Apigenin induces oxidative stress in mouse Sertoli TM4 cells

Background and Aim: Apigenin (API) is an estrogenic compound found in many plants. Sertoli cells reside in the testis and are a key target of environmental toxicants. This study aimed to examine the cytotoxicity, especially oxidative stress of API in mouse Sertoli TM4 cells. Materials and Methods: Mouse Sertoli TM4 cells were treated with 50 and 100 μM API for 48 h. Cell viability, lactate dehydrogenase (LDH) activities, glutathione reductase (GR) activities, production of reactive oxygen species (ROS), and malondialdehyde (MDA) levels were evaluated using various assays. Results: Treatment with API at both 50 and 100 μM decreased viability and GR activity but increased LDH activity, ROS production, and MDA levels in mouse Sertoli TM4 cells. Conclusion: Exposure to API induced oxidative stress in mouse Sertoli TM4 cells.

Lactate production can function to increase human epithelial cell iron concentration

Iron is an essential micronutrient required by every cell, inclusive of both prokaryotes and eukaryotes. Under conditions of limited iron availability, plants and microbes evolved mechanisms to acquire iron which include carbon metabolism reprogramming, with the activity of several enzymes involved in the Krebs cycle and the glycolytic pathway being stimulated by metal deficiency. Following release, resultant carboxylates/hydroxycarboxylates can function as ligands to complex iron and facilitate its solubilization and uptake, reversing the deficiency. Human epithelial tissue may produce lactate, a hydroxycarboxylate, during absolute and functional iron deficiency in an attempt to import metal to reverse limited availability. Here we investigate 1) if lactate can increase cell metal import, 2) if lactic dehydrogenase (LDH) activity in and lactate production by cells correspond to metal availability, and 3) if blood concentrations of LDH in a human cohort correlate with indices of iron homeostasis. Exposures of Caco-2 cells to both Na lactate and ferric ammonium citrate (FAC) increased metal import relative to FAC alone. Fumaric, isocitric, malic, and succinic acid exposure revealed that FAC co-incubation similarly increased iron import relative to FAC alone. Increased iron import following exposures to Na lactate and FAC elevated both ferritin and metal associated with mitochondria. LDH in Caco-2 cell scrapings did not change after exposure to deferoxamine but decreased with 24 hr exposure to FAC. Lactate levels in both the supernatants and cell scrapings revealed decreased levels at 4, 8, and 24 hr with FAC. In the National Health and Nutrition Examination Survey (NHANES 2005-2010), Spearman correlations demonstrated significant negative relationships between LDH concentrations and serum iron. We conclude that iron import in human cells can involve lactate, LDH activity can reflect the availability of this metal, and blood LDH concentrations can correlate with indices of iron homeostasis.

CPX-351, a Dual-Drug Liposomal Formulation, Alleviates the Cardiotoxicity of Daunorubicin and Cytarabine to Induced Human Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: CPX-351 (US: Vyxeos ®; EU: Vyxeos ® Liposomal), a dual-drug liposomal encapsulation of daunorubicin + cytarabine at a synergistic 1:5 molar ratio, is a standard of care for therapy-related acute myeloid leukemia (AML) or AML with myelodysplastic-related changes. Daunorubicin, an anthracycline, is a known cardiotoxicant. While liposomal formulations have shown promise in mitigating these effects (O'Brien, et al. Ann Oncol 2004), this potential advantage is difficult to assess clinically due to numerous confounding factors. Here, we sought to develop and employ an in vitro model to study the relative toxicity of CPX-351 versus free daunorubicin + cytarabine applied as a combination at the same concentrations. Model development: The hiPSC-derived cardiomyocytes used in this study were derived from fibroblasts obtained from a single adult Caucasian female donor with no known diseases, induced into a pluripotent state, and then reprogrammed into cardiomyocytes (CDI Datasheet 2018). They present many of the characteristics of healthy human cardiac muscle cells, including gene and protein expression and rhythmic beating. During model development, the prototypical compounds doxorubicin and liposomal doxorubicin were used as proof-of-concept to establish the translational value of the model because clinical data regarding their relative cardiotoxicity were available (O'Brien, et al. Ann Oncol 2004). The model recapitulated the cumulative toxicity of free doxorubicin and differentiated between liposomal and free drug. The in vitro model was then applied to compare the relative cardiotoxicity of CPX-351 versus the combination of free daunorubicin + cytarabine (1:5), which were applied to the cardiomyocytes for 24 hours on Days 1, 3, and 5 at concentrations ranging from 0 to 1,000 ng/mL daunorubicin (0 to 2,273 ng/mL cytarabine). Bright-field microscopy imaging, lactate dehydrogenase (LDH) release (reflects cell membrane integrity), ATP content (indicates metabolic activity), cell beat rate (indicates cardiomyocyte function), and cardiac biomarkers (FABP3, cardiac troponin I, NT-proBNP, and BNP) were evaluated on Days 2, 4, 6, and 8. Results: Qualitative review of the microscopic images suggested single dose- and cumulative dose-dependent cytotoxicity of free daunorubicin + cytarabine, especially at the highest doses on Day 8. These observations were confirmed by dose-dependent increases in single-day or cumulative LDH activity in the cell media and decreases in ATP content starting on Day 4. Specifically, after a single 24-hour exposure, LDH activity (a measure of plasma membrane damage) was comparable to that of saline-treated cardiomyocytes for both the free-drug combination and CPX-351. However, after repeated exposure to the free-drug combination, increasing drug concentration was associated with increasing LDH activity in the media, peaking at levels ~12 times the saline control on Day 8. In contrast, the LDH activity following repeated equivalent doses of CPX-351 was only ~3 times the saline control. Conversely, ATP content (a measure of cellular metabolic fitness) gradually decreased between Day 2 and Day 8. The ATP depletion (Day 8 vs Day 2) was more profound in cardiomyocytes exposed to the free-drug combination (−98.1%) than in those exposed to equivalent concentrations of CPX-351 (−38.5%). Following repeated exposure to the free-drug combination, the cell beat rate demonstrated a biphasic response consisting of an initial increase followed by a significant slowing and sometimes arrest of beating, demonstrating significant injury; this effect was not observed following repeated exposure to CPX-351. Finally, the cardiac biomarkers FABP3 and cardiac troponin I were significantly released from cardiomyocytes exposed to the free-drug combination, but not from those exposed to CPX-351, even after 3 repeated exposures. Conclusions: Overall, at equivalent concentrations administered on the same schedule, CPX-351 was considerably less toxic to hiPSC-derived cardiomyocytes than the free-drug combination of daunorubicin + cytarabine, as measured by viability (imaging, LDH release), metabolic health (ATP content), function (beating rate), and the cardiac biomarkers FABP3 and cardiac troponin I. Clinical data are needed to confirm the reduced cardiotoxicity observed with CPX-351 versus free drugs in this in vitro model. Disclosures Fortin: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. LaCroix: StemoniX: Current Employment; Jazz Pharmaceuticals: Consultancy. Wang: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cheung: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Dexmedetomidine Attenuates Hypoxia/Reoxygenation Injury of H9C2 Myocardial Cells by Upregulating miR-146a Expression via the MAPK Signal Pathway

<b><i>Introduction and Objective:</i></b> Dexmedetomidine (Dex) and a number of miRNAs contribute to ischemia/reperfusion injury. We aimed to explore the role of Dex and miR-146a on myocardial cells injured by hypoxia/reoxygenation (H/R). <b><i>Method:</i></b> H9C2 cells were injured by H/R. Cell viability was tested using the cell counting kit-8. Lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels were determined using commercial kits. Flow cytometry was performed to determine apoptosis rate and reactive oxygen species (ROS) level. Protein and mRNA levels were assessed using Western blot and qPCR. <b><i>Results:</i></b> miR-146a expression and cell viability of H9C2 cells were downregulated under the circumstance of H/R injury. The tendency could be reversed by Dex, which could also upregulate SOD activity and decrease apoptosis, LDH activity, MDA, 78-kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP) levels of H9C2 cells. GRP78, CHOP levels, and cell viability were negatively modulated by miR-146a. Dex elevated cell viability, catalase, MnSOD, and NAD(P)H dehydrogenase (NQO1) levels but suppressed apoptosis rate, GRP78, and CHOP levels by increasing miR-146a expression and downregulating ROS, phosphorylation of p38, and extracellular signal-regulated kinases 1/2 levels. By using SB203580 (SB), the p38 mitogen-activated protein kinase (MAPK) inhibitor, Dex or the inhibition of miR-146 upregulated cell viability but downregulated GRP78 and CHOP levels. <b><i>Conclusion:</i></b> Dex might regulate miR-146a expression, which could further modulate the endoplasmic reticulum stress and oxidative stress and eventually affect the cell viability and apoptosis of myocardial cells injured by H/R via the MAPK signal pathway.

Lactate dehydrogenase activity staining demonstrates time-dependent immune cell infiltration in human ex-vivo burn-injured skin

Burn injuries constitute one of the most serious accidental injuries. Increased metabolic rate is a hallmark feature of burn injury. Visualising lactate dehydrogenase (LDH) activity has been previously used to identify metabolic activity differences, hence cell viability and burn depth in burn skin. LDH activity was visualised in injured and uninjured skin from 38 sub-acute burn patients. LDH activity aided the identification of spatially correlating immunocompetent cells in a sub-group of six patients. Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI MSI) was used to describe relative lactate and pyruvate abundance in burned and uninjured tissue. LDH activity was significantly increased in the middle and deep regions of burnt skin compared with superficial areas in burnt skin and uninjured tissue and positively correlated with post-burn time. Regions of increased LDH activity showed high pyruvate and low lactate abundance when examined with DESI-MSI. Areas of increased LDH activity exhibited cellular infiltration, including CD3 + and CD4 + T-lymphocytes and CD68 + macrophages. Our data demonstrate a steady increase in functional LDH activity in sub-acute burn wounds linked to cellular infiltration. The cell types associated are related to tissue restructuring and inflammation. This region in burn wounds is likely the focus of dysregulated inflammation and hypermetabolism.

First-in-class topical therapeutic omilancor ameliorates disease severity and inflammation through activation of LANCL2 pathway in psoriasis

Psoriasis (PsO) is a complex immune-mediated disease that afflicts 100 million people. Omilancor is a locally-acting, small molecule that selectively activates the Lanthionine Synthetase C-like 2 (LANCL2) pathway, resulting in immunoregulatory effects at the intersection of immunity and metabolism. Topical omilancor treatment in an imiquimod-induced mouse model of PsO ameliorates disease severity, epidermal hyperplasia and acanthosis. Further, pharmacological activation of LANCL2 results in significant downregulation of proinflammatory markers including local reduction of IL17, and infiltration of proinflammatory cell subsets. These therapeutic effects were further validated in an IL-23 PsO model. This model reported increased preservation of homeostatic skin structure, accompanied by a decreased infiltration of proinflammatory T cell subsets. In CD4+ T cells and Th17 cells, the LANCL2 pathway regulates proinflammatory cytokine production, proliferation and glucose metabolism. Metabolically, the loss of Lancl2 resulted in increased glycolytic rates, lactate production and upregulated enzymatic activity of hexokinase and lactate dehydrogenase (LDH). Inhibition of LDH activity abrogated the increased proliferation rate in Lancl2−/− CD4+ T cells. Additionally, topical omilancor treatment decreased the metabolic upregulation in keratinocytes, keratinocyte hyperproliferation and expression of inflammatory markers. Omilancor is a promising topical, LANCL2-targeting therapeutic candidate for the treatment of PsO and other dermatology indications.

Micro-Physiological-Systems enable investigation of hypoxia induced pathological processes in human aortic valve cells and tissues

Aortic valve (AV) stenosis is characterized by tissue fibrosis and calcification. Fibrous thickening can result in reduced tissue oxygen supply leading to pathological valvular interstitial cell (VIC) differentiation and calcification. Static 2D VIC cultures and animal models are limited in the ability to reflect human AV calcification. Culturing of VICs in micro-physiological-systems (MPS) in a pulsatile flow and the establishment of a modular AV tissue incubation chamber (TIC) are new approaches to evaluate pathophysiological processes of AV disease. Therefore, a MPS able to adjust hypoxic conditions was applied for VIC culture. A significant increase of mRNA-expression of EGLN1 and HIF1α- regulated LDHA and HIF1α nuclear localisation were proven under hypoxia. AV tissue culture was established within a TIC and viability was monitored by Resazurin-reduction in the incubation medium and visualized by LDH-activity in tissue cryosections. Viability was compared between fluid and static incubated tissues revealing an advantageous effect of the fluidic assay condition. Consecutively, the application of MPS in AV research allows i) the investigation of VIC cultures with efficient oxygen regulation and ii) the culture of porcine or human AV tissues preserving viability and specifically reflecting in vivo parameters. These methods open up new possibilities beyond static 2D culture and facilitate a reduction of animal experiments in AV research.

Effects of aerobic physical rehabilitation on muscle fiber metabolism in patients with severe heart failure

Purpose To evaluate changes of functional state and metabolism of skeletal muscle fiber (MF) after aerobic physical rehabilitation (PR), based on original method; to study relationship between clinical data dynamics and indicators of skeletal muscle metabolism after PR. Methods 100 patients; heart failure (HF) with reduced ejection fraction (HFrEF), NYHA III, mean age 52±5,2 years, ejection fraction (EF) 27,3±4,2%, BMI 23,5±2,8 kg/m2. Before inclusion in PR program cardiopulmonary exercising test (CPET), echocardiography (EchoCG), quality of life (QOL), exercise tolerance (ET) were estimated. Physical training intensity was based on lactate threshold achievement during CPET. PR efficiency was estimated on the basis of peak oxygen uptake (VO2peak), EF, QOL and ET dynamics after 6 months PR. Shin muscle biopsy was performed at baseline and after 3–6 months PR. In muscle samples activity of alkaline phosphatase (AP), lactate dehydrogenase (LDH) and succinate dehydrogenase (SDG) was evaluated with plag-method. Data were statistically processed using software package “Statistika, 9.0”. Results After 6 months of training EF increased by 10,5±2,3%, ET increased by 9,7±0,5 points (p1,2 &lt;0,05), QOL changed by 24,8±3,5 points (significant regression of symptoms), VO2peak increased by 5,2±0,5 ml/min/kg (p3,4 &lt;0,05). MF diameter (dMF) after PR slightly decreased in 6 patients, in 2 patients it did not change. After 3–6 months of training AP activity increased by 24,2% (p&lt;0,05); LDH activity in glycolytic MF decreased by 24,4%, in oxidative MF it decreased by 6,0% only (p1 &lt;0,05, p2&gt;0,05). SDG activity in glycolytic MF increased by 20%, in oxidative MF it increased by 30% (p1 &lt;0,05, p2&lt;0,05). There was a positive relationship between heart failure functional class dynamics and dMF (r=0,4, p=0,05), increase in CPET parameters was associated with AP activity (r=0,5, p=0,05). Conclusion Aerobic physical rehabilitation in stable HF pts, selected on the basis of LT achievement, was effective in improving values of EF, QOL, VO2peak and ET. After 3–6 months PR dMF slightly decreased; Participating in the PR program decreased LDH activity in both oxidative and glycolytic muscle fibers; it also increased AP and SDG activity significantly. This may indicate an increase in oxidative metabolism activity and decrease in MF edema, improvement in skeletal muscles microcirculation. FUNDunding Acknowledgement Type of funding sources: None.

Assessment of Coagulopathy, Inflammation and LDH Activity in SARS-CoV-2-infected Patients

The patho-physiology of COVID19 is still not clear. This study investigated the status of coagulation, LDH activity, and inflammation in SARS-CoV-2 infected patients. One hundred and thirty-four newly diagnosed COVID19 infected patients (age ranged65-82years) attending Mullingar Regional Hospital, Mullingar, Republic of Ireland, volunteered to participate in this study. They all presented with a pulmonary disorder, pyrexia, vomiting, body pains, etc. SARS-CoV-2 confirmatory test was done with RT-PCR molecular test using Cepheid Genexpert System. The data of another 121 plasma samples of apparently normal, non-COVID19 infected individuals taken before the emergence of COVID19 served as controls. Levels of blood platelets was determined in the participants using Siemen ADVIA 2120 Haematological System, and plasma D-dimer was determined in the participants using Star Max-Stago–Automatic Coagulation Analyzer LDH activity, plasma ferritin, and C-reactive protein (CRP) were determined in the participants using Beckman AU680-Chemistry Analyser. SARS-CoV-2 –infected patients showed significantly (p<0.001) higher levels of D-dimer (1522.95+1395.45ng/ml), CRP (125.3+116.4mg/l), ferritin (488.5+514.9pg/l), and LDH activity (574.4+446.7iu/l) compared to controls (78.8+18.1ng/ml, 2.4+1.7mg/l, 61.3+58.2pg/l, 304.1+76.6iu/l respectively). The blood platelet count did not show significant (p>0.05) change in the COVID19 patients (252.2 x 109+101 x 109) compared to controls (256.4 x 109+63.2 x 109). Elevated LDH activity could indicate tissue breakdown in the SARS-Cov-2 infected patients. Hyper-coagulation and inflammation are imminent in the COVID19 patients. Adjuvant anticoagulant and anti-inflammatory therapies may be useful as part of therapeutic regimen in the SARS-CoV-2 infected patients. Keywords: COVID19, Coagulopathy, CRP and ferritin, LDH.

Platelet Derived Extracellular Vesicles Promote in Vitro Gingival Wound Healing.

Purpose: Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates like platelet lysate (PL). In this article, the use of extracellular vesicles (EVs) derived from PL and their combination with hyaluronic acid biomaterials (HA) in wound healing was investigated.Methods: EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro for biocompatibility (LDH activity and metabolic activity) and by a wound healing assay and gene expression analysis.Results: EVs and EVs-HA treatments were biocompatible and showed an increase in wound healing compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed in gingival keratinocytes and gingival fibroblasts after the treatment with EVs.Conclusion: EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet derived EVs emerge as promising candidates for oral regeneration with the possibility to combine them with biomaterials in order to enhance their application in clinical use.