Beta-Arrestin1 Prevents Preeclampsia by Downregulation of Mechanosensitive AT1-B2 Receptor Heteromers.

Preeclampsia is the most frequent pregnancy-related complication worldwide with no cure. While a number of molecular features have emerged, the underlying causal mechanisms behind the disorder remain obscure. Here, we find that increased complex formation between angiotensin II AT1 and bradykinin B2, two G protein-coupled receptors with opposing effects on blood vessel constriction, triggers symptoms of preeclampsia in pregnant mice. Aberrant heteromerization of AT1-B2 led to exaggerated calcium signaling and high vascular smooth muscle mechanosensitivity, which could explain the onset of preeclampsia symptoms at late-stage pregnancy as mechanical forces increase with fetal mass. AT1-B2 receptor aggregation was inhibited by beta-arrestin-mediated downregulation. Importantly, symptoms of preeclampsia were prevented by transgenic ARRB1 expression or a small-molecule drug. Because AT1-B2 heteromerization was found to occur in human placental biopsies from pregnancies complicated by preeclampsia, specifically targeting AT1-B2 heteromerization and its downstream consequences represents a promising therapeutic approach.

Transplanted allogeneic cardiac progenitor cells secrete GDF-15 and stimulate an active immune remodeling process in the ischemic myocardium.

BACKGROUND: Despite promising results in clinical studies, the mechanism for the beneficial effects of allogenic cell-based therapies remains unclear. Macrophages are not only critical mediators of inflammation but also critical players in cardiac remodeling. We hypothesized that transplanted allogenic rat cardiac progenitor cells (rCPCs) augment T-regulatory cells which ultimately promote proliferation of M2 like macrophages by an as-yet undefined mechanism. METHODS AND RESULTS: To test this hypothesis, we used crossover rat strains for exploring the mechanism of myocardial repair by allogenic CPCs. Human CPCs (hCPCs) were isolated from adult patients undergoing coronary artery bypass grafting, and rat CPCs (rCPCs) were isolated from male Wistar-Kyoto (WKY) rat hearts. Allogenic rCPCs suppressed the proliferation of T-cells observed in mixed lymphocyte reactions in vitro. Transplanted syngeneic or allogeneic rCPCs significantly increased cardiac function in a rat myocardial infarct (MI) model, whereas xenogeneic CPCs did not. Allogeneic rCPCs stimulated immunomodulatory responses by specifically increasing T-regulatory cells and M2 polarization, while maintaining their cardiac recovery potential and safety profile. Mechanistically, we confirmed the inactivation of NF-kB in Treg cells and increased M2 macrophages in the myocardium after MI by transplanted CPCs derived GDF15 and it's uptake by CD48 receptor on immune cells. CONCLUSION: Collectively, these findings strongly support the active immunomodulatory properties and robust therapeutic potential of allogenic CPCs in post-MI cardiac dysfunction.

Strategy of combining offline 2D LC-MS with LC-DIA-MS/MS to accurately identify chemical compounds and for quality control of Dioscorea septemloba Thunb.

INTRODUCTION: Dioscorea septemloba Thunb. (DST), the rhizome of Dioscorea spongiosa J. Q. Xi, M. Mizuno et W. L. Zhao or Fuzhou Dioscorea futschauensis Uline ex R. Kunth, has multiple biological activities. OBJECTIVES: We aimed to comprehensively characterize the chemical composition of DST and develop a quality control method. METHODS: Based on a UHPLC/Q-Orbitrap-MS platform, we developed an offline 2D LC-MS method (HILIC×RPLC) to characterize the chemical constituents in the 75% ethanol extract of DST at first. Secondly, a data-independent acquisition mode (DIA) was further established to conduct rapid qualitative analysis of compounds in DST from different habitats. Then, six differential compounds were screened out and selected as quantitative markers by UPLC-QQQ-MS to evaluate the content of DST from different habitats. RESULTS: In total, 137 compounds were identified in DST by combining offline 2D LC-MS with LC-DIA-MS/MS. Then, simultaneous targeted/non-targeted scanning technology was established based on the precursor ion list. Finally, six compounds, including dioscin, gracillin, pseduoprotodioscin, pseudoprotogracillin, protodioscin, and protogracillin, were accurately determined. The method validation showed a good linear relationship in the concentration range investigated (R2 > 0.999). The average recovery ranged from 86% to 107.5%, and LOD and LOQ were between 0.01 and 0.40 µg·mL-1 . CONCLUSION: Our strategy integrating offline 2D LC-MS and the DIA mode could effectively separate and identify compounds from DST, indicating it can be used in subsequent compounds characterization studies. At the same time, the quality of DST was comprehensively and systematically evaluated.

Stem Cell Therapy in Single-Ventricle Physiology: Recent Progress and Future Directions.

Current surgical and medical treatment options for single ventricle physiology conditions remain palliative. On the long term, despite treatment, the systemic ventricle has a significant risk of developing failure. There are unmet needs to develop novel treatment modalities to help ameliorate the ventricular dysfunction. Advances in the field of stem cell therapy have been promising for the treatment of heart failure. Numerous stem cell populations have been identified. Preclinical studies in small and large animal models provide evidence for effectiveness of this treatment modality and reveal several mechanisms of action by which stem cells exert their effect. Many clinical trials have been designed to further investigate the therapeutic potential that stem cell therapy may hold for pediatric populations with single ventricle physiology. In this review, we discuss the stem cell types used in these populations, some preclinical studies, and the clinical trials of stem cell therapy in single ventricle patients.

Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases.

Cardiovascular disease (CVD) is the leading cause of death in modern society. The adult heart innately lacks the capacity to repair and regenerate the damaged myocardium from ischemic injury. Limited understanding of cardiac tissue repair process hampers the development of effective therapeutic solutions to treat CVD such as ischemic cardiomyopathy. In recent years, rapid emergence of induced pluripotent stem cells (iPSC) and iPSC-derived cardiomyocytes presents a valuable opportunity to replenish the functional cells to the heart. The therapeutic effects of iPSC-derived cells have been investigated in many preclinical studies. However, the underlying mechanisms of iPSC-derived cell therapy are still unclear, and limited engraftment of iPSC-derived cardiomyocytes is well known. One facet of their mechanism is the paracrine effect of the transplanted cells. Microvesicles such as exosomes secreted from the iPSC-derived cardiomyocytes exert protective effects by transferring the endogenous molecules to salvage the injured neighboring cells by regulating apoptosis, inflammation, fibrosis, and angiogenesis. In this review, we will focus on the current advances in the exosomes from iPSC derivatives and discuss their therapeutic potential in the treatment of CVD.

Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice.

Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart.

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) triggers the growth-promoting mitogen-activated protein kinase (MAPK) pathway.

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.

ACE inhibition with captopril retards the development of signs of neurodegeneration in an animal model of Alzheimer's disease.

Increased generation of reactive oxygen species (ROS) is a significant pathological feature in the brains of patients with Alzheimer's disease (AD). Experimental evidence indicates that inhibition of brain ROS could be beneficial in slowing the neurodegenerative process triggered by amyloid-beta (Abeta) aggregates. The angiotensin II AT1 receptor is a significant source of brain ROS, and AD patients have an increased brain angiotensin-converting enzyme (ACE) level, which could account for an excessive angiotensin-dependent AT1-induced ROS generation. Therefore, we analyzed the impact of ACE inhibition on signs of neurodegeneration of aged Tg2576 mice as a transgenic animal model of AD. Whole genome microarray gene expression profiling and biochemical analyses demonstrated that the centrally active ACE inhibitor captopril normalized the excessive hippocampal ACE activity of AD mice. Concomitantly, the development of signs of neurodegeneration was retarded by six months of captopril treatment. The neuroprotective profile triggered by captopril was accompanied by reduced amyloidogenic processing of the amyloid precursor protein (APP), and decreased hippocampal ROS, which is known to enhance Abeta generation by increased activation of beta- and gamma-secretases. Taken together, our data present strong evidence that ACE inhibition with a widely used cardiovascular drug could interfere with Abeta-dependent neurodegeneration.

Generation of a human induced pluripotent stem cell line from a pediatric heart failure patient with de novo DNM1L mutation.

We generated a new iPSC line (LCHi003-A) from the pediatric dilated cardiomyopathy patient carrying the de novo mutation on DNM1L gene. The new iPSC line expressed high pluripotent markers and were capable to differentiate into trilineage.

BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot.

Tetralogy of Fallot (TOF) is one of the most prevalent congenital heart defects, with adverse cardiac remodeling and long-term cardiac complications. Here, searching for pathomechanisms, we find upregulated bublin coiled-coil protein (BBLN) in heart specimens of TOF patients with cyanosis, which positively correlates with cardiac remodeling pathways. Human BBLN, a protein with largely unknown function, promoted heart failure features, with increased mortality when overexpressed in mice, in a protein dosage-dependent manner. BBLN enhanced cardiac inflammation, fibrosis and necroptosis by calcium/calmodulin-dependent protein kinase II delta (CAMK2D) activation, whereas a BBLN mutant with impaired CAMK2D binding was inert. Downregulation of CAMK2D by an interfering RNA retarded BBLN-induced symptoms of heart failure. Endogenous BBLN was induced by hypoxia as a major TOF feature in human patients and by chronic pressure overload in mice, and its downregulation decreased CAMK2D hyperactivity, necroptosis and cardiovascular dysfunction. Thus, BBLN promotes CAMK2D-induced pathways to pathological cardiac remodeling, which are triggered by hypoxia in TOF.

DNA methylome and transcriptome profiling reveal key electrophysiology and immune dysregulation in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease. However, a detailed DNA methylation (DNAme) landscape has not yet been elucidated. Our study combined DNAme and transcriptome profiles for HCM myocardium and identify aberrant DNAme associated with altered myocardial function in HCM. The transcription of methylation-related genes did not significantly differ between HCM and normal myocardium. Nevertheless, the former had an altered DNAme profile compared with the latter. The hypermethylated and hypomethylated sites in HCM tissues had chromosomal distributions and functional enrichment of correlated genes differing from those of their normal tissue counterparts. The GO analysis of network underlying the genes correlated with DNAme alteration and differentially expressed genes (DEGs) shows functional clusters centred on immune cell function and muscle system processes. In KEGG analysis, only the calcium signalling pathway was enriched either by the genes correlated with changes in DNAme or DEGs. The protein-protein interactions (PPI) underlying the genes altered at both the DNAme and transcriptional highlighted two important functional clusters. One of these was related to the immune response and had the estrogen receptor-encoding ESR1 gene as its node. The other cluster comprised cardiac electrophysiology-related genes. Intelliectin-1 (ITLN1), a component of the innate immune system, was transcriptionally downregulated in HCM and had a hypermethylated site within 1500 bp upstream of the ITLN1 transcription start site. Estimates of immune infiltration demonstrated a relative decline in immune cell population diversity in HCM. A combination of DNAme and transcriptome profiles may help identify and develop new therapeutic targets for HCM.

Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway.

Cardiac fibrosis is a hallmark in late-stage familial dilated cardiomyopathy (DCM) patients, although the underlying mechanism remains elusive. Cardiac exosomes (Exos) have been reported relating to fibrosis in ischemic cardiomyopathy. Thus, we investigated whether Exos secreted from the familial DCM cardiomyocytes could promote fibrogenesis. Using human iPSCs differentiated cardiomyocytes we isolated Exos of angiotensin II stimulation conditioned media from either DCM or control (CTL) cardiomyocytes. Of interest, cultured cardiac fibroblasts had increased fibrogenesis following exposure to DCM-Exos rather than CTL-Exos. Meanwhile, injecting DCM-Exos into mouse hearts enhanced cardiac fibrosis and impaired cardiac function. Mechanistically, we identified the upregulation of miRNA-218-5p in the DCM-Exos as a critical contributor to fibrogenesis. MiRNA-218-5p activated TGF-ß signaling via suppression of TNFAIP3, a master inflammation inhibitor. In conclusion, our results illustrate a profibrotic effect of cardiomyocytes-derived Exos that highlights an additional pathogenesis pathway for cardiac fibrosis in DCM.

Augmentation of Histone Deacetylase 6 Activity Impairs Mitochondrial Respiratory Complex I in Ischemic/Reperfused Diabetic Hearts.

BACKGROUND: Diabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor α (TNFα) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and ß-oxidation. Here we examined how HDAC6 regulates TNFα production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts. METHODS: HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNFα and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups. RESULTS: Myocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNFα levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNFα with an anti-TNFα monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNFα levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNFα levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSIONS: Augmenting HDAC6 activity inhibits mCI activity by increasing TNFα levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes.

A single-dose toxicity study on non-radioactive iodinated hypericin for a targeted anticancer therapy in mice.

AIM: Hypericin (Hyp) and its radio-derivatives have been investigated in animal models with ischemic heart diseases and malignancies for diagnostic and therapeutic purposes. Before radioiodinated Hyp ((123)I-Hyp or (131)I-Hyp) can be considered as a clinically useful drug, vigorous evaluations on its chemotoxicity are necessary. In the present study, we examined the toxicity of a single dose of non-radioactive (127)I-Hyp in normal mice for 24 h and 14 d. METHODS: Studies were performed on 132 normal mice. (127)I -Hyp at a clinically relevant dose of 0.1 mg/kg body weight and a 100-times higher dose of 10 mg/kg was intravenously injected into 40 mice. The safety aspects of clinical manifestations, serological biochemistry, and histopathology were assessed. In another 72 mice, (127)I-Hyp was administered intravenously at assumed values to bracket the value of LD(50). The rest 20 mice were used in the control groups. RESULTS: At 24 h and 14 d following the injection of (127)I -Hyp at either 0.1 or 10 mg/kg, all mice tolerated well without mortality or any observable treatment-related symptoms. No significant differences were found in blood biochemical parameters between the test and control groups. All organs presented normal appearances upon histopathological inspection. The value of LD(50) of (127)I-Hyp in mice through intravenous injection was 20.26 mg/kg, with the 95% confidence interval between 18.90 and 21.55 mg/kg. CONCLUSION: The current study reveals a broad safety range of (127)I-Hyp, which not only supports the use of (123)I-Hyp or (131)I-Hyp in the necrosis targeting theragnostic strategy, but also serves as a valuable reference for exploring other possible applications for iodinated Hyp.

An efficient and sensitive method on the identification of unsaturated fatty acids in biosamples: Total lipid extract from bovine liver as a case study.

Unsaturated fatty acids (UFAs) are fatty acids with one or more carbon-carbon double bonds (C=C). They are building blocks of structurally complex lipids. According to the position of C=C bond in the UFAs, there are multiple isomers with different physiological activities. Therefore, an accurate understanding of UFAs is essential. Combining Paternò-Büchi (PB) reaction with tandem mass spectrometry (MS/MS) analysis has been successfully used to identify unsaturated lipids in bio-samples. However, there are still some challenges, such as low reactive rate and low MS response of PB products. In this study, 3-pyridinecarboxaldehyde (3-PYA) was selected as an efficient PB reagent for the first time. According to the nitrogen rule, introduction of nitrogen did not only make the target MS and MS/MS ions to be easily identified but also increase the ionization efficiency significantly. The developed method in this study demonstrated greater efficiency and sensitivity for the identification of UFAs in total lipid extracts from bovine liver compared with other commonly used PB reagents.

Multiple-model machine learning identifies potential functional genes in dilated cardiomyopathy.

Introduction: Machine learning (ML) has gained intensive popularity in various fields, such as disease diagnosis in healthcare. However, it has limitation for single algorithm to explore the diagnosing value of dilated cardiomyopathy (DCM). We aim to develop a novel overall normalized sum weight of multiple-model MLs to assess the diagnosing value in DCM. Methods: Gene expression data were selected from previously published databases (six sets of eligible microarrays, 386 samples) with eligible criteria. Two sets of microarrays were used as training; the others were studied in the testing sets (ratio 5:1). Totally, we identified 20 differently expressed genes (DEGs) between DCM and control individuals (7 upregulated and 13 down-regulated). Results: We developed six classification ML methods to identify potential candidate genes based on their overall weights. Three genes, serine proteinase inhibitor A3 (SERPINA3), frizzled-related proteins (FRPs) 3 (FRZB), and ficolin 3 (FCN3) were finally identified as the receiver operating characteristic (ROC). Interestingly, we found all three genes correlated considerably with plasma cells. Importantly, not only in training sets but also testing sets, the areas under the curve (AUCs) for SERPINA3, FRZB, and FCN3 were greater than 0.88. The ROC of SERPINA3 was significantly high (0.940 in training and 0.918 in testing sets), indicating it is a potentially functional gene in DCM. Especially, the plasma levels in DCM patients of SERPINA3, FCN, and FRZB were significant compared with healthy control. Discussion: SERPINA3, FRZB, and FCN3 might be potential diagnosis targets for DCM, Further verification work could be implemented.

Comparative efficacy and mechanism of action of cardiac progenitor cells after cardiac injury.

Successful cell therapy requires cells to resist the hostile ischemic myocardium, be retained to continue secreting cardioprotective growth factors/exosomes, and resist immunological host responses. Clinically relevant stem/progenitor cells in a rodent model of acute myocardial infarction (MI) demonstrated that neonatal cardiac mesenchymal stromal cells (nMSCs) provide the most robust cardiac functional recovery. Transplanted nMSCs significantly increased the number of tissue reparative macrophages and regulatory T-cells and decreased monocyte-derived inflammatory macrophages and neutrophils in the host myocardium. mRNA microarray and single-cell analyses combined with targeted depletion studies established CD47 in nMSCs as a key molecule responsible for cell retention in the myocardium through an antiphagocytic mechanism regulated by miR34a-5p. Gain and loss-of-function studies demonstrated that miR34a-5p also regulated the production of exosomes and cardioprotective paracrine factors in the nMSC secretome. In conclusion, miR34a-5p and CD47 play an important role in determining the composition of nMSCs' secretome and immune evasion, respectively.

Codelivery of Sustainable Antimicrobial Agents and Platelet-Derived Growth Factor via Biodegradable Nanofibers for Repair of Diabetic Infectious Wounds.

More than half of diabetic wounds demonstrate clinical signs of infection at presentation and lead to poor outcomes. This work develops coaxial sheath-core nanofibrous poly(lactide-co-glycolide) (PLGA) scaffolds that are loaded with bioactive antibiotics and platelet-derived growth factor (PDGF) for the repair of diabetic infectious wounds. PDGF and PLGA/antibiotic solutions were pumped, respectively, into two independent capillary tubings for coaxial electrospinning to prepare biodegradable sheath-core nanofibers. Spun nanofibrous scaffolds sustainably released PDGF, vancomycin, and gentamicin for 3 weeks. The scaffolds also reduced the phosphatase and tensin homologue content, enhanced the amount of angiogenesis marker (CD31) around the wound area, and accelerated healing in the early stage of infected diabetic wound repair. Antibiotic/biomolecule-loaded PLGA nanofibers may provide a very effective way to aid tissue regeneration at the sites of infected diabetic wounds.