Endothelial Progenitor Cells as Biomarkers of Cardiovascular Pathologies: A Narrative Review

Endothelial progenitor cells (EPC) may influence the integrity and stability of the vascular endothelium. The association of an altered total EPC number and function with cardiovascular diseases (CVD) and risk factors (CVF) was discussed; however, their role and applicability as biomarkers for clinical purposes have not yet been defined. Endothelial dysfunction is one of the key mechanisms in CVD. The assessment of endothelial dysfunction in vivo remains a major challenge, especially for a clinical evaluation of the need for therapeutic interventions or for primary prevention of CVD. One of the main challenges is the heterogeneity of this particular cell population. Endothelial cells (EC) can become senescent, and the majority of circulating endothelial cells (CEC) show evidence of apoptosis or necrosis. There are a few viable CECs that have properties similar to those of an endothelial progenitor cell. To use EPC levels as a biomarker for vascular function and cumulative cardiovascular risk, a correct definition of their phenotype, as well as an update on the clinical application and practicability of current isolation methods, are an urgent priority. Keywords: biomarker; cardiovascular disease; endothelial cells; progenitor

Endothelial Progenitor Cells as Biomarkers of Cardiovascular Pathologies: A Narrative Review

Endothelial progenitor cells (EPC) may influence the integrity and stability of the vascular endothelium. The association of an altered total EPC number and function with cardiovascular diseases (CVD) and risk factors (CVF) was discussed; however, their role and applicability as biomarkers for clinical purposes have not yet been defined. Endothelial dysfunction is one of the key mechanisms in CVD. The assessment of endothelial dysfunction in vivo remains a major challenge, especially for a clinical evaluation of the need for therapeutic interventions or for primary prevention of CVD. One of the main challenges is the heterogeneity of this particular cell population. Endothelial cells (EC) can become senescent, and the majority of circulating endothelial cells (CEC) show evidence of apoptosis or necrosis. There are a few viable CECs that have properties similar to those of an endothelial progenitor cell. To use EPC levels as a biomarker for vascular function and cumulative cardiovascular risk, a correct definition of their phenotype, as well as an update on the clinical application and practicability of current isolation methods, are an urgent priority

Myeloid Zinc Finger 1 (Mzf1) Differentially Modulates Murine Cardiogenesis by Interacting with an Nkx2.5 Cardiac Enhancer

Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations

Prostaglandin D2-supplemented “functional eicosanoid testing and typing” assay with peripheral blood leukocytes as a new tool in the diagnosis of systemic mast cell activation disease: an explorative diagnostic study

Background: Systemic mast cell activation disease (MCAD) is characterized by an enhanced release of mast cell-derived mediators, including eicosanoids, which induce a broad spectrum of clinical symptoms. Accordingly, the diagnostic algorithm of MCAD presupposes the proof of increased mast cell mediator release, but only a few mediators are currently established as routine laboratory parameters. We thus initiated an explorative study to evaluate in vitro typing of individual eicosanoid pattern of peripheral blood leukocytes (PBLs) as a new diagnostic tool in MCAD. Methods: Using the “functional eicosanoid testing and typing” (FET) assay, we investigated the balance (i.e. the complex pattern of formation, release and mutual interaction) of prostaglandin E2 (PGE2) and peptido-leukotrienes (pLT) release from PBLs of 22 MCAD patients and 20 healthy individuals. FET algorithms thereby consider both basal and arachidonic acid (AA)-, acetylsalicylic acid (ASA)-, and substance P (SP)-triggered release of PGE2 and pLT. The FET assay was further supplemented by analyzing prostaglandin D2 (PGD2), as mast cell-specific eicosanoid. Results: We observed marked PGE2-pLT imbalances for PBLs of MCAD patients, as indicated by a markedly enhanced mean FET value of 1.75 ± 0.356 (range: 1.14–2.36), compared to 0.53 ± 0.119 (range: 0.36-0.75) for healthy individuals. In addition, mean PGD2 release from PBLs of MCAD patients was significantly, 6.6-fold higher than from PBLs of healthy individuals (946 ± 302.2 pg/ml versus 142 ± 47.8 pg/ml; P < 0.001). In contrast to healthy individuals, PGD2 release from PBLs of MCAD patients was markedly triggered by SP (mean: 1896 ± 389.7 pg/ml; P < 0.001), whereas AA and ASA caused individually varying effects on both PGD2 and pLT release. Conclusions: The new in-vitro FET assay, supplemented with analysis of PGD2, demonstrated that the individual patterns of eicosanoid release from PBLs can unambiguously distinguish MCAD patients from healthy individuals. Notably, in our analyses, the FET value and both basal and triggered PGD2 levels were not significantly affected by MCAD-specific medication. Thus, this approach may serve as an in-vitro diagnostic tool to estimate mast cell activity and to support individualized therapeutic decision processes for patients suffering from MCAD

Detection of intracellular gene expression in live cells of murine, human and porcine origin using fluorescence-labeled nanoparticles.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells

Generation of three CRISPR/Cas9 edited human induced pluripotent stem cell lines (DHMi005-A-5, DHMi005-A-6 and DHMi005-A-7) carrying a Holt-Oram Syndrome patient-specific TBX5 mutation with known cardiac phenotype and a FLAG-tag after exon 9 of the TBX5 gene

TBX5 is a transcription factor (TF) playing essential role during cardiogenesis. It is well known that TF mutations possibly result in non- or additional binding of the DNA due to conformational changes of the protein. We introduced a Holt-Oram Syndrome (HOS) patient-specific TBX5 mutation c.920_C > A heterozygously in a healthy induced pluripotent stell cell (iPSC) line. This TBX5 mutation results in conformational changes of the protein and displayed ventricular septal defects in the patient itself. Additionally we introduced a FLAG-tag on the TBX5 mutation-carrying allele. The resulting heterozygous TBX5-FLAG iPSC lines are a powerful tool to investigate altered TF activity bonding

Analysis of prion protein aggregates in blood and brain from pre-clinical and clinical BSE cases

Prion diseases are infectious neurodegenerative diseases affecting humans and animals. The food-borne bovine spongiform encephalopathy (BSE) had serious impact on both economy and public health, respectively. To follow the pathogenesis of BSE, oral challenge studies were previously conducted, among others on the Isle of Riems, Germany (Balkema-Buschmann et al., 2011b). In the present work brain and plasma samples from this pathogenesis study were subjected to surface fluorescence distribution analysis (sFIDA). sFIDA is a diagnostic tool that exploits the aggregated state of the disease-related prion protein (PrP) as a biomarker for prion disorders. With the exception of one animal, all tested brain samples from clinical cattle exhibited a high titer of PrP particles. Moreover we could detect PrP aggregates already 16 and 24 months after infection. In contrast to our previous demonstration of PrP particles in blood plasma from scrapie sheep, however, no aggregates could be identified in plasma from pre-clinical and clinical cattle. This is in accordance with other studies suggesting a restriction of the BSE infection to the central nervous system

Genome Editing Redefines Precision Medicine in the Cardiovascular Field

Genome editing is a powerful tool to study the function of specific genes and proteins important for development or disease. Recent technologies, especially CRISPR/Cas9 which is characterized by convenient handling and high precision, revolutionized the field of genome editing. Such tools have enormous potential for basic science as well as for regenerative medicine. Nevertheless, there are still several hurdles that have to be overcome, but patient-tailored therapies, termed precision medicine, seem to be within reach. In this review, we focus on the achievements and limitations of genome editing in the cardiovascular field. We explore different areas of cardiac research and highlight the most important developments: (1) the potential of genome editing in human pluripotent stem cells in basic research for disease modelling, drug screening, or reprogramming approaches and (2) the potential and remaining challenges of genome editing for regenerative therapies. Finally, we discuss social and ethical implications of these new technologies