EGFR and MMP-9 are associated with neointimal hyperplasia in systemic-to-pulmonary shunts in children with complex cyanotic heart disease.

Systemic-to-pulmonary shunt malfunction contributes to morbidity in children with complex congenital heart disease after palliative procedure. Neointimal hyperplasia might play a role in the pathogenesis increasing risk for shunt obstruction. The aim was to evaluate the role of epidermal growth factor receptor (EGFR) and matrix-metalloproteinase 9 (MMP-9) in the formation of neointimal within shunts. Immunohistochemistry was performed with anti-EGFR and anti-MMP-9 on shunts removed at follow-up palliative or corrective procedure. Whole-genome single-nucleotide polymorphisms genotyping was performed on DNA extracted from patients´ blood samples and allele frequencies were compared between the group of patients with shunts displaying severe stenosis (≥ 40% of lumen) and the remaining group. Immunohistochemistry detected EGFR and MMP-9 in 24 of 31 shunts, located mainly in the luminal area. Cross-sectional area of EGFR and MMP-9 measured in median 0.19 mm2 (IQR 0.1-0.3 mm2) and 0.04 mm2 (IQR 0.03-0.09 mm2), respectively, and correlated positively with the area of neointimal measured on histology (r = 0.729, p < 0.001 and r = 0.0479, p = 0.018, respectively). There was a trend of inverse correlation between the dose of acetylsalicylic acid and the degree of EGFR, but not MMP-9, expression within neointima. Certain alleles in epidermal growth factor (EGF) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were associated with increased stenosis and neointimal hyperplasia within shunts. EGFR and MMP-9 contribute to neointimal proliferation in SP shunts of children with complex cyanotic heart disease. SP shunts from patients carrying certain risk alleles in the genes encoding for EGF and TIMP-1 displayed increased neointima.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Uncovering the molecular identity of cardiosphere-derived cells (CDCs) by single-cell RNA sequencing.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.

miR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations.

MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent Nkx2.5 enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.

Features of Human CD3+CD20+ T Cells.

Monoclonal Abs against CD20 reduce the number of relapses in multiple sclerosis (MS); commonly this effect is solely attributed to depletion of B cells. Recently, however, a subset of CD3(+)CD20(+) T cells has been described that is also targeted by the anti-CD20 mAb rituximab. Because the existence of cells coexpressing CD3 and CD20 is controversial and features of this subpopulation are poorly understood, we studied this issue in detail. In this study, we confirm that 3-5% of circulating human T cells display CD20 on their surface and transcribe both CD3 and CD20. We report that these CD3(+)CD20(+) T cells pervade thymus, bone marrow, and secondary lymphatic organs. They are found in the cerebrospinal fluid even in the absence of inflammation; in the cerebrospinal fluid of MS patients they occur at a frequency similar to B cells. Phenotypically, these T cells are enriched in CD8(+) and CD45RO(+) memory cells and in CCR7(-) cells. Functionally, they show a higher frequency of IL-4-, IL-17-, IFN-γ-, and TNF-α-producing cells compared with T cells lacking CD20. CD20-expressing T cells respond variably to immunomodulatory treatments given to MS patients: they are reduced by fingolimod, alemtuzumab, and dimethyl fumarate, whereas natalizumab disproportionally increases them in the blood. After depletion by rituximab, they show earlier and higher repopulation than CD20(+) B cells. Taken together, human CD3(+)CD20(+) T cells pervade lymphatic organs and the cerebrospinal fluid, have a strong ability to produce different cytokines, and respond to MS disease modifying drugs.

γ-Catenin acts as a tumor suppressor through context-dependent mechanisms in colorectal cancer.

PURPOSE: γ-Catenin is a protein closely related to ß-catenin. While the overexpression of ß-catenin has been linked with impaired prognosis and survival in various malignancies, both oncogenic and tumor suppressor functions have been described for γ-catenin. Thus, its role in cancer remains controversial. In this study, we examined the impact of γ-catenin expression on the malignant potential of colorectal cancer cells. METHODS: γ-Catenin was knocked down by short interfering RNA in the γ-catenin-proficient DLD-1 cell line and stably overexpressed in the γ-catenin-deficient cell line RKO. The effects of these molecular manipulations on the malignant potential of the cell lines were tested in vitro and in vivo in a xenograft tumor model. RESULTS: γ-Catenin contributed to Wnt signaling independent of the cellular context. Unlike its sister molecule ß-catenin, γ-catenin inhibited cellular invasion and anoikis in cells endogenously expressing γ-catenin. In line with this tumor suppressor function, its de novo expression in RKO cells inhibited proliferation via cell cycle arrest. In a xenograft tumor model, overexpression of γ-catenin starkly reduced tumor growth in vivo. CONCLUSIONS: This is the first report demonstrating a tumor-suppressive effect of γ-catenin in colorectal cancer both in vitro and in vivo. Detailed in vitro analysis revealed that effects of γ-catenin differ in γ-catenin proficient and deficient cells, indicating that its function in colorectal cancer is dependent on the cellular context. This finding adds to our understanding of γ-catenin and may have implications for future studies of catenin/Wnt targeted cancer therapies.

Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species.

The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells.

Tetralogy of Fallot and Hypoplastic Left Heart Syndrome - Complex Clinical Phenotypes Meet Complex Genetic Networks.

In many cases congenital heart disease (CHD) is represented by a complex phenotype and an array of several functional and morphological cardiac disorders. These malformations will be briefly summarized in the first part focusing on two severe CHD phenotypes, hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot (TOF). In most cases of CHD the genetic origin remains largely unknown, though the complexity of the clinical picture strongly argues against a dysregulation which can be attributed to a single candidate gene but rather suggests a multifaceted polygenetic origin with elaborate interactions. Consistent with this idea, genome-wide approaches using whole exome sequencing, comparative sequence analysis of multiplex families to identify de novo mutations and global technologies to identify single nucleotide polymorphisms, copy number variants, dysregulation of the transcriptome and epigenetic variations have been conducted to obtain information about genetic alterations and potential predispositions possibly linked to the occurrence of a CHD phenotype. In the second part of this review we will summarize and discuss the available literature on identified genetic alterations linked to TOF and HLHS.

Apolipoprotein E genotype, TNF-α 308G/A and risk for cardiac surgery associated-acute kidney injury in Caucasians.

OBJECTIVES: Acute kidney injury following cardiac surgery depicts a severe clinical problem that is strongly associated with adverse short- and long-term outcome. We analyzed two common genetic polymorphisms that have previously been linked to renal failure and inflammation, and have been supposed to be associated with cardiac surgery associated-acute kidney injury (CSA-AKI). METHODS: A total of 1415 consecutive patients who underwent elective cardiac surgery with CPB at our institution were prospectively enrolled. Patients were genotyped for Apolipoprotein E (ApoE E2,E3,E4) (rs429358 and rs7412) and TNF-α-308 G > A (rs1800629). RESULTS: Demographic characteristics and procedural data revealed no significant differences between genotypes. No association between ApoE (E2,E3,E4) and TNF-α-308 G > A genotypes and the RIFLE criteria could be detected. Several multiple linear regression analyses for postoperative creatinine increase revealed highly significant associations for aortic cross clamp time (p < 0.001), CPB-time (p < 0.001), norepinephrine (p < 0.001), left ventricular function (p = 0.004) and blood transfusion (p < 0.001). No associations were found for ApoE (E2,E3,E4) and TNF-α-308 G > A genotypes or baseline creatinine. When the sample size is 1415, the multiple linear regression test of R(2 )= 0 for seven covariates assuming normal distribution will have at least 99% power with significance level 0.05 to detect an R(2) of 0.108 or 0.107 as observed in the data. CONCLUSIONS: ApoE (E2,E3,E4) polymorphism and the TNF-α-308 G > A polymorphism are not associated with renal injury after CPB.

Insertion of a FLAG-tag sequence at the end of exon 9 of the TBX5 gene in three induced pluripotent stem cell lines (DHMi004-A-4, DHMi004-A-5, DHMi004-A-6) by CRISPR/Cas9 technology.

The identification of TBX5-related regulatory sequences in genes essential for heart development is hampered by the absence of antibodies which allow precipitation of TBX5:DNA complexes. Employing CRISPR/Cas9 technology, we have inserted a FLAG-tag sequence at the end of exon 9 of the TBX5 gene prior to the stop codon by homologous recombination. The translated TBX5-FLAG fusion protein of the three iPSC lines can effectively be precipitated by anti-FLAG antibodies and, thus, allow the detection of specific TBX5-binding sites and their associated genes.

A Laser-Driven Microrobot for Thermal Stimulation of Single Cells.

Here, the study presents a thermally activated cell-signal imaging (TACSI) microrobot, capable of photothermal actuation, sensing, and light-driven locomotion. The plasmonic soft microrobot is specifically designed for thermal stimulation of mammalian cells to investigate cell behavior under heat active conditions. Due to the integrated thermosensitive fluorescence probe, Rhodamine B, the system allows dynamic measurement of induced temperature changes. TACSI microrobots show excellent biocompatibility over 72 h in vitro, and they are capable of thermally activating single cells to cell clusters. Locomotion in a 3D workspace is achieved by relying on thermophoretic convection, and the microrobot speed is controlled within a range of 5-65 µm s-1 . In addition, light-driven actuation enables spatiotemporal control of the microrobot temperature up to a maximum of 60 °C. Using TACSI microrobots, this study targets single cells within a large population, and demonstrates thermal cell stimulation using calcium signaling as a biological output. Initial studies with human embryonic kidney 293 cells indicate a dose dependent change in intracellular calcium content within the photothermally controlled temperature range of 37-57 °C.

Correction of a deleterious TBX5 mutation in an induced pluripotent stem cell line (DHMi004-A-1) using a completely plasmid-free CRISPR/Cas 9 approach.

TBX5 is a transcription factor which plays an essential role at different checkpoints during cardiac differentiation. However, regulatory pathways affected by TBX5 still remain ill-defined. We have applied the CRISPR/Cas9 technology using a completely plasmid-free approach to correct a heterozygous causative "loss-of function" TBX5 mutation in an iPSC line (DHMi004-A), that has been established from a patient suffering from Holt-Oram syndrome (HOS). This isogenic iPSC line, DHMi004-A-1, represents a powerful in vitro tool to dissect the regulatory pathways affected by TBX5 in HOS.

Oligogenic Architecture of Rare Noncoding Variants Distinguishes 4 Congenital Heart Disease Phenotypes.

BACKGROUND: Congenital heart disease (CHD) is highly heritable, but the power to identify inherited risk has been limited to analyses of common variants in small cohorts. METHODS: We performed reimputation of 4 CHD cohorts (n=55 342) to the TOPMed reference panel (freeze 5), permitting meta-analysis of 14 784 017 variants including 6 035 962 rare variants of high imputation quality as validated by whole genome sequencing. RESULTS: Meta-analysis identified 16 novel loci, including 12 rare variants, which displayed moderate or large effect sizes (median odds ratio, 3.02) for 4 separate CHD categories. Analyses of chromatin structure link 13 of the genome-wide significant loci to key genes in cardiac development; rs373447426 (minor allele frequency, 0.003 [odds ratio, 3.37 for Conotruncal heart disease]; P=1.49×10-8) is predicted to disrupt chromatin structure for 2 nearby genes BDH1 and DLG1 involved in Conotruncal development. A lead variant rs189203952 (minor allele frequency, 0.01 [odds ratio, 2.4 for left ventricular outflow tract obstruction]; P=1.46×10-8) is predicted to disrupt the binding sites of 4 transcription factors known to participate in cardiac development in the promoter of SPAG9. A tissue-specific model of chromatin conformation suggests that common variant rs78256848 (minor allele frequency, 0.11 [odds ratio, 1.4 for Conotruncal heart disease]; P=2.6×10-8) physically interacts with NCAM1 (PFDR=1.86×10-27), a neural adhesion molecule acting in cardiac development. Importantly, while each individual malformation displayed substantial heritability (observed h2 ranging from 0.26 for complex malformations to 0.37 for left ventricular outflow tract obstructive disease) the risk for different CHD malformations appeared to be separate, without genetic correlation measured by linkage disequilibrium score regression or regional colocalization. CONCLUSIONS: We describe a set of rare noncoding variants conferring significant risk for individual heart malformations which are linked to genes governing cardiac development. These results illustrate that the oligogenic basis of CHD and significant heritability may be linked to rare variants outside protein-coding regions conferring substantial risk for individual categories of cardiac malformation.

Elucidation of the genetic causes of bicuspid aortic valve disease.

AIMS: The present study aims to characterize the genetic risk architecture of bicuspid aortic valve (BAV) disease, the most common congenital heart defect. METHODS AND RESULTS: We carried out a genome-wide association study (GWAS) including 2236 BAV patients and 11 604 controls. This led to the identification of a new risk locus for BAV on chromosome 3q29. The single nucleotide polymorphism rs2550262 was genome-wide significant BAV associated (P = 3.49 × 10-08) and was replicated in an independent case-control sample. The risk locus encodes a deleterious missense variant in MUC4 (p.Ala4821Ser), a gene that is involved in epithelial-to-mesenchymal transformation. Mechanistical studies in zebrafish revealed that loss of Muc4 led to a delay in cardiac valvular development suggesting that loss of MUC4 may also play a role in aortic valve malformation. The GWAS also confirmed previously reported BAV risk loci at PALMD (P = 3.97 × 10-16), GATA4 (P = 1.61 × 10-09), and TEX41 (P = 7.68 × 10-04). In addition, the genetic BAV architecture was examined beyond the single-marker level revealing that a substantial fraction of BAV heritability is polygenic and ∼20% of the observed heritability can be explained by our GWAS data. Furthermore, we used the largest human single-cell atlas for foetal gene expression and show that the transcriptome profile in endothelial cells is a major source contributing to BAV pathology. CONCLUSION: Our study provides a deeper understanding of the genetic risk architecture of BAV formation on the single marker and polygenic level.

Generation of a CRISPR/Cas edited human induced pluripotent stem cell line DHMi005-A-1 carrying a patient-specific disease-causing point mutation in the TBX5 gene.

A number of mutations in the human TBX5 gene have been described which cause Holt-Oram syndrome, a severe congenital disease associated with abnormalities in heart and upper limb development. We have used a prime-editing approach to introduce a patient-specific disease-causing TBX5 mutation (c.920_C > A) into an induced pluripotent stem cell (iPSC) line from a healthy donor. The resulting iPSC line provides a powerful tool to identify and analyze the biological and molecular impact of this specific TBX5 mutation in comparison to the isogenic control iPSC line during cardiac development.

Pulsatile ex vivo perfusion of human saphenous vein grafts under controlled pressure conditions increases MMP-2 expression.

BACKGROUND: The use of human saphenous vein grafts (HSVGs) as a bypass conduit is a standard procedure in the treatment of coronary artery disease while their early occlusion remains a major problem. METHODS: We have developed an ex vivo perfusion system, which uses standardized and strictly controlled hemodynamic parameters for the pulsatile and non-static perfusion of HSVGs to guarantee a reliable analysis of molecular parameters under different pressure conditions. Cell viability of HSVGs (n = 12) was determined by the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) into a purple formazan dye. RESULTS: Under physiological flow rates (10 mmHg) HSVGs remained viable for two weeks. Their exposure to arterial conditions (100 mmHg) was possible for one week without important reduction in viability. Baseline expression of matrix metalloproteinase-2 (MMP-2) after venous perfusion (2.2 ± 0.5, n = 5) was strongly up-regulated after exposure to arterial conditions for three days (19.8 ± 4.3) or five days (23.9 ± 6.1, p < 0.05). Zymographic analyses confirmed this increase on the protein level. Our results suggest that expression and activity of MMP-2 are strongly increased after exposure of HSVGs to arterial hemodynamic conditions compared to physiological conditions. CONCLUSION: Therefore, our system might be helpful to more precisely understand the molecular mechanisms leading to an early failure of HSVGs.

Aggrecan: a new biomarker for acute type A aortic dissection.

Acute type A aortic dissection (ATAAD) constitutes a life-threatening aortic pathology with significant morbidity and mortality. Without surgical intervention the usual mortality rate averages between 1 and 2% per hour. Thus, an early diagnosis of ATAAD is of pivotal importance to direct the affected patients to the appropriate treatment. Preceding tests to find an appropriate biomarker showed among others an increased aggrecan (ACAN) mRNA expression in aortic tissue of ATAAD patients. As a consequence, we investigated whether ACAN is a potential biomarker for diagnosing ATAAD. Mean ACAN protein concentration showed a significantly higher plasma concentration in ATAAD patients (38.59 ng/mL, n = 33) compared to plasma of patients with thoracic aortic aneurysms (4.45 ng/mL, n = 13), patients with myocardial infarction (11.77 ng/mL, n = 18) and healthy volunteers (8.05 ng/mL, n = 12). Cardiac enzymes like creatine kinase MB and cardiac troponin T showed no correlation with ACAN levels in ATAAD patients. Receiver-operator characteristics (ROC) curve analysis for ATAAD patients versus control subjects an optimum discrimination limit of ACAN plasma levels at 14.3 ng/mL with a corresponding sensitivity of 97% and specificity of 81%. According to our findings ACAN is a reliable potential biomarker in plasma samples to detect ATAAD with high sensitivity and specificity.

Congenital heart disease risk loci identified by genome-wide association study in European patients.

Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.

IGFBP-2 overexpression reduces the appearance of dysplastic aberrant crypt foci and inhibits growth of adenomas in chemically induced colorectal carcinogenesis.

Colon cancer patients frequently show increased levels of serum insulin-like growth factor-binding protein-2 (IGFBP-2), however, the pathogenetic relevance of this phenomenon for colorectal cancer is unclear. Therefore, we have used IGFBP-2 transgenic animals which overexpress IGFBP-2 systemically and locally in the intestine to study its role in chemically induced colorectal carcinogenesis. Mice received intraperitoneal injections of 1,2-dimethylhydrazine (DMH) (40 mg/kg body weight) once a week for 6 weeks to selectively induce aberrant crypt foci (ACF) and tumors in the colon. While tumor incidence was comparable in transgenic and control mice, the volume of adenomas in IGFBP-2 transgenic mice was reduced more than 2-fold. Furthermore, serum IGFBP-2 levels negatively correlated with tumor volume in the IGFBP-2 transgenic group. Histological examination showed that IGFBP-2 transgenic mice developed significantly less dysplastic ACF with a high potential to progress to advanced stages. The reduced tumor volume in IGFBP-2 transgenic animals was due to significantly reduced proliferative capacity, evidenced by a lower proportion of cells positive for Ki67. Our results demonstrate for the first time in an experimental model that IGFBP-2 overabundance prior to the onset and during colorectal carcinogenesis reduces tumor growth by inhibition of cell proliferation.

Myosin binding protein H-like (MYBPHL): a promising biomarker to predict atrial damage.

Myosin binding protein H-like (MYBPHL) is a protein associated with myofilament structures in atrial tissue. The protein exists in two isoforms that share an identical amino acid sequence except for a deletion of 23 amino acids in isoform 2. In this study, MYBPHL was found to be expressed preferentially in atrial tissue. The expression of isoform 2 was almost exclusively restricted to the atria and barely detectable in the ventricle, arteria mammaria interna, and skeletal muscle. After atrial damage induced by cryo- or radiofrequency ablation, MYBPHL was rapidly and specifically released into the peripheral circulation in a time-dependent manner. The plasma MYBPHL concentration remained substantially elevated up to 24 hours after the arrival of patients at the intensive care unit. In addition, the recorded MYBPHL values were strongly correlated with those of the established biomarker CK-MB. In contrast, an increase in MYBPHL levels was not evident in patients undergoing aortic valve replacement or transcatheter aortic valve implantation. In these patients, the values remained virtually constant and never exceeded the concentration in the plasma of healthy controls. Our findings suggest that MYBPHL can be used as a precise and reliable biomarker to specifically predict atrial myocardial damage.