including inefficient cell production, long duration of cell culture, and tumorigenic potential.5 Recently, the ability to directly reprogram accessible human cells into disease-relevant cell types through cellular reprogramming has opened novel avenues for basic research and regenerative medicine. Direct reprogramming avoids residual pluripotent stem cells in the transplanted populations, eliminating the risk of teratoma formation. Moreover, higher yields and faster kinetics of EC production by direct reprogramming of nonvascular cells would lower the costs and reduce the delivery time to patients, thereby permitting the use of autologous cells even potentially for subacute conditions. However, transdifferentiation potential by direct reprogramming has been minimal6,7 and often consists of heterogeneous populations of reprogrammed cells.8Direct reprogramming of fibroblasts to generate ECs by the use of ETS family of TFs has been previously attempted. ETS TF are implicated in hematoendothelial specification at the embryonic phase.9 ETS TF drive the expression of genes associated with EC development and function.10 ETV2 is a member of ETS TF family, which is transiently expressed during embryonic development and is absent in adult ECs.11,12 Recent studies reported that ETV2 directly reprograms mature amniotic cells into functional ECs avoiding the poor proliferation and lineage instability associated with pluripotent stem cell–derived EC.9,13 However, the strategy for generating amniotic cell-derived ECs is complex and requires long-term culture, which is not applicable for even subacute vascular injury, is impractical and costly and could require immunosuppression given the allogeneic cell source. Although human adult fibroblasts are readily accessible for EC generation, facilitating autologous therapeutic angiogenesis, earlier studies have shown that it is difficult to induce ECs from fibroblasts.13 Recently, 2 studies examined the effects of ETV2 on reprogramming fibroblasts into ECs. Han et al14 reported that a combination of 5 TFs (FOXO1, ETV2, KLF2, TAL1, and LMO2) directly reprogrammed mouse fibroblasts into ECs. Reprogramming failed when ETV2 was omitted. However, ETV2 alone was not capable of converting mouse fibroblasts into ECs.14 In contrast, recently, Morita et al10 reported that ETV2 alone could convert HDFs into ECs. Of note, the gene level of ETV2, which is minimally or not expressed in mammalian postnatal ECs11,15 and implicated in certain vascular abnormalities,12 was expressed at high levels in the converted ECs.10In this issue of Circulation Research, Lee et al4 tackled some of these limitations to successfully reprogram adult HDFs into functional ECs using ETV2 as single TF. They endeavored to reprogram HDFs to ECs starting with a pool of 6 vasculogenic/endothelial TFs (ETV2, FOXC2, MEF2C, SOX17, SMAD1, and HEY1).4 They observed that overexpression of these endothelial TFs converted HDFs into EC lineage. When they omitted ETV2 factor from the 6 factors, they found a significant reduction in expression of major endothelial genes compared with the combination, including ETV2. Finally, they showed that overexpression of ETV2 alone best induces the expression of EC markers (CDH5, KDR, PECAM1, CD34, and TEK) in HDFs. ETV2 efficiently reprogramed HDFs into functional reprogrammed ECs (rECs) with high efficiency (30%) without transitioning through a pluripotent state. Importantly, these data revealed a novel role of ETV2 in fate changes of differentiated HDFs into ECs.4 The authors identified 2 stages of rECs: early and late rECs (Figures 1 and 2) based on EC/fibroblast gene expression and cell features. They observed that early rECs, which appeared within a week after transduction of ETV2, are immature and consisted of cells with mixed signatures of ECs and fibroblasts. Functionally, the early rECs were capable of taking up acetylated low-density lipoprotein, formed tubular structures and contributed to vessel formation, suggesting that these cells may potentially be applied to future autologous cell-based therapy of vascular diseases in patients. Moreover, the short duration of cell culture for early rEC generation is attractive for potential clinical applications. In addition, Lee et al4 also reprogrammed late rECs by a second round of stimuli of early rEC with ETV2 plus valproic acid, a class 1 histone deacetylase inhibitor. They determined the late rECs show dramatically increased PECAM1 expression and exhibit all of the phenotypic characteristics of genuine ECs (human umbilical vein endothelial cells and human microvascular endothelial cells). The identification of late rECs provides possibilities for understanding the underlying mechanisms of maturation of reprogrammed ECs as well as a cell source for drug discovery. These novel findings are intriguing and may pave the way for enhancing the generation of reprogrammed ECs from fibroblasts and other somatic cells for therapeutic purposes.Download figureDownload PowerPointFigure 1. Summary of direct reprogramming of human dermal fibroblasts (HDFs) into endothelial cells by E-26 variant 2 (ETV2). Ac-LDL indicates acetylated low-density lipoprotein; ANGPT, angiopoietin; FGF, fibroblast growth factor; HMVECs, human microvascular endothelial cells; HUVECs, human umbilical vein endothelial cells; MMPs, matrix metalloproteinases; NO, nitric oxide; rECs, reprogrammed endothelial cells; VEGFA, vascular endothelial growth factor A; and VPA, valproic acid.Download figureDownload PowerPointFigure 2. Summary of direct reprogramming of human dermal fibroblasts (HDFs) into endothelial cells by E-26 variant 2 (ETV2). Ac-LDL indicates acetylated low-density lipoprotein; ANGPT, angiopoietin; Dox, doxycycline; FGF, fibroblast growth factor; HMVECs, human microvascular endothelial cells; HUVECs, human umbilical vein endothelial cells; MMPs, matrix metalloproteinases; NO, nitric oxide; rECs, reprogrammed endothelial cells; VEGFA, vascular endothelial growth factor A; and VPA, valproic acid.On the basis of current and previous findings, ETV2 is clearly crucial for reprogramming of HDFs into ECs. Although the data reported in this article provide a platform for better understanding of EC reprogramming, there are several questions that remain incompletely answered. The underlying molecular and epigenetic mechanisms of ETV2-activated EC genes and suppressed fibroblast genes remain to be determined. Although the authors used a doxycycline-inducible system to initiate reprogramming, the stability of the reprogrammed ECs after the withdrawal of doxycycline remains to be ascertained as do the epigenetic landscape and epigenetic memories of the reprogrammed ECs. The subspecification of reprogrammed cells (arterial versus venous) also remains to be determined as well as understanding the aging of the mature reprogrammed EC. Another unresolved question is how closely reprogrammed cells resemble their target cell counterparts? Moreover, the effect of ETVs on reprogramming of HDFs into ECs in vivo needs to be examined as this may have even greater potential for regenerative medicine applications. Other safety issues would still need to be addressed, including the risk of genomic integration of lentiviral constructs leading to spontaneous transformation during cell expansion in culture. For avoiding insertional mutagenesis, it is critical to develop nonintegrating episomal vectors or substitute a nongenetic method for lentiviral vectors for ETV2-induced reprogramming HDFs into ECs.Despite these questions, which are applicable generally to entire field of direct cellular reprogramming, the current work of Lee et al4 provides strong evidence that ETV2-reprogrammed early rECs are functional, as evidenced by the fact that transplantation of these cells improved blood perfusion recovery and vessel formation in mouse ischemic hind limbs. The current work greatly advances the effort toward direct cellular reprogramming including that of ECs. Finally, the insights offered by Lee et al4 may inform new strategies for reprogramming other human nonvascular cells into ECs. Early rEC reprogrammed from HDFs by ETV2 may provide great potential for regenerative medicine and therapy of vascular injury. Recently, exosome-secreted paracrine angiogenic factors have been implicated in regenerative medicine.16,17 Future studies elucidating whether ETV2-reprogrammed early rEC-derived exosomes regulate their angiogenesis/neovascularization properties and what contents in the exosome play critical role on those beneficial effects may shed further light on the mechanisms of action of reprogrammed ECs.Sources of FundingWork described in this article was, in part, supported by National Institute of Health grants HL091983, HL126186, and HL053354 (R. Kishore); American Heart Association-Scientist Development Grant 14SDG20480104 (S.K. Verma); and American Heart Association-Scientist Development Grant SDG16390004 (Z. Cheng).DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Douglas W. Losordo, MD, Caladrius Biosciences, 106 Allen Rd, Fourth Floor, Basking Ridge, NJ 07920. E-mail [email protected]References1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126:663–676. doi: 10.1016/j.cell.2006.07.024.CrossrefMedlineGoogle Scholar2. Srivastava D, DeWitt N. In vivo cellular reprogramming: the next generation.Cell. 2016; 166:1386–1396. doi: 10.1016/j.cell.2016.08.055.CrossrefMedlineGoogle Scholar3. Batty JA, Lima JA, Kunadian V. Direct cellular reprogramming for cardiac repair and regeneration.Eur J Heart Fail. 2016; 18:145–156. doi: 10.1002/ejhf.446.CrossrefMedlineGoogle Scholar4. Lee S, Park C, Han JW, et al. Direct reprogramming of human dermal fibroblasts into endothelial cells using ER71/ETV2.Circ Res. 2017; 120:848–861. doi: 10.1161/CIRCRESAHA.116.309833.LinkGoogle Scholar5. Cohen DE, Melton D. Turning straw into gold: directing cell fate for regenerative medicine.Nat Rev Genet. 2011; 12:243–252. doi: 10.1038/nrg2938.CrossrefMedlineGoogle Scholar6. Fadini GP, Losordo D, Dimmeler S. Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use.Circ Res. 2012; 110:624–637. doi: 10.1161/CIRCRESAHA.111.243386.LinkGoogle Scholar7. O'Neill TJ, Wamhoff BR, Owens GK, Skalak TC. Mobilization of bone marrow-derived cells enhances the angiogenic response to hypoxia without transdifferentiation into endothelial cells.Circ Res. 2005; 97:1027–1035.LinkGoogle Scholar8. Graf T, Stadtfeld M. Heterogeneity of embryonic and adult stem cells.Cell Stem Cell. 2008; 3:480–483. doi: 10.1016/j.stem.2008.10.007.CrossrefMedlineGoogle Scholar9. De Val S, Black BL. Transcriptional control of endothelial cell development.Dev Cell. 2009; 16:180–195. doi: 10.1016/j.devcel.2009.01.014.CrossrefMedlineGoogle Scholar10. Morita R, Suzuki M, Kasahara H, Shimizu N, Shichita T, Sekiya T, Kimura A, Sasaki K, Yasukawa H, Yoshimura A. ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells.Proc Natl Acad Sci U S A. 2015; 112:160–165. doi: 10.1073/pnas.1413234112.CrossrefMedlineGoogle Scholar11. Park C, Lee TJ, Bhang SH, et al. Injury-mediated vascular regeneration requires endothelial ER71/ETV2.Arterioscler Thromb Vasc Biol. 2016; 36:86–96. doi: 10.1161/ATVBAHA.115.306430.LinkGoogle Scholar12. Hayashi M, Pluchinotta M, Momiyama A, Tanaka Y, Nishikawa S, Kataoka H. Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice.Exp Hematol. 2012; 40:738–750.e11. doi: 10.1016/j.exphem.2012.05.012.CrossrefMedlineGoogle Scholar13. Ginsberg M, James D, Ding BS, et al. Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFβ suppression.Cell. 2012; 151:559–575. doi: 10.1016/j.cell.2012.09.032.CrossrefMedlineGoogle Scholar14. Han JK, Chang SH, Cho HJ, Choi SB, Ahn HS, Lee J, Jeong H, Youn SW, Lee HJ, Kwon YW, Cho HJ, Oh BH, Oettgen P, Park YB, Kim HS. Direct conversion of adult skin fibroblasts to endothelial cells by defined factors.Circulation. 2014; 130:1168–1178. doi: 10.1161/CIRCULATIONAHA.113.007727.LinkGoogle Scholar15. Kataoka H, Hayashi M, Nakagawa R, Tanaka Y, Izumi N, Nishikawa S, Jakt ML, Tarui H, Nishikawa S. ETV2/ER71 induces vascular mesoderm from Flk1+PDGFRα+ primitive mesoderm.Blood. 2011; 118:6975–6986. doi: 10.1182/blood-2011-05-352658.CrossrefMedlineGoogle Scholar16. Kishore R, Khan M. More than tiny sacks: stem cell exosomes as cell-free modality for cardiac repair.Circ Res. 2016; 118:330–343. doi: 10.1161/CIRCRESAHA.115.307654.LinkGoogle Scholar17. Kishore R, Khan M. Cardiac cell-derived exosomes: changing face of regenerative biology.Eur Heart J. 2016; 38:212–215.Google Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByMathur T, Tronolone J and Jain A (2021) Comparative Analysis of Blood‐Derived Endothelial Cells for Designing Next‐Generation Personalized Organ‐on‐Chips, Journal of the American Heart Association, 10:22, Online publication date: 16-Nov-2021. Vilà-González M, Kelaini S, Magee C, Caines R, Campbell D, Eleftheriadou M, Cochrane A, Drehmer D, Tsifaki M, O'Neill K, Pedrini E, Yang C, Medina R, McDonald D, Simpson D, Zampetaki A, Zeng L, Grieve D, Lois N, Stitt A and Margariti A (2018) Enhanced Function of Induced Pluripotent Stem Cell-Derived Endothelial Cells Through ESM1 Signaling, Stem Cells, 10.1002/stem.2936, 37:2, (226-239), Online publication date: 1-Feb-2019. March 3, 2017Vol 120, Issue 5 Advertisement Article InformationMetrics © 2017 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.117.310573PMID: 28254795 Originally publishedMarch 3, 2017 Keywordsembryonic stem cellsteratomaadultendothelial cellsfibroblastsEditorialsPDF download Advertisement

Purpose The need for novel approaches to cardiovascular drug development served as the impetus to convene an open meeting of experts from the pharmaceutical industry and academia to assess the challenges and develop solutions for drug discovery in cardiovascular disease. Methods The Novel Cardiovascular Therapeutics Summit first reviewed recent examples of ongoing or recently completed programs translating basic science observations to targeted drug development, highlighting successes (protein convertase sutilisin/kexin type 9 [PCSK9] and neprilysin inhibition) and targets still under evaluation (cholesteryl ester transfer protein [CETP] inhibition), with the hope of gleaning key lessons to successful drug development in the current era. Participants then reviewed the use of innovative approaches being explored to facilitate rapid and more cost-efficient evaluations of drug candidates in a short timeframe. Results We summarize observations gleaned from this summit and offer insight into future cardiovascular drug development. Conclusions The rapid development in genetic and high-throughput drug evaluation technologies, coupled with new approaches to rapidly evaluate potential cardiovascular therapies with in vitro techniques, offer opportunities to identify new drug targets for cardiovascular disease, study new therapies with better efficiency and higher throughput in the preclinical setting, and more rapidly bring the most promising therapies to human testing. However, there must be a critical interface between industry and academia to guide the future of cardiovascular drug development. The shared interest among academic institutions and pharmaceutical companies in developing promising therapies to address unmet clinical needs for patients with cardiovascular disease underlies and guides innovation and discovery platforms that are significantly altering the landscape of cardiovascular drug development.