Thymosin ß4 and MRTF-A mitigate vessel regression despite cardiovascular risk factors.

Since clinical revascularization techniques of coronary or peripheral artery disease (CAD/PAD) focus on macrovessels of the heart, the microcirculatory compartment largely goes unnoticed. However, cardiovascular risk factors not only drive large vessel atherosclerosis, but also microcirculatory rarefaction, an instance unmet by current therapeutic schemes. Angiogenic gene therapy has the potential to reverse capillary rarefaction, but only if the disease-causing inflammation and vessel-destabilization are addressed. This review summarizes the current knowledge with regard to capillary rarefaction due to cardiovascular risk factors. Moreover, the potential of Thymosin ß4 (Tß4) and its downstream signal, myocardin-related transcription factor-A (MRTF-A), to counteract capillary rarefaction are discussed.

AAV-Mediated Somatic Gene Editing for Cardiac and Skeletal Muscle in a Large Animal Model.

Here we describe a protocol to produce a recombinant adeno-associated viral vector (rAAV)-based system to deliver the CRISPR-Cas9 complex into porcine skeletal muscle and myocardial cells. We initially describe the genomic composition of the rAAV-CRISPR vectors used in our lab. Furthermore, we give a step-by-step instruction into the production of recombinant viral vectors with high yields and purity. Lastly we describe the minimally invasive injection regimes to target the myocardium in a pig.

Endothelial Retargeting of AAV9 In Vivo.

Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2CNN AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2CNN AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE-/- mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2CNN AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2CNN AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2CNN AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2CNN AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2CNN AAV9-gRNAeNOS . Therefore, G2CNN AAV9 may enable gene transfer in vascular and atherosclerosis models.

Vascular Tissue Specific miRNA Profiles Reveal Novel Correlations with Risk Factors in Coronary Artery Disease.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Non-coding RNAs have already been linked to CVD development and progression. While microRNAs (miRs) have been well studied in blood samples, there is little data on tissue-specific miRs in cardiovascular relevant tissues and their relation to cardiovascular risk factors. Tissue-specific miRs derived from Arteria mammaria interna (IMA) from 192 coronary artery disease (CAD) patients undergoing coronary artery bypass grafting (CABG) were analyzed. The aims of the study were 1) to establish a reference atlas which can be utilized for identification of novel diagnostic biomarkers and potential therapeutic targets, and 2) to relate these miRs to cardiovascular risk factors. Overall, 393 individual miRs showed sufficient expression levels and passed quality control for further analysis. We identified 17 miRs-miR-10b-3p, miR-10-5p, miR-17-3p, miR-21-5p, miR-151a-5p, miR-181a-5p, miR-185-5p, miR-194-5p, miR-199a-3p, miR-199b-3p, miR-212-3p, miR-363-3p, miR-548d-5p, miR-744-5p, miR-3117-3p, miR-5683 and miR-5701-significantly correlated with cardiovascular risk factors (correlation coefficient >0.2 in both directions, p-value (p < 0.006, false discovery rate (FDR) <0.05). Of particular interest, miR-5701 was positively correlated with hypertension, hypercholesterolemia, and diabetes. In addition, we found that miR-629-5p and miR-98-5p were significantly correlated with acute myocardial infarction. We provide a first atlas of miR profiles in IMA samples from CAD patients. In perspective, these miRs might play an important role in improved risk assessment, mechanistic disease understanding and local therapy of CAD.

Early Bronchoscopy Improves Extubation Rates after Out-of-Hospital Cardiac Arrest: A Retrospective Cohort Analysis.

BACKGROUND: Patients suffering from out-of-hospital cardiac arrest (OHCA) frequently receive a bronchoscopy after being admitted to the ICU. We investigated the optimal timing and the outcome in these patients. METHODS: All patients who suffered from OHCA and were treated in our ICU from January 2013 to December 2018 were retrospectively analyzed. The data were collected from the patients' medical files, and included duration of mechanical ventilation, antibiotics, microbiological test results and neurological outcome. The outcome was the effect of early bronchoscopy (≤48 h after administration) on the rate of intubated patients on day five and day seven. RESULTS: From January 2013 to December 2018, 190 patients were admitted with OHCA. Bronchoscopy was performed in 111 patients out of the 164 patients who survived the first day. Late bronchoscopy >48 h was associated with higher rates of intubation on day five (OR 4.94; 95% CI 1.2-36.72, 86.7% vs. 55.0%, p = 0.036) and day seven (OR 4.96; 95% CI 1.38-24.69; 80.0% vs. 43.3%, p = 0.019). CONCLUSION: This study shows that patients who suffered from OHCA might have a better outcome if they receive a bronchoscopy early after hospital admission. Our data suggests an association of early bronchoscopy with a shorter intubation period.

AntimiR-132 Attenuates Myocardial Hypertrophy in an Animal Model of Percutaneous Aortic Constriction.

BACKGROUND: Pathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure. OBJECTIVES: This study aimed to establish a novel porcine model of pressure-overload-induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model. METHODS: This study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28. RESULTS: At d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 µm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132-treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2). CONCLUSIONS: The inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin.

Left Ventricular Pressure Volume Loop Measurements Using Conductance Catheters to Assess Myocardial Function in Mice.

Left ventricular catheterization in mice allows for in-depth assessment of myocardial function in healthy and diseased animals with the advent of pressure volume loop recordings greatly enhancing the technique. While a powerful tool, proper execution of the procedure is paramount to ensure reproducibility and reliability of the results obtained. Here, we describe the technique of left ventricular catheterization using the Scisense conductance catheter system by Transonic; however, the basic method applies to all murine catheter systems. We furthermore indicate possible pitfalls during the procedure and how to avoid them.

Sphingosine-1-Phosphate Attenuates Lipopolysaccharide-Induced Pericyte Loss via Activation of Rho-A and MRTF-A.

The high mortality seen in sepsis is caused by a systemic hypotension in part owing to a drastic increase in vascular permeability accompanied by a loss of pericytes. As has been shown previously, pericyte retention in the perivascular niche during sepsis can enhance the integrity of the vasculature and promote survival via recruitment of adhesion proteins such as VE-cadherin and N-cadherin. Sphingosine-1-phosphate (S1P) represents a lipid mediator regulating the deposition of the crucial adhesion molecule VE-cadherin at sites of interendothelial adherens junctions and of N-cadherin at endothelial-pericyte adherens junctions. Furthermore, in septic patients, S1P plasma levels are decreased and correlate with mortality in an indirectly proportional way. In the present study, we investigated the potential of S1P to ameliorate a lipopolysaccharide-induced septic hypercirculation in mice. Here we establish S1P as an antagonist of pericyte loss, vascular hyperpermeability, and systemic hypotension, resulting in an increased survival in mice. During sepsis S1P preserved VE-cadherin and N-cadherin deposition, mediated by a reduction of Src and cadherin phosphorylation. At least in part, this effect is mediated by a reduction of globular actin and a subsequent increase in nuclear translocation of MRTF-A (myocardin-related transcription factor A). These findings indicate that S1P may counteract pericyte loss and microvessel disassembly during sepsis and additionally emphasize the importance of pericyte-endothelial interactions to stabilize the vasculature.

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.

Rabgap1 promotes recycling of active ß1 integrins to support effective cell migration.

Integrin function depends on the continuous internalization of integrins and their subsequent endosomal recycling to the plasma membrane to drive adhesion dynamics, cell migration and invasion. Here we assign a pivotal role for Rabgap1 (GAPCenA) in the recycling of endocytosed active ß1 integrins to the plasma membrane. The phosphotyrosine-binding (PTB) domain of Rabgap1 binds to the membrane-proximal NPxY motif in the cytoplasmic domain of ß1 integrin subunits on endosomes. Silencing Rabgap1 in mouse fibroblasts leads to the intracellular accumulation of active ß1 integrins, alters focal adhesion formation, and decreases cell migration and cancer cell invasion. Functionally, Rabgap1 facilitates active ß1 integrin recycling to the plasma membrane through attenuation of Rab11 activity. Taken together, our results identify Rabgap1 as an important factor for conformation-specific integrin trafficking and define the role of Rabgap1 in ß1-integrin-mediated cell migration in mouse fibroblasts and breast cancer cells.

Atherosclerosis and the Capillary Network; Pathophysiology and Potential Therapeutic Strategies.

Atherosclerosis and associated ischemic organ dysfunction represent the number one cause of mortality worldwide. While the key drivers of atherosclerosis, arterial hypertension, hypercholesterolemia and diabetes mellitus, are well known disease entities and their contribution to the formation of atherosclerotic plaques are intensively studied and well understood, less effort is put on the effect of these disease states on microvascular structure an integrity. In this review we summarize the pathological changes occurring in the vascular system in response to prolonged exposure to these major risk factors, with a particular focus on the differences between these pathological alterations of the vessel wall in larger arteries as compared to the microcirculation. Furthermore, we intend to highlight potential therapeutic strategies to improve microvascular function during atherosclerotic vessel disease.

LCP1 preferentially binds clasped αMß2 integrin and attenuates leukocyte adhesion under flow.

Integrins are α/ß heterodimers that interconvert between inactive and active states. In the active state the α/ß cytoplasmic domains recruit integrin-activating proteins and separate the transmembrane and cytoplasmic (TMcyto) domains (unclasped TMcyto). Conversely, in the inactive state the α/ß TMcyto domains bind integrin-inactivating proteins, resulting in the association of the TMcyto domains (clasped TMcyto). Here, we report the isolation of integrin cytoplasmic tail interactors using either lipid bicelle-incorporated integrin TMcyto domains (α5, αM, αIIb, ß1, ß2 and ß3 integrin TMcyto) or a clasped, lipid bicelle-incorporated αMß2 TMcyto. Among the proteins found to preferentially bind clasped rather than the isolated αM and ß2 subunits was L-plastin (LCP1, also known as plastin-2), which binds to and maintains the inactive state of αMß2 integrin in vivo and thereby regulates leukocyte adhesion to integrin ligands under flow. Our findings offer a global view on cytoplasmic proteins interacting with different integrins and provide evidence for the existence of conformation-specific integrin interactors.

Induced pluripotent stem cell derived cardiac models: effects of Thymosin ß4.

INTRODUCTION: The establishment of induced pluripotent stem cells (iPSCs) and cardiomyocytes differentiated from them generated a new platform to study pathophysiological processes and to generate drug screening platforms and iPSC-derived tissues as therapeutic agents. Although major advances have been made in iPSC-reprogramming, cardiac differentiation and EHT production, reprogramming efficiency and the maturity of iPSC-CMs need to be further improved. AREAS COVERED: In this review, the authors summarize the current state of the field of iPSC research, the methodology of cardiac differentiation of iPSCs, the use of iPSC-CMs as disease models and toxicity screening platforms, and the potential of EHTs as therapeutic agents. The authors furthermore highlight the mechanisms by which Thymosin ß4 might enhance the production of iPSC-CMs and EHTs to improve their maturity and performance. EXPERT OPINION: iPSCs derived cardiomyocytes and EHTs represent a still young research field with many problems and pitfalls that need to be resolved to realize the full potential of iPSC-CMs and EHTs. Given that Thymosin ß4 directly enhances cardiac differentiation while also promoting angiogenic sprouting and vessel maturation, Tß4 might be of particular interest as a novel agent in tackling the difficulty of iPSC-CMs and engineered heart tissue grafts.

Tß4 Increases Neovascularization and Cardiac Function in Chronic Myocardial Ischemia of Normo- and Hypercholesterolemic Pigs.

Translations of new therapeutic options for cardiovascular disease from animal studies into a clinical setting have been hampered, in part by an improper reflection of a relevant patient population in animal models. In this study, we investigated the impact of thymosin ß4 (Tß4), which promotes collateralization and capillarization, during hypercholesterolemia, a known risk factor of coronary artery disease. Initial in vitro results highlighted an improved endothelial cell function upon Tß4 treatment under control conditions and during hypercholesterolemic stress (scratch area [pixels]: oxidized low-density lipoprotein [oxLDL], 191,924 ± 7,717; and oxLDL + Tß4, 105,621 ± 11,245). To mimic the common risk factor of hypercholesterolemia in vivo, pigs on regular (NC) or high-fat (HC) diet underwent chronic myocardial ischemia followed by recombinant adeno-associated virus (rAAV)-mediated transduction of Tß4 or LacZ as a control. We show that Tß4 overexpression improves capillarization and collateralization (collaterals: NC + rAAV.LacZ, 2.1 ± 0.5; NC + rAAV.Tß4, 6.7 ± 0.5; HC + rAAV.LacZ, 3.0 ± 0.3; and HC + rAAV.Tß4, 6.0 ± 0.4), ultimately leading to an improved myocardial function in both diet groups (ejection fraction [EF] at day 56 [%]: NC + rAAV.LacZ, 26 ± 1.1; NC + rAAV.Tß4, 45 ± 1.5; HC + rAAV.LacZ, 26 ± 2.5; and HC + rAAV.Tß4, 41 ± 2.6). These results demonstrate the potency of Tß4 in a patient-relevant large animal model of chronic myocardial ischemia.

Translational Aspects of Adeno-Associated Virus-Mediated Cardiac Gene Therapy.

Recombinant adeno-associated virus vectors (rAAVs) represent a reliable tool for basic and translational research, while rAAVs are also making strides in early clinical trials as vehicles for gene transfer. Their low immunogenicity, tissue tropism, and relative safety due to their low rate of genomic integration represent key features, making rAAVs promising instruments as vectors for future gene therapy approaches. Specifically, for cardiovascular gene therapy, rAAVs appear superior to other vector systems such as lenti- and adenoviral vectors due to the ease of accomplishing long-term cardiac expression of target genes and the reduced risk of provoking immune responses or triggering malignancies through genomic integration. However, major obstacles remain to be resolved if rAAVs are to achieve their full potential as gene therapy vectors in clinical trials. The main hurdles prohibiting their sustained success are their limited capacity to carry transgenes of larger sizes, the prevalence of neutralizing antibodies in the general population, and their tissue specificity, which leaves room for improvement. This review discusses the properties of rAAV that make them useful tools in experimental studies and the treatment of cardiovascular disease in patients.

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Steerable Induction of the Thymosin ß4/MRTF-A Pathway via AAV-Based Overexpression Induces Therapeutic Neovascularization.

Viral vectors have been frequently used in a variety of preclinical animal models to deliver genetic constructs into tissues. Among the vectors used, adeno-associated viral vectors (AAVs) may be targeted to specific tissues, depending on the serotype used. Moreover, they show robust expression for prolonged periods of time and have a low immunogenic potential. Furthermore, AAVs, unlike other vector systems, only display a low rate of genomic integration. However, to ensure efficient transgene production, expression is typically driven by constitutively active promoters, such as the cytomegalovirus (CMV) promoter. Tetracyclin responsive promoters represent a promising alternative to unregulated promoters. The present study compares AAVs encoding either constitutively active CMV or tet-off promoter regions in the preclinical models of hindlimb and chronic myocardial ischemia. Therapeutically, mediators regulating vessel maturation, specifically thymosin beta 4 (Tß4) and the downstream signaling molecule myocardin-related transcription factor A (MRTF-A) as well as the endothelial activator angiopoietin-2 (Ang2) were overexpressed via AAVs using both promotors. In the model of rabbit hindlimb ischemia, temporary (tet-off) expression of Tß4 improved capillary density, collateralization, and perfusion in the ischemic hindlimb, with no detectable difference to constitutive Tß4 overexpression. Similarly, constitutive overexpression of MRTF-A alone was able to improve capillarization, collateralization and perfusion. Temporary expression of Ang2 for 7 days further increased capillary density and pericyte coverage compared with MRTF-A alone, without further improving collateralization or perfusion. In the pig model of chronic myocardial ischemia constitutive expression of Tß4 for 4 weeks induced capillary and collateral growth similarly to a pulsed expression (2 day expression per week for 3 weeks). Taken together these findings demonstrate for two models of preclinical interventions that temporary gene expression may lead to similar results as constitutive expression, highlighting the potential of controlled temporary gene expression for induction of vascular growth as a therapeutic approach.

Thymosin ß4 Improves Differentiation and Vascularization of EHTs.

Induced pluripotent stem cells (iPSC) constitute a powerful tool to study cardiac physiology and represents a promising treatment strategy to tackle cardiac disease. However, iPSCs remain relatively immature after differentiation. Additionally, engineered heart tissue (EHT) has been investigated as a therapy option in preclinical disease models with promising results, although their vascularization and functionality leave room for improvement. Thymosin ß4 (Tß4) has been shown to promote the differentiation of progenitor cell lines to cardiomyocytes while it also induces angiogenic sprouting and vascular maturation. We examined the potential impact of Tß4 to enhance maturation of cardiomyocytes from iPSCs. Assessing the expression of transcription factors associated with cardiac differentiation, we were able to demonstrate the increased generation of cells displaying cardiomyocyte characteristics in vitro. Furthermore, we demonstrated, in a zebrafish model of embryonic vascular development, that Tß4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing Tß4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with Tß4 promotes vascularization and contractility in EHTs, highlighting Tß4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes.

Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI.

The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C- macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.