Prognostic impact of interstitial lung abnormalities in lung cancer: a systematic review and meta-analysis.

Background: Newly identified as a radiological concept, interstitial lung abnormalities (ILA) is emerging as a prognostic factor for lung cancer. Yet, debates persist regarding the prognostic significance of ILA in lung cancer. Our inaugural meta-analysis aimed to investigate the correlation between ILA and lung cancer outcomes, offering additional insights for clinicians in predicting patient prognosis. Methods: Articles meeting the criteria were found through PubMed, the Cochrane Library, EMBASE, and Web of Science by February 29, 2024. The outcomes evaluated were the survival rates such as overall survival (OS), disease-free survival (DFS), progression-free survival (PFS), and cancer-specific survival (CSS). Results: A total of 12 articles with 4416 patients were included in this meta-analysis. The pooled results showed that lung cancer patients with interstitial lung abnormalities had an inferior OS (n=11; HR=2.22; 95% CI=1.68-2.95; P<0.001; I2 = 72.0%; Ph<0.001), PFS (n=3; HR=1.59; 95% CI=1.08-2.32; P=0.017; I2 = 0%; Ph=0.772), and CSS (n=2; HR=4.00; 95% CI=1.94-8.25; P<0.001; I2 = 0%; Ph=0.594) than those without, however, the ILA was not significantly associated with the DFS (n=2; HR=2.07; 95% CI=0.94-7.02; P=0.066; I2 = 90.4%; Ph=0.001). Moreover, lung cancer patients with ILA were significantly correlated with male (OR=2.43; 95% CI=1.48-3.98; P<0.001), smoking history (OR=2.11; 95% CI=1.37-3.25; P<0.001), advanced age (OR=2.50; 95% CI=1.56-4.03; P<0.001), squamous carcinoma (OR=0.42; 95% CI=0.24-0.71; P=0.01), and EGFR mutation (OR=0.50; 95% CI=0.32-0.78; P=0.002). The correlation between ILA and race, stage, ALK, however, was not significant. Conclusion: ILA was a availability factors of prognosis in patients with lung cancers. These findings highlight the importance of early pulmonary fibrosis, namely ILA for prognosis in patients with lung cancer, and provide a partial rationale for future clinical work.

Bone Marrow and Wharton's Jelly Mesenchymal Stromal Cells are Ineffective for Myocardial Repair in an Immunodeficient Rat Model of Chronic Ischemic Cardiomyopathy.

BACKGROUND: Although cell therapy provides benefits for outcomes of heart failure, the most optimal cell type to be used clinically remains unknown. Most of the cell products used for therapy in humans require in vitro expansion to obtain a suitable number of cells for treatment; however, the clinical background of the donor and limited starting material may result in the impaired proliferative and reparative capacity of the cells expanded in vitro. Wharton's jelly mesenchymal cells (WJ MSCs) provide a multitude of advantages over adult tissue-derived cell products for therapy. These include large starting tissue material, superior proliferative capacity, and disease-free donors. Thus, WJ MSC if effective would be the most optimal cell source for clinical use. OBJECTIVES: This study evaluated the therapeutic efficacy of Wharton's jelly (WJ) and bone marrow (BM) mesenchymal stromal cells (MSCs) in chronic ischemic cardiomyopathy in rats. METHODS: Human WJ MSCs and BM MSCs were expanded in vitro, characterized, and evaluated for therapeutic efficacy in a immunodeficient rat model of ischemic cardiomyopathy. Cardiac function was evaluated with hemodynamics and echocardiography. The extent of cardiac fibrosis, hypertrophy, and inflammation was assessed with histological analysis. RESULTS: In vitro analysis revealed that WJ MSCs and BM MSCs are morphologically and immunophenotypically indistinguishable. Nevertheless, the functional analysis showed that WJ MSCs have a superior proliferative capacity, less senescent phenotype, and distinct transcriptomic profile compared to BM MSC. WJ MSCs and BM MSC injected in rat hearts chronically after MI produced a small, but not significant improvement in heart structure and function. Histological analysis showed no difference in the scar size, collagen content, cardiomyocyte cross-sectional area, and immune cell count. CONCLUSIONS: Human WJ and BM MSC have a small but not significant effect on cardiac structure and function when injected intramyocardially in immunodeficient rats chronically after MI.

Transient Cell Cycle Induction in Cardiomyocytes to Treat Subacute Ischemic Heart Failure.

BACKGROUND: The regenerative capacity of the heart after myocardial infarction is limited. Our previous study showed that ectopic introduction of 4 cell cycle factors (4F; CDK1 [cyclin-dependent kinase 1], CDK4 [cyclin-dependent kinase 4], CCNB [cyclin B1], and CCND [cyclin D1]) promotes cardiomyocyte proliferation in 15% to 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction in mice. METHODS: Using temporal single-cell RNA sequencing, we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure. RESULTS: Temporal bulk and single-cell RNA sequencing and further biochemical validations of mature human induced pluripotent stem cell-derived cardiomyocytes treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 hours after infection with 4F, which was associated mainly with sarcomere disassembly and metabolic reprogramming (n=3/time point/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic nonintegrating lentivirus (NIL) encoding 4F; each is driven by a TNNT2 (cardiac troponin T isoform 2) promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially 1 week after myocardial infarction in rats (n=10/group) or pigs (n=6-7/group). Four weeks after injection, TNNT2-4Fpolycistronic-NIL-treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus-treated animals. At 4 months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group). CONCLUSIONS: This study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors owing to the use of a novel transient and cardiomyocyte-specific viral construct.

Effect of intravenous cell therapy in rats with old myocardial infarction.

Mounting evidence shows that cell therapy provides therapeutic benefits in experimental and clinical settings of chronic heart failure. However, direct cardiac delivery of cells via transendocardial injection is logistically complex, expensive, entails risks, and is not amenable to multiple dosing. Intravenous administration would be a more convenient and clinically applicable route for cell therapy. Thus, we determined whether intravenous infusion of three widely used cell types improves left ventricular (LV) function and structure and compared their efficacy. Rats with a 30-day-old myocardial infarction (MI) received intravenous infusion of vehicle (PBS) or 1 of 3 types of cells: bone marrow mesenchymal stromal cells (MSCs), cardiac mesenchymal cells (CMCs), and c-kit-positive cardiac cells (CPCs), at a dose of 12 × 106 cells. Rats were followed for 35 days after treatment to determine LV functional status by serial echocardiography and hemodynamic studies. Blood samples were collected for Hemavet analysis to determine inflammatory cell profile. LV ejection fraction (EF) dropped ≥ 20 points in all hearts at 30 days after MI and deteriorated further at 35-day follow-up in the vehicle-treated group. In contrast, deterioration of EF was halted in rats that received MSCs and attenuated in those that received CMCs or CPCs. None of the 3 types of cells significantly altered scar size, myocardial content of collagen or CD45-positive cells, or Hemavet profile. This study demonstrates that a single intravenous administration of 3 types of cells in rats with chronic ischemic cardiomyopathy is effective in attenuating the progressive deterioration in LV function. The extent of LV functional improvement was greatest with CPCs, intermediate with CMCs, and least with MSCs.

After the storm: an objective appraisal of the efficacy of c-kit+ cardiac progenitor cells in preclinical models of heart disease.

The falsification of data related to c-kit+ cardiac progenitor cells (CPCs) by a Harvard laboratory has been a veritable tragedy. Does this fraud mean that CPCs are not beneficial in models of ischemic cardiomyopathy? At least 50 studies from 26 laboratories independent of the Harvard group have reported beneficial effects of CPCs in mice, rats, pigs, and cats. The mechanism of action remains unclear. Our group has shown that CPCs do not engraft in the diseased heart, do not differentiate into new cardiac myocytes, do not regenerate dead myocardium, and thus work via paracrine mechanisms. A casualty of the misconduct at Harvard has been the SCIPIO trial, a collaboration between the Harvard group and the group in Louisville. The retraction of the SCIPIO paper was caused exclusively by issues with data generated at Harvard, not those generated in Louisville. In the retraction notice, the Lancet editors stated: "Although we do not have any reservations about the clinical work in Louisville that used the preparations from Anversa's laboratory in good faith, the lack of reliability regarding the laboratory work at Harvard means that we are now retracting this paper". We must be careful not to dismiss all work on CPCs because of one laboratory's misconduct. An unbiased review of the literature supports the therapeutic potential of CPCs for heart failure at the preclinical level.

Rapid Lipid Modification of Endothelial Cell Membranes in Cardiac Ischemia/Reperfusion Injury: a Novel Therapeutic Strategy to Reduce Infarct Size.

PURPOSE: Plasma membranes constitute a gathering point for lipids and signaling proteins. Lipids are known to regulate the location and activity of signaling proteins under physiological and pathophysiological conditions. Membrane lipid therapies (MLTs) that gradually modify lipid content of plasma membranes have been developed to treat chronic disease; however, no MLTs have been developed to treat acute conditions such as reperfusion injury following myocardial infarction (MI) and percutaneous coronary intervention (PCI). A fusogenic nanoliposome (FNL) that rapidly incorporates exogenous unsaturated lipids into endothelial cell (EC) membranes was developed to attenuate reperfusion-induced protein signaling. We hypothesized that administration of intracoronary (IC) FNL-MLT interferes with EC membrane protein signaling, leading to reduced microvascular dysfunction and infarct size (IS). METHODS: Using a myocardial ischemia/reperfusion swine model, the efficacy of FNL-MLT in reducing IS following a 60-min coronary artery occlusion was tested. Animals were randomized to receive IC Ringer's lactate solution with or without 10 mg/mL/min of FNLs for 10 min prior to reperfusion (n = 6 per group). RESULTS: The IC FNL-MLT reduced IS (25.45 ± 16.4% vs. 49.7 ± 14.1%, P < 0.02) and enhanced regional myocardial blood flow (RMBF) in the ischemic zone at 15 min of reperfusion (2.13 ± 1.48 mL/min/g vs. 0.70 ± 0.43 mL/min/g, P < 0.001). The total cumulative plasma levels of the cardiac injury biomarker cardiac troponin I (cTnI) were trending downward but were not significant (999.3 ± 38.7 ng/mL vs. 1456.5 ± 64.8 ng/mL, P = 0.1867). However, plasma levels of heart-specific fatty acid binding protein (hFABP), another injury biomarker, were reduced at 2 h of reperfusion (70.3 ± 38.0 ng/mL vs. 137.3 ± 58.2 ng/mL, P = 0.0115).  CONCLUSION: The IC FNL-MLT reduced IS compared to vehicle in this swine model. The FNL-MLT maybe a promising adjuvant to PCI in the treatment of acute MI.

Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity.

The limited availability of human heart tissue and its complex cell composition are major limiting factors for the reliable testing of drug efficacy and toxicity. Recently, we developed functional human and pig heart slice biomimetic culture systems that preserve the viability and functionality of 300 µm heart slices for up to 6 days. Here, we tested the reliability of this culture system for testing the cardiotoxicity of anti-cancer drugs. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) with known different mechanisms of cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and gene expression. Slices incubated with any of these drugs for 48 h showed diminished in viability as well as loss of cardiomyocyte structure and function. Mechanistically, RNA sequencing of doxorubicin-treated tissues demonstrated a significant downregulation of cardiac genes and upregulation of oxidative stress responses. Trastuzumab treatment downregulated cardiac muscle contraction-related genes consistent with its clinically known effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes, in line with its mechanism of action. Similar to hiPS-derived-cardiomyocytes, heart slices recapitulated the expected toxicity of doxorubicin and trastuzumab, however, slices were superior in detecting sunitinib cardiotoxicity and mechanism in the clinically relevant concentration range of 0.1-1 µM. These results indicate that heart slice culture models have the potential to become a reliable platform for testing and elucidating mechanisms of drug cardiotoxicity.

The Effect of Cardiogenic Factors on Cardiac Mesenchymal Cell Anti-Fibrogenic Paracrine Signaling and Therapeutic Performance.

Intrinsic cardiogenic factor expression, a proxy for cardiomyogenic lineage commitment, may be an important determinant of donor cell cardiac reparative capacity in cell therapy applications; however, whether and how this contributes to their salutary effects remain largely ambiguous. Methods: The current study examined the consequences of enhanced cardiogenic factor expression, via lentiviral delivery of GMT (GATA4, MEF2C, and TBX5), on cardiac mesenchymal cell (CMC) anti-fibrogenic paracrine signaling dynamics, in vitro, and cardiac reparative capacity, in vivo. Proteome cytokine array analyses and in vitro cardiac fibroblast activation assays were performed using conditioned medium derived from either GMT- or GFP control-transduced CMCs, to respectively assess cardiotrophic factor secretion and anti-fibrogenic paracrine signaling aptitude. Results: Relative to GFP controls, GMT CMCs exhibited enhanced secretion of cytokines implicated to function in pathways associated with matrix remodeling and collagen catabolism, and more ably impeded activated cardiac fibroblast Col1A1 synthesis in vitro. Following their delivery in a rat model of chronic ischemic cardiomyopathy, conventional echocardiography was unable to detect a therapeutic advantage with either CMC population; however, hemodynamic analyses identified a modest, yet calculable supplemental benefit in surrogate measures of global left ventricular contractility with GMT CMCs relative to GFP controls. This phenomenon was neither associated with a decrease in infarct size nor an increase in viable myocardium, but with only a marginal decrease in regional myocardial collagen deposition. Conclusion: Overall, these results suggest that CMC cardiomyogenic lineage commitment biases cardiac repair and, further, that enhanced anti-fibrogenic paracrine signaling potency may underlie, in part, their improved therapeutic utility.

Epigenetically modified cardiac mesenchymal stromal cells limit myocardial fibrosis and promote functional recovery in a model of chronic ischemic cardiomyopathy.

Preclinical investigations support the concept that donor cells more oriented towards a cardiovascular phenotype favor repair. In light of this philosophy, we previously identified HDAC1 as a mediator of cardiac mesenchymal cell (CMC) cardiomyogenic lineage commitment and paracrine signaling potency in vitro-suggesting HDAC1 as a potential therapeutically exploitable target to enhance CMC cardiac reparative capacity. In the current study, we examined the effects of pharmacologic HDAC1 inhibition, using the benzamide class 1 isoform-selective HDAC inhibitor entinostat (MS-275), on CMC cardiomyogenic lineage commitment and CMC-mediated myocardial repair in vivo. Human CMCs pre-treated with entinostat or DMSO diluent control were delivered intramyocardially in an athymic nude rat model of chronic ischemic cardiomyopathy 30 days after a reperfused myocardial infarction. Indices of cardiac function were assessed by echocardiography and left ventricular (LV) Millar conductance catheterization 35 days after treatment. Compared with naïve CMCs, entinostat-treated CMCs exhibited heightened capacity for myocyte-like differentiation in vitro and superior ability to attenuate LV remodeling and systolic dysfunction in vivo. The improvement in CMC therapeutic efficacy observed with entinostat pre-treatment was not associated with enhanced donor cell engraftment, cardiomyogenesis, or vasculogenesis, but instead with more efficient inhibition of myocardial fibrosis and greater increase in myocyte size. These results suggest that HDAC inhibition enhances the reparative capacity of CMCs, likely via a paracrine mechanism that improves ventricular compliance and contraction and augments myocyte growth and function.

Repeated Administrations of Cardiac Progenitor Cells Are Superior to a Single Administration of an Equivalent Cumulative Dose.

BACKGROUND: We have recently found that 3 repeated doses (12×106 each) of c-kitPOS cardiac progenitor cells (CPCs) were markedly more effective than a single dose of 12×106 cells in alleviating postinfarction left ventricular dysfunction and remodeling. However, since the single-dose group received only one third of the total number of CPCs given to the multiple-dose group, it is unknown whether the superior therapeutic efficacy was caused by repeated treatments per se or by administration of a higher total number of CPCs. This issue has major clinical implications because multiple cell injections in patients pose significant challenges, which would be obviated by using 1 large injection. Accordingly, we determined whether the beneficial effects of 3 repeated CPC doses can be recapitulated by 1 large dose containing the same total number of cells. METHODS AND RESULTS: Rats with a 30-day-old myocardial infarction received 3 echo-guided intraventricular infusions, 35 days apart, of vehicle-vehicle-vehicle, 36×106 CPCs-vehicle-vehicle, or 3 equal doses of 12×106 CPCs. Infusion of a single, large dose of CPCs (36×106 cells) produced an initial improvement in left ventricular function, but no further improvement was observed after the second and third infusions (both vehicle). In contrast, each of the 3 doses of CPCs (12×106) caused a progressive improvement in left ventricular function, the cumulative magnitude of which was greater than with a single dose. Unlike the single dose, repeated doses reduced collagen content and immune cell infiltration. CONCLUSIONS: Three repeated doses of CPCs are superior to 1 dose even though the total number of cells infused is the same, possibly because of greater antifibrotic and anti-inflammatory actions.

Myocardial Reparative Properties of Cardiac Mesenchymal Cells Isolated on the Basis of Adherence.

BACKGROUND: The authors previously reported that the c-kit-positive (c-kitPOS) cells isolated from slowly adhering (SA) but not from rapidly adhering (RA) fractions of cardiac mesenchymal cells (CMCs) are effective in preserving left ventricular (LV) function after myocardial infarction (MI). OBJECTIVES: This study evaluated whether adherence to plastic alone, without c-kit sorting, was sufficient to isolate reparative CMCs. METHODS: RA and SA CMCs were isolated from mouse hearts, expanded in vitro, characterized, and evaluated for therapeutic efficacy in mice subjected to MI. RESULTS: Morphological and phenotypic analysis revealed that murine RA and SA CMCs are indistinguishable; nevertheless, transcriptome analysis showed that they possess fundamentally different gene expression profiles related to factors that regulate post-MI LV remodeling and repair. A similar population of SA CMCs was isolated from porcine endomyocardial biopsy samples. In mice given CMCs 2 days after MI, LV ejection fraction 28 days later was significantly increased in the SA CMC group (31.2 ± 1.0% vs. 24.7 ± 2.2% in vehicle-treated mice; p < 0.05) but not in the RA CMC group (24.1 ± 1.2%). Histological analysis showed reduced collagen deposition in the noninfarcted region in mice given SA CMCs (7.6 ± 1.5% vs. 14.5 ± 2.8% in vehicle-treated mice; p < 0.05) but not RA CMCs (11.7 ± 1.7%), which was associated with reduced infiltration of inflammatory cells (14.1 ± 1.6% vs. 21.3 ± 1.5% of total cells in vehicle and 19.3 ± 1.8% in RA CMCs; p < 0.05). Engraftment of SA CMCs was negligible, which implies a paracrine mechanism of action. CONCLUSIONS: We identified a novel population of c-kit-negative reparative cardiac cells (SA CMCs) that can be isolated with a simple method based on adherence to plastic. SA CMCs exhibited robust reparative properties and offered numerous advantages, appearing to be more suitable than c-kitPOS cardiac progenitor cells for widespread clinical therapeutic application.

Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy.

RATIONALE: The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent. OBJECTIVE: To determine whether 3 CPC infusions have greater efficacy than 1 infusion. METHODS AND RESULTS: Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region. CONCLUSIONS: This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.

Long-Term Outcome of Administration of c-kit(POS) Cardiac Progenitor Cells After Acute Myocardial Infarction: Transplanted Cells Do not Become Cardiomyocytes, but Structural and Functional Improvement and Proliferation of Endogenous Cells Persist for at Least One Year.

RATIONALE: Cardiac progenitor cells (CPCs) improve left ventricular remodeling and function after acute or chronic myocardial infarction. However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown. OBJECTIVE: To assess the outcome of CPC therapy at 1 year. METHODS AND RESULTS: Female rats underwent a 90-minute coronary occlusion; 4 hours after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved left ventricular remodeling and regional and global left ventricular function. No tumors were observed. Some transplanted (Y-chromosome(POS)) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4-8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative response of endogenous cells, resulting in increased formation of endothelial cells and Y-chromosome(NEG) CPCs for 12 months and increased formation, for at least 7 months, of small cells that expressed cardiomyocytic proteins (α-sarcomeric actin) but did not have a mature cardiomyocyte phenotype. CONCLUSIONS: The beneficial effects of CPCs on left ventricular remodeling and dysfunction are sustained for at least 1 year and thus are likely to be permanent. Because transplanted CPCs do not differentiate into mature myocytes, their major mechanism of action must involve paracrine actions. These paracrine mechanisms could be very prolonged because some CPCs engraft, proliferate, and persist at 1 year. This is the first report that transplantation of any cell type in the heart induces a proliferative response that lasts at least 1 year. The results strongly support the safety and clinical utility of CPC therapy.

Effect of the stop-flow technique on cardiac retention of c-kit positive human cardiac stem cells after intracoronary infusion in a porcine model of chronic ischemic cardiomyopathy.

It is commonly thought that the optimal method for intracoronary administration of cells is to stop coronary flow during cell infusion, in order to prolong cell/vascular wall contact, enhance adhesion, and promote extravasation of cells into the interstitial space. However, occlusion of a coronary artery with a balloon involves serious risks of vascular damage and/or dissection, particularly in non-stented segments such as those commonly found in patients with heart failure. It remains unknown whether the use of the stop-flow technique results in improved donor cell retention. Acute myocardial infarction was produced in 14 pigs. One to two months later, pigs received 10 million indium-111 oxyquinoline (oxine)-labeled c-kit(pos) human cardiac stem cells (hCSCs) via intracoronary infusion with (n = 7) or without (n = 7) balloon inflation. Pigs received cyclosporine to prevent acute graft rejection. Animals were euthanized 24 h later and hearts harvested for radioactivity measurements. With the stop-flow technique, the retention of hCSCs at 24 h was 5.41 ± 0.80 % of the injected dose (n = 7), compared with 4.87 ± 0.62 % without coronary occlusion (n = 7), (P = 0.60). When cells are delivered intracoronarily in a clinically relevant porcine model of chronic ischemic cardiomyopathy, the use of the stop-flow technique does not result in greater myocardial cell retention at 24 h compared with non-occlusive infusion. These results have practical implications for the design of cell therapy trials. Our observations suggest that the increased risk of complications secondary to coronary manipulation and occlusion is not warranted.

Effects of Intracoronary Infusion of Escalating Doses of Cardiac Stem Cells in Rats With Acute Myocardial Infarction.

BACKGROUND: Although c-kit(pos) cardiac stem cells (CSCs) preserve left ventricular (LV) function and structure after myocardial infarction, CSC doses have been chosen arbitrarily, and the dose-effect relationship is unknown. METHODS AND RESULTS: Rats underwent a 90-minute coronary occlusion followed by 35 days of reperfusion. Vehicle or CSCs at 5 escalating doses (0.3×10(6), 0.75×10(6), 1.5×10(6), 3.0×10(6), and 6.0×10(6) cells/heart) were given intracoronarily 4 h after reperfusion. The lowest dose (0.3×10(6)) had no effect on LV function and morphology, whereas 0.75, 1.5, and 3.0×10(6) significantly improved regional and global LV function (echocardiography and hemodynamic studies). These 3 doses had similar effects on echocardiographic parameters (infarct wall thickening fraction, LV end-systolic and end-diastolic volumes, LV ejection fraction) and hemodynamic variables (LV end-diastolic pressure, LV dP/dtmax, preload adjusted maximal power, end-systolic elastance, preload recruitable stroke work) and produced similar reductions in apoptosis, scar size, infarct wall thinning, and LV expansion index and similar increases in viable myocardium in the risk region (morphometry). Infusion of 6.0×10(6) CSCs markedly increased postprocedural mortality. Green fluorescent protein and 5-bromo-2'-deoxyuridine staining indicated that persistence of donor cells and formation of new myocytes were negligible with all doses. CONCLUSIONS: Surprisingly, in this rat model of acute myocardial infarction, the dose-response relationship for intracoronary CSCs is flat. A minimal dose between 0.3 and 0.75×10(6) is necessary for efficacy; above this threshold, a 4-fold increase in cell number does not produce greater improvement in LV function or structure. Further increases in cell dose are harmful.

Safety of intracoronary infusion of 20 million C-kit positive human cardiac stem cells in pigs.

BACKGROUND: There is mounting interest in using c-kit positive human cardiac stem cells (c-kit(pos) hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs. METHODS: Right atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment. RESULTS: Compared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion. CONCLUSIONS: Intracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.

Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy.

BACKGROUND: Relevant preclinical models are necessary for further mechanistic and translational studies of c-kit+ cardiac stem cells (CSCs). The present study was undertaken to determine whether intracoronary CSCs are beneficial in a porcine model of chronic ischemic cardiomyopathy. METHODS AND RESULTS: Pigs underwent a 90-minute coronary occlusion followed by reperfusion. Three months later, autologous CSCs (n=11) or vehicle (n=10) were infused into the infarct-related artery. At this time, all indices of left ventricular (LV) function were similar in control and CSC-treated pigs, indicating that the damage inflicted by the infarct in the 2 groups was similar; 1 month later, however, CSC-treated pigs exhibited significantly greater LV ejection fraction (echocardiography) (51.7±2.0% versus 42.9±2.3%, P<0.01), systolic thickening fraction in the infarcted LV wall, and maximum LV dP/dt, as well as lower LV end-diastolic pressure. Confocal microscopy showed clusters of small α-sarcomeric actin-positive cells expressing Ki67 in the scar of treated pigs, consistent with cardiac regeneration. The origin of these cycling myocytes from the injected cells was confirmed in 4 pigs that received enhanced green fluorescent protein -labeled CSCs, which were positive for the cardiac markers troponin I, troponin T, myosin heavy chain, and connexin-43. Some engrafted CSCs also formed vascular structures and expressed α-smooth muscle actin. CONCLUSIONS: Intracoronary infusion of autologous CSCs improves regional and global LV function and promotes cardiac and vascular regeneration in pigs with old myocardial infarction (scar). The results mimic those recently reported in humans (Stem Cell Infusion in Patients with Ischemic CardiOmyopathy [SCIPIO] trial) and establish this porcine model of ischemic cardiomyopathy as a useful and clinically relevant model for studying CSCs.

The heme oxygenase 1 inducer (CoPP) protects human cardiac stem cells against apoptosis through activation of the extracellular signal-regulated kinase (ERK)/NRF2 signaling pathway and cytokine release.

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H(2)O(2), concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

Transplantation of expanded bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) improves left ventricular function and remodelling after myocardial infarction.

Adult bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) exhibit a Sca-1(+)/Lin(-)/CD45(-) phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (1 × 10(6)) of freshly isolated, non-expanded VSEL-SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL-SCs in the marrow is very low, we examined whether VSEL-SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n = 11), 1 × 10(5) enhanced green fluorescent protein (EGFP)-labelled expanded untreated VSEL-SCs (group II, n = 7), or 1 × 10(5) EGFP-labelled expanded VSEL-SCs pre-incubated in a cardiogenic medium (group III, n = 8). At 35 days after myocardial infarction (MI), mice treated with pre-incubated VSEL-SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle-treated controls. In contrast, transplantation of expanded but untreated VSEL-SCs did not produce appreciable reparative benefits. Scattered EGFP(+) cells expressing α-sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)-1, or von Willebrand factor were present in VSEL-SC-treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL-SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic growth factors and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre-incubation confers therapeutic efficacy does not involve differentiation into new cardiac cells. These results support the potential therapeutic utility of VSEL-SCs for cardiac repair.