Site-specific genetic and functional signatures of aortic endothelial cells at aneurysm predilection sites in healthy and AngII ApoE-/- mice.

Aortic aneurysm is characterized by a pathological dilation at specific predilection sites of the vessel and potentially results in life-threatening vascular rupture. Herein, we established a modified "Häutchen method" for the local isolation of endothelial cells (ECs) from mouse aorta to analyze their spatial heterogeneity and potential role in site-specific disease development. When we compared ECs from aneurysm predilection sites of healthy mice with adjacent control segments we found regulation of genes related to extracellular matrix remodeling, angiogenesis and inflammation, all pathways playing a critical role in aneurysm development. We also detected enhanced cortical stiffness of the endothelium at these sites. Gene expression of ECs from aneurysms of the AngII ApoE-/- model when compared to sham animals mimicked expression patterns from predilection sites of healthy animals. Thus, this work highlights a striking genetic and functional regional heterogeneity in aortic ECs of healthy mice, which defines the location of aortic aneurysm formation in disease.

Pharmacological Gq inhibition induces strong pulmonary vasorelaxation and reverses pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease with limited survival. Herein, we propose the pharmacological inhibition of Gq proteins as a novel concept to counteract pulmonary vasoconstriction and proliferation/migration of pulmonary artery smooth muscle cells (PASMCs) in PAH. We demonstrate that the specific pan-Gq inhibitor FR900359 (FR) induced a strong vasorelaxation in large and small pulmonary arteries in mouse, pig, and human subjects ex vivo. Vasorelaxation by FR proved at least as potent as the currently used triple therapy. We also provide in vivo evidence that local pulmonary application of FR prevented right ventricular systolic pressure increase in healthy mice as well as in mice suffering from hypoxia (Hx)-induced pulmonary hypertension (PH). In addition, we demonstrate that chronic application of FR prevented and also reversed Sugen (Su)Hx-induced PH in mice. We also demonstrate that Gq inhibition reduces proliferation and migration of PASMCs in vitro. Thus, our work illustrates a dominant role of Gq proteins for pulmonary vasoconstriction as well as remodeling and proposes direct Gq inhibition as a powerful pharmacological strategy in PH.

Forward programming of hiPSCs via the transcription factor ETV2: rapid, reproducible, and cost-effective generation of highly enriched, functional endothelial cells.

AIMS: Endothelial cell (EC) dysfunction plays a key role in the initiation and progression of cardiovascular disease. However, studying these disorders in ECs from patients is challenging, hence the use of human induced pluripotent stem cells (hiPSCs) and their in vitro differentiation into ECs represents a very promising approach. Still, the generation of hiPSC-derived ECs (hECs) remains demanding as a cocktail of growth factors and an intermediate purification step are required for hEC enrichment. Therefore, we probed the utility of a forward programming approach using transgenic hiPSC lines. METHODS AND RESULTS: We have used the transgenic hiPSC line PGP1 ETV2 iso2 to explore the in vitro differentiation of hECs via doxycycline-dependent induction of the transcription factor ETV2 and compared these with a standard differentiation protocol for hECs using non-transgenic control hiPSCs. The transgenic hECs were highly enriched without an intermediate purification step and expressed - as non-transgenic hECs and HUVECs - characteristic EC markers. The viability and yield of transgenic hECs were strongly improved by applying EC growth medium during differentiation. This protocol was successfully applied in two more transgenic hiPSC lines yielding reproducible results with low line-to-line variability. Transgenic hECs displayed typical functional properties, such as tube formation and LDL uptake, and a more mature phenotype than non-transgenic hECs. Transgenic hiPSCs preferentially differentiated into the arterial lineage, this was further enhanced by adding a high VEGF concentration to the medium. We also demonstrate that complexing lentivirus with magnetic nanoparticles and application of a magnetic field enables efficient transduction of transgenic hECs. CONCLUSIONS: We have established a highly efficient, cost-effective, and reproducible differentiation protocol for the generation of functional hECs via forward programming. The transgenic hECs can be genetically modified and are a powerful tool for disease modelling, tissue engineering, and translational purposes.

Electrically Conductive Collagen-PEDOT:PSS Hydrogel Prevents Post-Infarct Cardiac Arrhythmia and Supports hiPSC-Cardiomyocyte Function.

Myocardial infarction (MI) causes cell death, disrupts electrical activity, triggers arrhythmia, and results in heart failure, whereby 50-60% of MI-associated deaths manifest as sudden cardiac deaths (SCD). The most effective therapy for SCD prevention is implantable cardioverter defibrillators (ICDs). However, ICDs contribute to adverse remodeling and disease progression and do not prevent arrhythmia. This work develops an injectable collagen-PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate) hydrogel that protects infarcted hearts against ventricular tachycardia (VT) and can be combined with human induced pluripotent stem cell (hiPSC)-cardiomyocytes to promote partial cardiac remuscularization. PEDOT:PSS improves collagen gel formation, micromorphology, and conductivity. hiPSC-cardiomyocytes in collagen-PEDOT:PSS hydrogels exhibit near-adult sarcomeric length, improved contractility, enhanced calcium handling, and conduction velocity. RNA-sequencing data indicate enhanced maturation and improved cell-matrix interactions. Injecting collagen-PEDOT:PSS hydrogels in infarcted mouse hearts decreases VT to the levels of healthy hearts. Collectively, collagen-PEDOT:PSS hydrogels offer a versatile platform for treating cardiac injuries.

Right ventricular cardiomyocyte expansion accompanies cardiac regeneration in newborn mice after large left ventricular infarcts.

Cauterization of the root of the left coronary artery (LCA) in the neonatal heart on postnatal day 1 (P1) resulted in large, reproducible lesions of the left ventricle (LV), and an attendant marked adaptive response in the right ventricle (RV). The response of both chambers to LV myocardial infarction involved enhanced cardiomyocyte (CM) division and binucleation, as well as LV revascularization, leading to restored heart function within 7 days post surgery (7 dps). By contrast, infarction of P3 mice resulted in cardiac scarring without a significant regenerative and adaptive response of the LV and the RV, leading to subsequent heart failure and death within 7 dps. The prominent RV myocyte expansion in P1 mice involved an acute increase in pulmonary arterial pressure and a unique gene regulatory response, leading to an increase in RV mass and preserved heart function. Thus, distinct adaptive mechanisms in the RV, such as CM proliferation and RV expansion, enable marked cardiac regeneration of the infarcted LV at P1 and full functional recovery.

Electrophysiological Properties of Tetraploid Cardiomyocytes Derived from Murine Pluripotent Stem Cells Generated by Fusion of Adult Somatic Cells with Embryonic Stem Cells.

Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.

The focal adhesion protein ß-parvin controls cardiomyocyte shape and sarcomere assembly in response to mechanical load.

Physiological and pathological cardiac stress induced by exercise and hypertension, respectively, increase the hemodynamic load for the heart and trigger specific hypertrophic signals in cardiomyocytes leading to adaptive or maladaptive cardiac hypertrophy responses involving a mechanosensitive remodeling of the contractile cytoskeleton. Integrins sense load and have been implicated in cardiac hypertrophy, but how they discriminate between the two types of cardiac stress and translate mechanical loads into specific cytoskeletal signaling pathways is not clear. Here, we report that the focal adhesion protein ß-parvin is highly expressed in cardiomyocytes and facilitates the formation of cell protrusions, the serial assembly of newly synthesized sarcomeres, and the hypertrophic growth of neonatal rat ventricular cardiomyocytes (NRVCs) in vitro. In addition, physiological mechanical loading of NRVCs by either the application of cyclic, uni-axial stretch, or culture on physiologically stiff substrates promotes NRVC elongation in a ß-parvin-dependent manner, which is achieved by binding of ß-parvin to α/ß-PIX, which in turn activates Rac1. Importantly, loss-of-function studies in mice also revealed that ß-parvin is essential for the exercise-induced cardiac hypertrophy response in vivo. Our results identify ß-parvin as a novel mechano-responsive signaling hub in hypertrophic cardiomyocytes that drives cell elongation in response to physiological mechanical loads.

Histone Deacetylase 6 Inhibitor JS28 Prevents Pathological Gene Expression in Cardiac Myocytes.

Background Epigenetic modulators have been proposed as promising new drug targets to treat adverse remodeling in heart failure. Here, we evaluated the potential of 4 epigenetic drugs, including the recently developed histone deacetylase 6 (HDAC6) inhibitor JS28, to prevent endothelin-1 induced pathological gene expression in cardiac myocytes and analyzed the chromatin binding profile of the respective inhibitor targets. Methods and Results Cardiac myocytes were differentiated and puromycin-selected from mouse embryonic stem cells and treated with endothelin-1 to induce pathological gene expression (938 differentially expressed genes, q<0.05). Dysregulation of gene expression was at least in part prevented by epigenetic inhibitors, including the pan-BRD (bromodomain-containing protein) inhibitor bromosporine (290/938 genes), the BET (bromodomain and extraterminal) inhibitor JQ1 (288/938), the broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid (227/938), and the HDAC6 inhibitor JS28 (210/938). Although the 4 compounds were similarly effective toward pathological gene expression, JS28 demonstrated the least adverse effects on physiological gene expression. Genome-wide chromatin binding profiles revealed that HDAC6 binding sites were preferentially associated with promoters of genes involved in RNA processing. In contrast, BRD4 binding was associated with genes involved in core cardiac myocyte functions, for example, myocyte contractility, and showed enrichment at enhancers and intronic regions. These distinct chromatin binding profiles of HDAC6 and BRD4 might explain the different effects of their inhibitors on pathological versus physiological gene expression. Conclusions In summary, we demonstrated, that the HDAC6 inhibitor JS28 effectively prevented the adverse effects of endothelin-1 on gene expression with minor impact on physiological gene expression in cardiac myocytes. Selective HDAC6 inhibition by JS28 appears to be a promising strategy for future evaluation in vivo and potential translation into clinical application.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells.

Connexins (Cx) are a large family of membrane proteins that can form intercellular connections, so-called gap junctions between adjacent cells. Cx43 is widely expressed in mammals and has a variety of different functions, such as the propagation of electrical conduction in the cardiac ventricle. Despite Cx43 knockout models, many questions regarding the biology of Cx43 in health and disease remain unanswered. Herein we report the establishment of a Cre-inducible Cx43 overexpression system in murine embryonic stem (ES) cells. This enables the investigation of the impact of Cx43 overexpression in somatic cells. We utilized a double reporter system to label Cx43-overexpressing cells via mCherry fluorescence and exogenous Cx43 via fusion with P2A peptide to visualize its distribution pattern. We proved the functionality of our systems in ES cells, HeLa cells, and 3T3-fibroblasts and demonstrated the formation of functional gap junctions based on dye diffusion and FRAP experiments. In addition, Cx43-overexpressing ES cells could be differentiated into viable cardiomyocytes, as shown by the formation of cross striation and spontaneous beating. Analysis revealed faster and more rhythmic beating of Cx43-overexpressing cell clusters. Thus, our Cx43 overexpression systems enable the investigation of Cx43 biology and function in cardiomyocytes and other somatic cells.

High-Throughput Screening Platform in Postnatal Heart Cells and Chemical Probe Toolbox to Assess Cardiomyocyte Proliferation.

Restoring lost heart muscle is an attractive goal for cardiovascular regenerative medicine. One appealing strategy is the therapeutic stimulation of cardiomyocyte proliferation, which inter alia remains challenging due to available assay technologies capturing the complex biology. Here, a high-throughput-formatted phenotypic assay platform was established using rodent whole heart-derived cells to preserve the cellular environment of cardiomyocytes. Several readouts allowed the quantification of cycling cardiomyocytes, including a transgenic H2B-mCherry system for unequivocal, automated detection of cardiomyocyte nuclei. A chemical genetics approach revealed pronounced species differences and furnished pan-kinase inhibitors 5 and 36 as potent and robust inducers of endoreplication and acytokinetic mitosis. Combined profiling of the commonly used p38 MAPK inhibitors SB203580 (1), SB239063 (2) and a novel set of skepinone-L (6) derivatives pointed to off-target effects beyond p38 that might be critical for effective cardiomyocyte cytokinesis. Kinome-focused screening eventually furnished TG003 (38) as a novel candidate for stimulating cardiomyocyte proliferation.

Imaging of Gαq Proteins in Mouse and Human Organs and Tissues.

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics.

Bone marrow CD73+ mesenchymal stem cells display increased stemness in vitro and promote fracture healing in vivo.

Mesenchymal stem cells (MSCs) are multipotent and considered to be of great potential for regenerative medicine. We could show recently (Breitbach, Kimura et al. 2018) that a subpopulation of MSCs as well as sinusoidal endothelial cells (sECs) in the bone marrow (BM) of CD73-EGFP reporter mice could be labeled in vivo. We took advantage of this model to explore the plasticity and osteogenic potential of CD73-EGFP+ MSCs in vitro and their role in the regenerative response upon bone lesion in vivo. Herein we show that isolated CD73-EGFP+ MSCs displayed more pronounced stemness and stronger in vitro differentiation capacity into the osteogenic lineage compared to CD73-EGFP- MSCs. In a bone fracture model, endogenous BM-resident CD73-EGFP+ MSCs were found to migrate to the fracture site and differentiate into cartilage and bone cells. Our analysis also showed that CD73-EGFP+ sECs contributed to the neovascularization of the fracture site. In addition, grafting of CD73-EGFP+ MSCs into acute bone lesions revealed their capacity to differentiate into chondrocytes and osteocytes in vivo and their contribution to callus formation in the regeneration process of fracture healing. Thus, CD73+ MSCs display enhanced stemness and osteogenic differentiation potential in vitro and in vivo illustrating a prominent role of the CD73+ MSC subpopulation to promote fracture repair.

Trophectoderm cell failure leads to peri-implantation lethality in Trpm7-deficient mouse embryos.

Early embryogenesis depends on proper control of intracellular homeostasis of ions including Ca2+ and Mg2+. Deletion of the Ca2+ and Mg2+ conducting the TRPM7 channel is embryonically lethal in mice but leaves compaction, blastomere polarization, blastocoel formation, and correct specification of the lineages of the trophectoderm and inner cell mass unaltered despite that free cytoplasmic Ca2+ and Mg2+ is reduced at the two-cell stage. Although Trpm7-/- embryos are able to hatch from the zona pellucida, no expansion of Trpm7-/- trophoblast cells can be observed, and Trpm7-/- embryos are not identifiable in utero at E6.5 or later. Given the proliferation and adhesion defect of Trpm7-/- trophoblast stem cells and the ability of Trpm7-/- ESCs to develop to embryos in tetraploid embryo complementation assays, we postulate a critical role of TRPM7 in trophectoderm cells and their failure during implantation as the most likely explanation of the developmental arrest of Trpm7-deficient mouse embryos.

Inhibition of Vascular Growth by Modulation of the Anandamide/Fatty Acid Amide Hydrolase Axis.

OBJECTIVE: Pathological angiogenesis is a hallmark of various diseases characterized by local hypoxia and inflammation. These disorders can be treated with inhibitors of angiogenesis, but current compounds display a variety of side effects and lose efficacy over time. This makes the identification of novel signaling pathways and pharmacological targets involved in angiogenesis a top priority. Approach and Results: Here, we show that inactivation of FAAH (fatty acid amide hydrolase), the enzyme responsible for degradation of the endocannabinoid anandamide, strongly impairs angiogenesis in vitro and in vivo. Both, the pharmacological FAAH inhibitor URB597 and anandamide induce downregulation of gene sets for cell cycle progression and DNA replication in endothelial cells. This is underscored by cell biological experiments, in which both compounds inhibit proliferation and migration and evoke cell cycle exit of endothelial cells. This prominent antiangiogenic effect is also of pathophysiological relevance in vivo, as laser-induced choroidal neovascularization in the eye of FAAH-/- mice is strongly reduced. CONCLUSIONS: Thus, elevation of endogenous anandamide levels by FAAH inhibition represents a novel antiangiogenic mechanism.

Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy.

An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.

Macrocyclic Gq Protein Inhibitors FR900359 and/or YM-254890-Fit for Translation?

Guanine nucleotide-binding proteins (G proteins) transduce extracellular signals received by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. While GPCRs represent the largest class of drug targets, G protein inhibition has only recently been recognized as a novel strategy for treating complex diseases such as asthma, inflammation, and cancer. The structurally similar macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent selective inhibitors of the Gq subfamily of G proteins. FR and YM differ in two positions, FR being more lipophilic than YM. Both compounds are utilized as pharmacological tools to block Gq proteins in vitro and in vivo. However, no detailed characterization of FR and YM has been performed, which is a prerequisite for the compounds' translation into clinical application. Here, we performed a thorough study of both compounds' physicochemical, pharmacokinetic, and pharmacological properties. Chemical stability was high across a large range of pH values, with FR being somewhat more stable than YM. Oral bioavailability and brain penetration of both depsipeptides were low. FR showed lower plasma protein binding and was metabolized significantly faster than YM by human and mouse liver microsomes. FR accumulated in lung after chronic intratracheal or intraperitoneal application, while YM was more distributed to other organs. Most strikingly, the previously observed longer residence time of FR resulted in a significantly prolonged pharmacologic effect as compared to YM in a methacholine-induced bronchoconstriction mouse model. These results prove that changes within a molecule which seem marginal compared to its structural complexity can lead to crucial pharmacological differences.