Antibacterial activity of juglone @ chitosan nanoemulsion against Staphylococcus aureus and its effect on pork shelf life.

Food poisoning caused by Staphylococcus aureus (S. aureus) contaminated meat has received a lot of attention. Although juglone has anti-S. aureus properties, its limited water solubility prevents it from being used in food manufacturing. Juglone @ chitosan nanoemulsion (NJ) was produced for the first time in order to increase its solubility. At the same time, it was applied to the pork model. According to the findings, NJ's particle size was 119.30 nm, its polymer dispersity index (PDI) value was 0.290, and its zeta potential was -57.3 mV. And it's stable over a 7-day storage period. The cell shape and membrane integrity of S. aureus were significantly damaged by NJ. At the same time, NJ showed extreme vigor for biofilm removal. The inclusion of NJ coating significantly reduced S. aureus, total volatile base nitrogen (TVB-N), total viable count (TVC), thiobarbituric acid reactants (TBARS), and pH in the sample when using the pork feeding model. NJ, meantime, halted the sensory evaluation's fall in meat score. Additionally, NJ demonstrated good biocompatibility in mouse acute toxicity tests. The aforementioned findings demonstrate that NJ is anticipated to become an anti-S. aureus and a novel method for coating pork preservation.

Stability and emetic activity of enterotoxin like X (SElX) with high carrier rate of food poisoning Staphylococcus aureus.

In order to analyze and clarify the thermal stability of food poisoning Staphylococcus aureus (S. aureus) enterotoxin-like X (SElX) and the biological characteristics of digestive enzymes, and to evaluate the risk of S. aureus carrying selx gene in food poisoning, the selx gene carrying rates of 165 strains isolated from 95 food poisoning events from 2006 to 2019 were first statistically analyzed. Subsequently, the purified recombinant SElX protein was digested and heated, and the superantigen activity was verified with mouse spleen cells and peripheral blood mononuclear cells of kittens. At the same time, the emetic activity and toxicity of SElX were evaluated using the kitten vomiting animal model, mice toxin model and in vitro cell models. The results showed the selx gene carrying rate of 165 food poisoning S. aureus strains was 90.30 %. SElX had significant resistance to heat treatment and pepsin digestion (pH = 4.0 and pH = 4.5), and had good superantigen activity and emetic activity. However, there is no significant lethal effect on mice and no significant toxicity to cells. Importantly, we found that SElX had an inhibitory effect on acidic mucus of goblet cells in various segments of the small intestine. The present study investigated the stability of SElX, and confirmed the emetic activity of SElX by establishing a kitten vomiting model for the first time, suggesting that SElX is a high risk toxin of food poisoning, which will provide new ideas for the prevention and control of S. aureus food poisoning.

Antibacterial Activity of Juglone Revealed in a Wound Model of Staphylococcus aureus Infection.

A serious problem currently facing the field of wound healing is bacterial infection, especially Staphylococcus aureus (S. aureus) infection. Although the application of antibiotics has achieved good effects, their irregular use has resulted in the emergence of drug-resistant strains. It is thus the purpose of this study to analyze whether the naturally extracted phenolic compound, juglone, can inhibit S. aureus in wound infection. The results show that the minimum inhibitory concentration (MIC) of juglone against S. aureus was 1000 µg/mL. Juglone inhibited the growth of S. aureus by inhibiting membrane integrity and causing protein leakage. At sub-inhibitory concentrations, juglone inhibited biofilm formation, the expression of α-hemolysin, the hemolytic activity, and the production of proteases and lipases of S. aureus. When applied to infected wounds in Kunming mice, juglone (50 µL juglone with a concentration of 1000 µg/mL) significantly inhibited the number of S. aureus and had a significant inhibitory effect on the expression of inflammatory mediators (TNF-α, IL-6 and IL-1ß). Moreover, the juglone-treated group promoted wound healing. At the same time, in animal toxicity experiments, juglone had no obvious toxic effects on the main tissues and organs of mice, indicating that juglone has good biocompatibility and has the potential to be used in the treatment of wounds infected with S. aureus.

Lysine Inhibits Hemolytic Activity of Staphylococcus aureus and Its Application in Food Model Contaminated with Staphylococcus aureus.

Alpha-hemolysin (Hla) is one of the important exotoxins of Staphylococcus aureus (S. aureus) and can be used as a target to reduce the virulence of S. aureus. This study explored the inhibitory effect of Lysine (Lys) on Hla and its application in food safety. Lys significantly inhibited the expression of Hla at sub-inhibitory concentrations and directly interacted with Hla to interfere with its oligomerization and thus significantly inhibited its hemolytic activity. Notably, Lys attenuated S. aureus damage to mouse small intestine and Caco-2 cells and delayed mouse mortality. In the food model, Lys inhibited the expression of Hla of S. aureus and had no significant effect on the sensory score. Moreover, Lys had no obvious damage effect on the main organs of mice, which indicated that Lys has good biocompatibility and has the potential to be used in the food industry as an anti-S. aureus preparation.

Widely targeted metabolomics analysis of enriched secondary metabolites and determination of their corresponding antioxidant activities in Elaeagnus angustifolia var. orientalis (L.)Kuntze fruit juice enhanced by Bifidobacterium animalis subsp. Lactis HN-3 fermentation.

Elaeagnus angustifolia var. orientalis (L.)Kuntze fruit contains a large number of naturally occurring molecules present as glycoside, methylated, and methyl ester conjugates, which should be hydolysed or transformed to become bioactive forms. For this purpose, Bifidobacterium animalis subsp. lactis HN-3 was selected to ferment Elaeagnus angustifolia var. orientalis (L.)Kuntze fruit juice (EOJ). After fermentation, the total phenolic content (TPC) and antioxidant capacity of the EOJ increased significantly compared to the non-fermented EOJ. Using widely-targeted metabolomics analysis, polyphenolic compounds involved in the flavonoid biosynthetic pathway were determined to be up-regulated in the fermented EOJ. In addition, the metabolites generated by 8 deglycosidation, 5 demethylation, 5 hydrogenation, and 28 other reactions were detected in higher concentrations in the fermented EOJ compared to the non-fermented EOJ. Interestingly, these up-regulated metabolites have higher antioxidant and other biological activities than their metabolic precursors, which provide a theoretical basis for the development of Bifidobacterium-fermented plant products with stronger functional activities.

Effects of Heme Oxygenase-1 on c-Kit-Positive Cardiac Cells.

Heme oxygenase-1 (HO-1) is one of the most powerful cytoprotective proteins known. The goal of this study was to explore the effects of HO-1 in c-kit-positive cardiac cells (CPCs). LinNEG/c-kitPOS CPCs were isolated and expanded from wild-type (WT), HO-1 transgenic (TG), or HO-1 knockout (KO) mouse hearts. Compared with WT CPCs, cell proliferation was significantly increased in HO-1TG CPCs and decreased in HO-1KO CPCs. HO-1TG CPCs also exhibited a marked increase in new DNA synthesis during the S-phase of cell division, not only under normoxia (21% O2) but after severe hypoxia (1% O2 for 16 h). These properties of HO-1TG CPCs were associated with nuclear translocation (and thus activation) of Nrf2, a key transcription factor that regulates antioxidant genes, and increased protein expression of Ec-SOD, the only extracellular antioxidant enzyme. These data demonstrate that HO-1 upregulates Ec-SOD in CPCs and suggest that this occurs via activation of Nrf2, which thus is potentially involved in the crosstalk between two antioxidants, HO-1 in cytoplasm and Ec-SOD in extracellular matrix. Overexpression of HO-1 in CPCs may improve the survival and reparative ability of CPCs after transplantation and thus may have potential clinical application to increase efficacy of cell therapy.

Exercise-induced late preconditioning in mice is triggered by eNOS-dependent generation of nitric oxide and activation of PKCε and is mediated by increased iNOS activity.

The purpose of this study was to assess whether short-term, mild exercise induces protection against myocardial infarction and, if so, what role the eNOS-PKCε-iNOS axis plays. Mice were subjected to 2 bouts/day of treadmill exercise (60 min at 15 m/min) for 2 consecutive days. At 24 h after the last bout of exercise, mice were subjected to a 30-min coronary artery occlusion and 24 h of reperfusion. In the exercise group (group III, wild-type mice), infarct size (25.5 ± 8.8% of risk region) was significantly (P < 0.05) reduced compared with the control groups (sham exercise, group II [63.4 ± 7.8%] and acute myocardial infarction, group I [58.6 ± 7.0%]). This effect was abolished by pretreatment with the NOS inhibitor L-NA (group VI, 56.1 ± 16.2%) and the PKC inhibitor chelerythrine (group VIII, 57.9 ± 12.5%). Moreover, the late PC effect of exercise was completely abrogated in eNOS-/- mice (group XIII, 61.0 ± 11.2%). The myocardial phosphorylated eNOS at Ser-1177 was significantly increased at 30 min after treadmill training (exercise group) compared with sham-exercised hearts. PKCε translocation was significantly increased at 30 min after exercise in WT mice but not in eNOS-/- mice. At 24 h after exercise, iNOS protein was upregulated compared with sham-exercised hearts. The protection of late PC was abrogated in iNOS-/- mice (group XVI, 56.4 ± 12.9%) and in wildtype mice given the selective iNOS inhibitor 1400 W prior to ischemia (group X 62.0 ± 8.8% of risk region). We conclude that 1) even short, mild exercise induces a delayed PC effect that affords powerful protection against infarction; 2) this cardioprotective effect is dependent on activation of eNOS, eNOS-derived NO generation, and subsequent PKCε activation during PC; 3) the translocation of PKCε is dependent on eNOS; 4) the protection 24 h later is dependent on iNOS activity. Thus, eNOS is the trigger and iNOS the mediator of PC induced by mild exercise.

Single dose of synthetic microRNA-199a or microRNA-149 mimic does not improve cardiac function in a murine model of myocardial infarction.

Intramyocardial injection of synthetic microRNAs (miRs) has recently been reported to be beneficial after myocardial infarction (MI). We conducted a randomized blinded study to evaluate the efficacy and reproducibility of this strategy in a mouse model of reperfused MI using rigorous methodology. Mice undergoing a 60-min coronary occlusion followed by reperfusion were randomly assigned to control miR, hsa-miR-199a-3p, hsa-miR-149-3p, or hsa-miR-149-5p mimic treatment. Intramyocardial injections of miRs were performed in the border zone right after reperfusion. At 8 weeks after MI, there were no significant differences in ejection fraction (EF) among groups (EF = 27.1 ± 0.4% in control group [n = 6] and 25.9 ± 0.5%, 26.0 ± 0.8%, and 26.6 ± 0.6% in hsa-miR-199a-3p, hsa-miR-149-3p, or hsa-miR-149-5p groups, respectively [n = 9 each]). Net change (delta) in EF at 8 weeks compared with day 3 after MI was - 4.1% in control and - 3.2%, - 2.4%, and - 0.4% in the miR-treated groups (P = NS). Assessment of cardiac function by hemodynamic studies (a method independent of echocardiography) confirmed that there was no difference in left ventricular systolic or diastolic function among groups. Consistent with the functional data, histological analysis showed no difference in scar size, cardiomyocyte area, capillary density, collagen content, or apoptosis among groups. In conclusion, this randomized, blinded study demonstrates that intramyocardial injection of a single dose of synthetic hsa-miR-199a-3p, hsa-miR-149-3p, or hsa-miR-149-5p mimic does not improve cardiac function or remodeling in a murine model of reperfused MI. The strategy of using synthetic miR mimics for cardiac repair after MI needs to be evaluated with rigorous preclinical studies before its potential clinical translation.

Cardiac Mesenchymal Cells Cultured at Physiologic Oxygen Tension Have Superior Therapeutic Efficacy in Heart Failure Caused by Myocardial Infarction.

Stem/progenitor cells are usually cultured at atmospheric O2 tension (21%); however, since physiologic O2 tension in the heart is ∼5%, using 21% O2 may cause oxidative stress and toxicity. Cardiac mesenchymal cells (CMCs), a newly discovered and promising type of progenitor cells, are effective in improving left ventricle (LV) function after myocardial infarction (MI). We have previously shown that, compared with 21% O2, culture at 5% O2 increases CMC proliferation, telomerase activity, telomere length, and resistance to severe hypoxia in vitro. However, it is unknown whether these beneficial effects of 5% O2 in vitro translate into greater therapeutic efficacy in vivo in the treatment of heart failure. Thus, murine CMCs were cultured at 21% or 5% O2. Mice with heart failure caused by a 60-min coronary occlusion followed by 30 days of reperfusion received vehicle, 21% or 5% O2 CMCs via echocardiography-guided intraventricular injection. After 35 days, the improvement in LV ejection fraction effected by 5% O2 CMCs was > 3 times greater than that afforded by 21% O2 CMCs (5.2 vs. 1.5 units, P < 0.01). Hemodynamic studies (Millar catheter) yielded similar results both for load-dependent (LV dP/dt) and load-independent (end-systolic elastance) indices. Thus, two independent approaches (echo and hemodynamics) demonstrated the therapeutic superiority of 5% O2 CMCs. Further, 5% O2 CMCs, but not 21% O2 CMCs, significantly decreased scar size, increased viable myocardium, reduced LV hypertrophy and dilatation, and limited myocardial fibrosis both in the risk and non-infarcted regions. Taken together, these results show, for the first time, that culturing CMCs at physiologic (5%) O2 tension provides superior therapeutic efficacy in promoting cardiac repair in vivo. This concept may enhance the therapeutic potential of CMCs. Further, culture at 5% O2 enables greater numbers of cells to be produced in a shorter time, thereby reducing costs and effort and limiting cell senescence. Thus, the present study has potentially vast implications for the field of cell therapy.

Comparison of Repeated Doses of C-kit-Positive Cardiac Cells versus a Single Equivalent Combined Dose in a Murine Model of Chronic Ischemic Cardiomyopathy.

Using a murine model of chronic ischemic cardiomyopathy caused by an old myocardial infarction (MI), we have previously found that three doses of 1 × 106 c-kit positive cardiac cells (CPCs) are more effective than a single dose of 1 × 106 cells. The goal of this study was to determine whether the beneficial effects of three doses of CPCs (1 × 106 cells each) can be fully replicated by a single combined dose of 3 × 106 CPCs. Mice underwent a 60-min coronary occlusion; after 90 days of reperfusion, they received three echo-guided intraventricular infusions at 5-week intervals: (1) vehicle × 3; (2) one combined dose of CPCs (3 × 106) and vehicle × 2; or (3) three doses of CPCs (1 × 106 each). In the combined-dose group, left ventricular ejection fraction (LVEF) improved after the 1st CPC infusion, but not after the 2nd and 3rd (vehicle) infusions. In contrast, in the multiple-dose group, LVEF increased after each CPC infusion; at the final echo, LVEF averaged 35.2 ± 0.6% (p < 0.001 vs. the vehicle group, 27.3 ± 0.2%). At the end of the study, the total cumulative change in EF from pretreatment values was numerically greater in the multiple-dose group (6.6 ± 0.6%) than in the combined-dose group (4.8 ± 0.8%), although the difference was not statistically significant (p = 0.08). Hemodynamic studies showed that several parameters of LV function in the multiple-dose group were numerically greater than in the combined-dose group (p = 0.08 for the difference in LVEF). Compared with vehicle, cardiomyocyte cross-sectional area was reduced only in the multiple-dose group (-32.7%, 182.6 ± 15.1 µm2 vs. 271.5 ± 27.2 µm2, p < 0.05, in the risk region and -28.5%, 148.5 ± 12.1 µm2 vs. 207.6 ± 20.5 µm2, p < 0.05, in the noninfarcted region). LV weight/body weight ratio and LV weight/tibia length ratios were significantly reduced in both cell treated groups vs. the vehicle group, indicating the attenuation of LV hypertrophy; however, the lung weight/body weight ratio was significantly reduced only in the multiple-dose group, suggesting decreased pulmonary congestion. Taken together, these results indicate that in mice with chronic ischemic cardiomyopathy, the beneficial effects of three doses of CPCs on LV function and hypertrophy cannot be fully replicated with a single dose, notwithstanding the fact that the total number of cells delivered with one or three doses is the same. Thus, it is the multiplicity of doses, and not the total number of cells, that accounts for the superiority of the repeated-dose paradigm. This study supports the idea that the efficacy of cell therapy in heart failure can be augmented by repeated administrations.

Echocardiography-guided percutaneous left ventricular intracavitary injection as a cell delivery approach in infarcted mice.

In the field of cell therapy for heart disease, a new paradigm of repeated dosing of cells has recently emerged. However, the lack of a repeatable cell delivery method in preclinical studies in rodents is a major obstacle to investigating this paradigm. We have established and standardized a method of echocardiography-guided percutaneous left ventricular intracavitary injection (echo-guided LV injection) as a cell delivery approach in infarcted mice. Here, we describe the method in detail and address several important issues regarding it. First, by integrating anatomical and echocardiographic considerations, we have established strategies to determine a safe anatomical window for injection in infarcted mice. Second, we summarize our experience with this method (734 injections). The overall survival rate was 91.4%. Third, we examined the efficacy of this cell delivery approach. Compared with vehicle treatment, cardiac mesenchymal cells (CMCs) delivered via this method improved cardiac function assessed both echocardiographically and hemodynamically. Furthermore, repeated injections of CMCs via this method yielded greater cardiac function improvement than single-dose administration. Echo-guided LV injection is a feasible, reproducible, relatively less invasive and effective delivery method for cell therapy in murine models of heart disease. It is an important approach that could move the field of cell therapy forward, especially with regard to repeated cell administrations.

Physiological Oxygen Tension Enhances Competence and Functional Properties of Murine Cardiac Mesenchymal Cells.

Cardiac mesenchymal cells (CMCs), a newly-discovered and promising type of progenitor cells, are effective in improving cardiac function in rodents after myocardial infarction. Stem/progenitor cells are usually cultured at atmospheric O2 tension (21%); however, the physiologic O2 tension in the heart is ~5%, raising the concern that 21% O2 may cause toxicity due to oxidative stress. Thus, we compared mouse CMCs cultured at 21% or 5% O2 beginning at passage 2. At passage 5, CMCs underwent severe hypoxic stress (1% O2 for 24 h). Compared with CMCs cultured at 21% O2, culture at 5% O2 consistently improved cell morphology throughout 5 passages, markedly decreased cell size, increased cell number, shortened cell doubling time, and dramatically reduced lactate dehydrogenase release from CMCs into culture media after hypoxic stress. Furthermore, culture at 5% O2 increased telomerase activity and telomere length, implying that 21% O2 tension impairs telomerase activity, resulting in telomere shortening and decreased cell proliferation. Thus far, almost all preclinical and clinical studies of cell therapy for the heart disease have used atmospheric (21%) O2 to culture cells. Our data challenge this paradigm. Our results demonstrate that, compared with 21% O2, 5% O2 tension greatly enhances the competence and functional properties of CMCs. The increased proliferation rate at 5% O2 means that target numbers of CMCs can be achieved with much less time and cost. Furthermore, since this increased proliferation may continue in vivo after CMC transplantation, and since cells grown at 5% O2 are markedly resistant to severe hypoxic stress, and thus may be better able to survive after transplantation into scarred regions of the heart where O2 is very low, culture at 5% O2 may enhance the reparative properties of CMCs (and possibly other cell types). In conclusion, our data support a change in the methods used to culture CMCs and possibly other progenitor cells.

Comparison of One and Three Intraventricular Injections of Cardiac Progenitor Cells in a Murine Model of Chronic Ischemic Cardiomyopathy.

Repeated doses of c-kit+ cardiac progenitor cells (CPCs) are superior to a single dose in improving LV function in rats with old myocardial infarction (MI). However, this concept needs testing in different species to determine whether it is generalizable. We used a new murine model of chronic ischemic cardiomyopathy whose unique feature is that cell therapy was started late (3 months) after MI. Mice received three echo-guided intraventricular infusions, 5 weeks apart, of vehicle, CPCs × 1, or CPCs × 3. Echocardiography demonstrated that the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (CPCs), but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group LVEF continued to improve, so that the final value was greater than in vehicle or single-dose groups (P < 0.05). Hemodynamic studies showed that compared with vehicle, both preload-dependent and preload-independent functional parameters were significantly increased in the multiple-dose group but not in the single-dose group. Thus, two independent methods of functional assessment (echocardiography and hemodynamic studies) consistently demonstrated the superiority of three doses of CPCs vs. one dose. Compared with the single-dose group, the multiple-dose group exhibited less LV hypertrophy, as evidenced by a greater reduction in LV/body weight ratio and cardiomyocyte cross-sectional area. Furthermore, unlike the single dose, three CPC doses reduced myocardial inflammatory cells in the risk region. This is the first study of echo-guided intraventricular infusion of CPCs in mice with chronic ischemic cardiomyopathy. The results demonstrate that the beneficial effects of three CPC doses are greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate cell therapy. Our findings indicate that this concept applies not only to rat models but also to murine models. The generalizability of this strategy greatly enhances its importance and provides a rationale for large animal studies. Graphical abstract.

Serum Magnesium and Fractional Exhaled Nitric Oxide in Relation to the Severity in Asthma-Chronic Obstructive Pulmonary Disease Overlap.

Serum concentrations of magnesium and manganese may be associated with increased chronic obstructive pulmonary disease exacerbation risk. However, associations with other aspects of asthma-chronic obstructive pulmonary disease overlap, pulmonary function test results and health status, have been studied less extensively. The aim of this study was to investigate the associations between serum concentrations of trace elements and T lymphocyte subsets, FeNO, and COPD-related questionnaire scores in individuals with ACO and the potential impact of these parameters on lung function. All the patients met the diagnostic criteria of ACO and were divided into two groups (group A, mild-moderate; group B, severe-very severe) by their specific characteristics. Pulmonary function testing and serum Mg and serum Mn and FeNO were measured. Four hundred sixty-five patients were screened, and 42 were included. Group A had significantly higher Mg and Fe concentrations than group B. No significant differences were seen in the serum concentration of any other trace element between the two groups. Serum Mg and Mn were correlated with FEV1% predicted (p < 0.01). Group A had a significantly higher FeNO concentration than group B (p = 0.005). The scores on CAT (p = 0.011) and mMRC (p = 0.008) in group A were lower than in group B. The low-FeNO group had a significantly lower concentration of serum Mg than the high-FeNO group (p = 0.03). Pulmonary function declined faster (p < 0.05) in the low-FeNO group than the high-FeNO group. Serum Mg concentration may indicate protective effects against lung function loss in ACO. This implies that FeNO might be a biomarker for identifying individuals with ACO who might benefit from inhaled corticosteroid therapy. Serum Mg and FeNO were associated with ACO severity. However, their role in guiding personalised treatment of individuals with ACO needs to be further investigated.

Inducible cardiac-specific overexpression of cyclooxygenase-2 (COX-2) confers resistance to ischemia/reperfusion injury.

The role of cyclooxygenase-2 (COX-2) in cardiovascular biology remains controversial. Although COX-2 has been reported to mediate the protective actions of late preconditioning, other studies show that it is also an important mediator of inflammation, toxic shock, and apoptosis, resulting in significant dysfunction and injury in several tissues. To determine whether increased myocardial COX-2, in itself, is protective, cardiac-specific, inducible (Tet-off) COX-2 transgenic (iCOX-2 TG) mice were generated by crossbreeding α-MyHC-tTA transgenic mice (tetracycline transactivator [tTA]) with CMV/TRE-COX-2 transgenic mice. Three months after COX-2 induction, mice were subjected to a 30-min coronary occlusion and 24 h of reperfusion. Three different lines (L5, L7, and L8) of iCOX-2 TG mice were studied; in all three lines, infarct size was markedly reduced compared with WT mice: L5 TG/TG 23.4 ± 5.8 vs. WT/WT 48.5 ± 6.1% of risk region; L7 TG/TG 23.2 ± 6.2 vs. WT/WT 53.3 ± 3.6%; and L8 TG/TG 23.5 ± 2.8 vs. WT/WT 52.7 ± 4.6% (P < 0.05 for each). COX-2 inhibition with NS-398 completely abolished the cardioprotection provided by COX-2 overexpression. This study for the first time utilizes an inducible cardiac-specific COX-2 overexpression system to examine the role of this enzyme in ischemia/reperfusion injury in vivo. We demonstrate that induced cardiac-specific overexpression of COX-2 exerts a potent cardioprotective effect against myocardial infarction in mice, and that chronic COX-2 overexpression is not associated with any apparent deleterious effects. We also show that PGE2 levels are upregulated in COX-2 overexpressing cardiac tissue, confirming increased enzyme activity. Finally, we have developed a valuable genetic tool to further our understanding of the role of COX-2 in ischemia/reperfusion injury and other settings. The concept that COX-2 is chronically protective has important therapeutic implications for studies of long-term gene therapy aimed at increasing myocardial COX-2 content as well as other COX-2- based strategies.

Cardiac mesenchymal cells from diabetic mice are ineffective for cell therapy-mediated myocardial repair.

Although cell therapy improves cardiac function after myocardial infarction, highly variable results and limited understanding of the underlying mechanisms preclude its clinical translation. Because many heart failure patients are diabetic, we examined how diabetic conditions affect the characteristics of cardiac mesenchymal cells (CMC) and their ability to promote myocardial repair in mice. To examine how diabetes affects CMC function, we isolated CMCs from non-diabetic C57BL/6J (CMCWT) or diabetic B6.BKS(D)-Leprdb/J (CMCdb/db) mice. When CMCs were grown in 17.5 mM glucose, CMCdb/db cells showed > twofold higher glycolytic activity and a threefold higher expression of Pfkfb3 compared with CMCWT cells; however, culture of CMCdb/db cells in 5.5 mM glucose led to metabolic remodeling characterized by normalization of metabolism, a higher NAD+/NADH ratio, and a sixfold upregulation of Sirt1. These changes were associated with altered extracellular vesicle miRNA content as well as proliferation and cytotoxicity parameters comparable to CMCWT cells. To test whether this metabolic improvement of CMCdb/db cells renders them suitable for cell therapy, we cultured CMCWT or CMCdb/db cells in 5.5 mM glucose and then injected them into infarcted hearts of non-diabetic mice (CMCWT, n = 17; CMCdb/db, n = 13; Veh, n = 14). Hemodynamic measurements performed 35 days after transplantation showed that, despite normalization of their properties in vitro, and unlike CMCWT cells, CMCdb/db cells did not improve load-dependent and -independent parameters of left ventricular function. These results suggest that diabetes adversely affects the reparative capacity of CMCs and that modulating CMC characteristics via culture in lower glucose does not render them efficacious for cell therapy.

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 doses of cardiac mesenchymal cells are therapeutically superior to a single dose in mice with old myocardial infarction.

We have recently demonstrated that repeated administrations of c-kitPOS cardiac progenitor cells (CPCs) have cumulative beneficial effects in rats with old myocardial infarction (MI), resulting in markedly greater improvement in left ventricular (LV) function compared with a single administration. To determine whether this paradigm applies to other species and cell types, mice with a 3-week-old MI received one or three doses of cardiac mesenchymal cells (CMCs), a novel cell type that we have recently described. CMCs or vehicle were infused percutaneously into the LV cavity, 14 days apart. Compared with vehicle-treated mice, the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (consisting of CMCs) but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group, LV EF improved after each CMC infusion, so that at the end of the study, LV EF averaged 35.5 ± 0.7% vs. 32.7 ± 0.6% in the single-dose group (P < 0.05). The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of CMCs were negligible in both groups, indicating paracrine effects. These results demonstrate that, in mice with ischemic cardiomyopathy, the beneficial effects of three doses of CMCs are significantly greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy. Thus, the repeated-treatment paradigm is not limited to c-kit POS CPCs or to rats, but applies to other cell types and species. The generalizability of this concept dramatically augments its significance.

TNF receptor signaling inhibits cardiomyogenic differentiation of cardiac stem cells and promotes a neuroadrenergic-like fate.

Despite expansion of resident cardiac stem cells (CSCs; c-kit+Lin-) after myocardial infarction, endogenous repair processes are insufficient to prevent adverse cardiac remodeling and heart failure (HF). This suggests that the microenvironment in post-ischemic and failing hearts compromises CSC regenerative potential. Inflammatory cytokines, such as tumor necrosis factor-α (TNF), are increased after infarction and in HF; whether they modulate CSC function is unknown. As the effects of TNF are specific to its two receptors (TNFRs), we tested the hypothesis that TNF differentially modulates CSC function in a TNFR-specific manner. CSCs were isolated from wild-type (WT), TNFR1-/-, and TNFR2-/- adult mouse hearts, expanded and evaluated for cell competence and differentiation in vitro in the absence and presence of TNF. Our results indicate that TNF signaling in murine CSCs is constitutively related primarily to TNFR1, with TNFR2 inducible after stress. TNFR1 signaling modestly diminished CSC proliferation, but, along with TNFR2, augmented CSC resistance to oxidant stress. Deficiency of either TNFR1 or TNFR2 did not impact CSC telomerase activity. Importantly, TNF, primarily via TNFR1, inhibited cardiomyogenic commitment during CSC differentiation, and instead promoted smooth muscle and endothelial fates. Moreover, TNF, via both TNFR1 and TNFR2, channeled an alternate CSC neuroadrenergic-like fate (capable of catecholamine synthesis) during differentiation. Our results suggest that elevated TNF in the heart restrains cardiomyocyte differentiation of resident CSCs and may enhance adrenergic activation, both effects that would reduce the effectiveness of endogenous cardiac repair and the response to exogenous stem cell therapy, while promoting adverse cardiac remodeling.