Genetic Lineage Tracing of Pericardial Cavity Macrophages in the Injured Heart.

BACKGROUND: Macrophages play an important role in cardiac repair after myocardial infarction (MI). In addition to the resident macrophages and blood-derived monocytes, Gata6+ cavity macrophages located in the pericardial space were recently reported to relocate to the injured myocardium and prevent cardiac fibrosis. However, there is no direct genetic evidence to support it. METHODS: We used dual recombinases (Cre and Dre) to specifically label Gata6+ pericardial macrophages (GPCMs) in vivo. For functional study, we generated genetic systems to specifically ablate GPCMs by induced expression of DTR (diphtheria toxin receptor) or knockout of Gata6 (GATA binding protein 6) gene in GPCMs. We used these genetic systems to study GPCMs in pericardium intact MI model. RESULTS: Dual recombinases-mediated genetic system targeted GPCMs specifically and efficiently. Lineage tracing study revealed accumulation of GPCMs on the surface of MI heart without deep penetration into the myocardium. We did not detect significant change of cardiac fibrosis or function of MI hearts after cell ablation or Gata6 knockout in GPCMs. CONCLUSIONS: GPCMs minimally invade the injured heart after MI. Nor do they prevent cardiac fibrosis and exhibit reparative function on injured heart. This study also underlines the importance of using specific genetic tool for studying in vivo cell fates and functions.

Distinct Phenotypes Induced by Different Degrees of Transverse Aortic Constriction in C57BL/6N Mice.

The transverse aortic constriction (TAC) model surgery is a widely used disease model to study pressure overload-induced cardiac hypertrophy and heart failure in mice. The severity of adverse cardiac remodeling of the TAC model is largely dependent on the degree of constriction around the aorta, and the phenotypes of TAC are also different in different mouse strains. Few studies focus on directly comparing phenotypes of the TAC model with different degrees of constriction around the aorta, and no study compares the difference in C57BL/6N mice. In the present study, C57BL/6N mice aged 10 weeks were subjected to sham, 25G TAC, 26G TAC, and 27G TAC surgery for 4 weeks. We then analyzed the different phenotypes induced by 25G TAC, 26G TAC, and 27G TAC in c57BL/6N mice in terms of pressure gradient, cardiac hypertrophy, cardiac function, heart failure situation, survival condition, and cardiac fibrosis. All C57BL/6N mice subjected to TAC surgery developed significantly hypertrophy. Mice subjected to 27G TAC had severe cardiac dysfunction, severe cardiac fibrosis, and exhibited characteristics of heart failure at 4 weeks post-TAC. Compared with 27G TAC mice, 26G TAC mice showed a much milder response in cardiac dysfunction and cardiac fibrosis compared to 27G TAC, and a very small fraction of the 26G TAC group exhibited characteristics of heart failure. There was no obvious cardiac dysfunction, cardiac fibrosis, and characteristics of heart failure observed in 25G TAC mice. Based on our results, we conclude that the 25G TAC, 26G TAC, and 27G TAC induced distinct phenotypes in C57BL/6N mice.

Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction.

Characterizing the stem cells responsible for lung repair and regeneration is important for the treatment of pulmonary diseases. Recently, a unique cell population located at the bronchioalveolar-duct junctions has been proposed to comprise endogenous stem cells for lung regeneration. However, the role of bronchioalveolar stem cells (BASCs) in vivo remains debated, and the contribution of such cells to lung regeneration is not known. Here we generated a genetic lineage-tracing system that uses dual recombinases (Cre and Dre) to specifically track BASCs in vivo. Fate-mapping and clonal analysis showed that BASCs became activated and responded distinctly to different lung injuries, and differentiated into multiple cell lineages including club cells, ciliated cells, and alveolar type 1 and type 2 cells for lung regeneration. This study provides in vivo genetic evidence that BASCs are bona fide lung epithelial stem cells with deployment of multipotency and self-renewal during lung repair and regeneration.

Diosgenin Protects Rats from Myocardial Inflammatory Injury Induced by Ischemia-Reperfusion.

BACKGROUND Diosgenin, a phytosteroid sapogenin, has anti-inflammatory properties shown to reduce myocardial ischemia-reperfusion injury (MIRI). However, the specific mechanism by which this is achieved is not clear. This study investigated the protective effects of diosgenin on myocardial ischemia/reperfusion (I/R) and the potential anti-inflammatory mechanisms. MATERIAL AND METHODS Healthy adult male SD rats, body weight (b.w.) 250-280 g, were used to model ischemia-reperfusion injury (IRI) and were administered diosgenin (50 mg/kg and 100 mg/kg b.w.) intragastrically for 4 consecutive weeks before surgery. The left anterior descending artery (LAD) was ligated to induce myocardial ischemia for 30 min and reperfusion for 30 min, 60 min, and 120 min while relevant indicators were detected. RESULTS Both 50 mg and 100 mg diosgenin oral administration increased left ventricular developed pressure (LVDP) and maximum changing rate of ventricular pressure (±dp/dtmax), decreased left ventricular end-diastolic pressure (LVEDP), and myocardial enzyme markers. TTC staining suggested that diosgenin reduced myocardial infarct size in the rat model. Pathological results showed that myocardial ischemia and inflammation were alleviated by diosgenin. In addition, the increased expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) in serum, and myeloperoxidase (MPO) in myocardium were significantly suppressed by diosgenin administration. Diosgenin further inhibited the phosphorylation of transcription factor NF-κB and modulated the expression of downstream inflammatory cytokines by regulating the activation of p38-MAPK and JNK pathways. CONCLUSIONS Results demonstrate diosgenin plays an anti-inflammatory role in the protection of MIRI through regulation of p38-MAPK and JNK pathways and phosphorylation of NF-κB.

Identification of a hybrid myocardial zone in the mammalian heart after birth.

Noncompaction cardiomyopathy is characterized by the presence of extensive trabeculations, which could lead to heart failure and malignant arrhythmias. How trabeculations resolve to form compact myocardium is poorly understood. Elucidation of this process is critical to understanding the pathophysiology of noncompaction disease. Here we use genetic lineage tracing to mark the Nppa+ or Hey2+ cardiomyocytes as trabecular and compact components of the ventricular wall. We find that Nppa+ and Hey2+ cardiomyocytes, respectively, from the endocardial and epicardial zones of the ventricular wall postnatally. Interposed between these two postnatal layers is a hybrid zone, which is composed of cells derived from both the Nppa+ and Hey2+ populations. Inhibition of the fetal Hey2+ cell contribution to the hybrid zone results in persistence of excessive trabeculations in postnatal heart. Our findings indicate that the expansion of Hey2+ fetal compact component, and its contribution to the hybrid myocardial zone, are essential for normal formation of the ventricular walls.Fetal trabecular muscles in the heart undergo a poorly described morphogenetic process that results into a solidified compact myocardium after birth. Tian et al. show that cardiomyocytes in the fetal compact layer also contribute to this process, forming a hybrid myocardial zone that is composed of cells derived from both trabecular and compact layers.

Eupatilin inhibits the apoptosis in H9c2 cardiomyocytes via the Akt/GSK-3ß pathway following hypoxia/reoxygenation injury.

Eupatilin, a pharmacologically active flavone derived from the Artemisia plant species, has been reported to have anti-oxidant, anti-inflammatory, anti-allergic, and neuroprotective activities against cerebral ischemia/reperfusion (I/R). However, the role of eupatilin in myocardial I/R injury remains unclear. In the present study, we aimed to investigate the potential molecular mechanisms against hypoxia/reoxygenation (H/R) induced cardiomyocytes apoptosis in vitro. Our results showed that eupatilin markedly improved the cell viability and decreased lactate dehydrogenase (LDH) release. Eupatilin also suppressed oxidative stress and apoptosis in H9c2 cells after myocardial I/R injury. Furthermore, eupatilin obviously increased the phosphorylation of Akt and GSK-3ß in H9c2 cells. Our results suggested that eupatilin could provide significant cardioprotection against myocardial I/R injury, and the potential mechanisms might involve inhibition of cardiomyocyte apoptosis through activating the Akt/GSK-3ß signaling pathway.

Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes.

Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit(+) cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit(+) CSCs.

SALVIANOLIC ACID B ALLEVIATING MYOCARDIUM INJURY IN ISCHEMIA REPERFUSION RATS.

BACKGROUND: Salvia miltiorrhiza (SM) Bunge is one of the widely-used Chinese medicinal herbs. Salvianolic acid B (Sal B), a bioactive compound isolated from the Chinese herb Radix Salviae Miltiorrhizae, has been reported to exhibit anti-inflammatory and anti-oxidantive effects. MATERIAL AND METHOD: To study the cardioprotective effects of salvianolic acid B (Sal B) on acute myocardial ischemia reperfusion (MIR) injury rats, on the basis of this investigation, the possible mechanism of salvianolic acid B was elucidated. Male Sprague- Dawley rats (200-220 g) were randomly divided into five groups: sham-operated, MIR, MIR + Sal B (10 mg/kg/day, orally), MIR + Sal B (20 mg/kg/ day, orally) and MIR + Sal B (30 mg/kg/ day, orally). Before operation, the foregoing groups were pretreated with homologous drug once a day for 7 days, respectively. After twelve hours in MIR, the cardioprotective effects of SPJ were evaluated by infarct size, biochemical values, and the antioxidative and antiapoptotic relative gene expressions. RESULTS: Sal B significantly improved heart function and decreased infarct size; remarkably decreased levels of serum TNF-α and IL-Ιß levels, increased contents of myocardium antioxidant enzymes activities; western blot results showed that Sal B ameliorate the increased Bax and caspase-3 protins expressions and decreased Bcl-2 proteins expression and ratios of Bcl-2 to Bax. CONCLUSION: In ischemic myocardium, oxidative stress caused the overgeneration and accumulation of reactive oxygen species (ROS), which was central of cardiac ischemic injury. Sal B exerted beneficially cardioprotective effects on myocardial ischemia injury rats, mainly scavenging oxidative stress-triggered overgeneration and accumulation of ROS, alleviating myocardial ischemia injury and cardiac cell death. List of abbreviations: salvianolic acid B (Sal B); myocardial ischemia reperfusion (MIR); reactive oxygen species (ROS); Left ventricular end-diastolic pressure (LVEDP); left ventricular end-diastolic volume (LVEDV); Malondialdehyde (MDA); superoxide dismutase (SOD); catalase (CAT); Glutathione peroxidase (GSH-Px); glutathione reductase (GR).

Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic-reperfusion rats.

Salvia miltiorrhiza has strong antioxidative activity. They may have a strong potential as cardioprotective agents in ischemic-reperfusion injury. Experiments were carried out in Sprague-Dawley rats with myocardium ischemia reperfusion (IR). Myocardial injuries during IR were determined by changes in electrocardiogram analysis of arrhythmias, antioxidant enzyme activities, AST, CK-MB, lactate dehydrogenase (LDH) levels, and myocyte apoptosis. Results showed that S. miltiorrhiza aqueous extract (SAME) pre-treatment significantly decreased the ST-segment (ΣST120) and myocardium MDA, AST, CK-MB, lactate dehydrogenase (LDH) levels, increased myocardium antioxidant enzyme activities, and inhibit myocardium cell apoptosis. Furthermore, the SAME pre-treatment significantly upregulated p-JAK2 and p-STAT3 protein expression, decreased myocardium TNF-α and IL-6 concentrations in IR rats. The levels of TNF-α and IL-6 were positively correlated with the changes in myocardium p-JAK2 and p-STAT3 protein expression levels in IR rats. It can be concluded that the SAME pre-treatment has anti-ischemic and anti-apoptosis activity in heart IR rats. SAME pre-treatment protects heart against IR injury, at least in part, through its stimulating effects on injury-induced deactivation of JAK2/STAT3 signaling pathway.

Effects of ischemic preconditioning on myocardium Caspase-3, SOCS-1, SOCS-3, TNF-α and IL-6 mRNA expression levels in myocardium IR rats.

The aim of this study was to characterise the effects of ischemic preconditioning (IP) on heart function parameters (ΔST and ΔT), activities of serum creatine kinase (CK), lactate dehydrogenase (LDH), and levels of serum nitric oxide (NO), malondialdehyde (MDA), and myocardium Caspase-3 mRNA, SOCS-1, SOCS-3, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression levels and Apoptosis index in myocardium IR rats. Results showed that ΔST and ΔST values in IP group were markedly lower than those in IR group. Compared with IR group, IP significantly (p < 0.01) decreased serum CK (0.83 ± 0.09 vs 1.36 ± 0.15), LDH (5613 ± 462 vs 7106 ± 492) activities and MDA (11.32 ± 1.05 vs 15.49 ± 1.26) level, increased the serum NO (86.39 ± 7.03 vs 53.77 ± 4.27) level in IR group. The IP induced a significant decreased in myocardium Caspase-3 mRNA (0.303 ± 0.021 vs 0.515 ± 0.022) gene expression (p < 0.01) compared to IR model group. The IP induced a significant decreased in myocardium SOCS-1 (0.241 ± 0.031 vs 0.596 ± 0.036), SOCS-3 (0.258 ± 0.031 vs 0.713 ± 0.057), TNF-α (0.137 ± 0.011 vs 0.427 ± 0.035) and IL-6 (0.314 ± 0.021 vs 0.719 ± 0.064) mRNA gene expression (p < 0.01) compared to IR model group. We conclude that IP is effective in the therapy of heart disease. These findings may have implications for the clinical development of preconditioning-based therapies for ischemic heart disease.

Fosinoprilat alleviates lipopolysaccharide (LPS)-induced inflammation by inhibiting TLR4/NF-κB signaling in monocytes.

OBJECTIVE: To evaluate the effect of the fosinoprilat on lipopolysacharides (LPS) induced inflammation in monocytes in vitro. METHODS: Human mononuclear THP1 cells were cultured in complete medium, treated with or without LPS and different concentrations (0,0.25,0.5,1,5,and 10µmol/L) of fosinoprilat. Toll-like receptor (TLR4) mRNA expression was detected by real-time RT-PCR and TLR4 protein level on the surface of monocyte was determined by flow cytometry. Nuclear factor-kappa B (NF-κB) protein level was detected by Western blotting. Cultured supernatant of the THP1 cells in different groups were analyzed by ELISA to detect the levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF-α). RESULTS: Both the mRNA and surface protein level of the TLR4 in the THP1 cells were enhanced by the LPS treatment and down-regulated by pretreatment of the fosinoprilat. Accordingly, LPS-induced NF-κB protein was decreased by the fosinoprilat treatment. The increasing secretion of IL-1ß, IL-6 and TNF-α induced by LPS could also be attenuated by the fosinoprilat treatment. CONCLUSION: The inhibitory effect of the fosinoprilat on the TRL4/NF-κB signaling pathway reveals a potential anti-inflammatory and anti-atherosclerosis target.

Expression of sPLA2-V, estrogen and its target protein in myocardium tissue.

In this study, we examined the effects of Prostaglandin E1 and tea polysaccharides (TP) on serum estrogen and FSH levels, myocardium sPLA2-V positive levels, and sPLA2-V protein expression in the rats fed on hypercholesterolemic diet. Hyperlipidemic rats were treated with Prostaglandin E1 and TP. Serum estrogen and FSH levels were significantly enhanced by Prostaglandin E1 and TP, whereas myocardium sPLA2-V positive rate and protein expression levels were decreased compared to the HCD group. Our results suggest that Prostaglandin E1 and TP exert strong heart-protective effects and therefore can be used to reduce the risk of heart disorders.

Evaluation of the antioxidant potential of Salvia miltiorrhiza ethanol extract in a rat model of ischemia-reperfusion injury.

The present study was undertaken to evaluate the protection potential of ethanol extract of Salvia miltiorrhiza (SMEE) against oxidative injury in the ischemia-reperfusion (I/R) model of rats in vivo. Rats were divided into six groups of 10 rats each. Group I/R model and sham were fed with a standard rat chow, groups SMEE I and SMEE II were fed with a standard rat chow and 400 or 800 mg/kg b.w. ethanol extract for 12 days before the beginning of I/R studies. Positive control group was fed with a standard rat chow and salvianolic acid B (55 mg/kg b.w.) or tanshinone II-A (55 mg/kg b.w.) for 12 days before the beginning of I/R studies. To produce I/R, the left anterior descending artery (LAD) was occluded in anesthetized rats for 15 min, followed by 120 min reperfusion. Infarct sizes were found significantly decreased in SMEE-treated and positive control groups compared to I/R model group. Serum AST, LDH and CK-MB activities were significantly reduced and myocardium Na+-K+ ATPase, Ca2+-Mg2+ ATPase activities and antioxidant enzyme activities (SOD, CAT, GSH-Px) were markedly increased in SMEE-treated and salvianolic acid B or tanshinone II-A positive control groups compared to the I/R model group. Pretreatment of S. miltiorrhiza ethanol extract and salvianolic acid B or tanshinone II-A dose-dependently reduced significantly myocardium MDA level, ROS and NOS activities and enhanced myocardium GSH level in I/R rats compared to I/R rats model. In conclusion, we clearly demonstrated that S. miltiorrhiza ethanol extract pretreatment can decrease oxidative injury in rats subjected to myocardial I/R.