Cardiac progenitor cell therapy: mechanisms of action

Abstract Heart failure (HF) is an end-stage of many cardiac diseases and one of the main causes of death worldwide. The current management of this disease remains suboptimal. The adult mammalian heart was considered a post-mitotic organ. However, several reports suggest that it may possess modest regenerative potential. Adult cardiac progenitor cells (CPCs), the main players in the cardiac regeneration, constitute, as it may seem, a heterogenous group of cells, which remain quiescent in physiological conditions and become activated after an injury, contributing to cardiomyocytes renewal. They can mediate their beneficial effects through direct differentiation into cardiac cells and activation of resident stem cells but majorly do so through paracrine release of factors. CPCs can secrete cytokines, chemokines, and growth factors as well as exosomes, rich in proteins, lipids and non-coding RNAs, such as miRNAs and YRNAs, which contribute to reparation of myocardium by promoting angiogenesis, cardioprotection, cardiomyogenesis, anti-fibrotic activity, and by immune modulation. Preclinical studies assessing cardiac progenitor cells and cardiac progenitor cells-derived exosomes on damaged myocardium show that administration of cardiac progenitor cells-derived exosomes can mimic effects of cell transplantation. Exosomes may become new promising therapeutic strategy for heart regeneration nevertheless there are still several limitations as to their use in the clinic. Key questions regarding their dosage, safety, specificity, pharmacokinetics, pharmacodynamics and route of administration remain outstanding. There are still gaps in the knowledge on basic biology of exosomes and filling them will bring as closer to translation into clinic

Photobiomodulation with Red and Near-Infrared Light Improves Viability and Modulates Expression of Mesenchymal and Apoptotic-Related Markers in Human Gingival Fibroblasts

Photobiomodulation (PBM), also called low-level laser treatment (LLLT), has been considered a promising tool in periodontal treatment due to its anti-inflammatory and wound healing properties. However, photobiomodulation’s effectiveness depends on a combination of parameters, such as energy density, the duration and frequency of the irradiation sessions, and wavelength, which has been shown to play a key role in laser-tissue interaction. The objective of the study was to compare the in vitro effects of two different wavelengths—635 nm and 808 nm—on the human primary gingival fibroblasts in terms of viability, oxidative stress, inflammation markers, and specific gene expression during the four treatment sessions at power and energy density widely used in dental practice (100 mW, 4 J/cm2). PBM with both 635 and 808 nm at 4 J/cm2 increased the cell number, modulated extracellular oxidative stress and inflammation markers and decreased the susceptibility of human primary gingival fibroblasts to apoptosis through the downregulation of apoptotic-related genes (P53, CASP9, BAX). Moreover, modulation of mesenchymal markers expression (CD90, CD105) can reflect the possible changes in the differentiation status of irradiated fibroblasts. The most pronounced results were observed following the third irradiation session. They should be considered for the possible optimization of existing low-level laser irradiation protocols used in periodontal therapies

New Gene Markers for Metabolic Processes and Homeostasis in Porcine Buccal Pouch Mucosa during Cells Long Term-Cultivation—A Primary Culture Approach

The oral mucosal tissue is a compound structure composed of morphologically and physiologically different cell types. The morphological modification involves genetically determined lifespan, which may be recognized as the balance between cell survival and apoptosis. Although the biochemical processes and pathways in oral mucosa, with special regards to drug transport, delivery, and metabolism, are well known, the cellular physiological homeostasis in this tissue requires further investigation. The porcine buccal pouch mucosal cells (BPMCs) collected from 20 pubertal crossbred Landrace gilts, were used in this study. Immediately after recovery, the oral mucosa was separated micro-surgically, and treated enzymatically. The dispersed cells were transferred into primary in vitro culture systems for a long-term cultivation of 30 days. After each step of in vitro culture (IVC), the cells were collected for isolation of total RNA at 24 h, 7, 15, and 30 days of IVC. While the expression was analyzed for days 7, 15, and 30, the 24th hour was used as a reference for outcome calibration. The gene expression profile was determined using Affymetrix microarray assays and necessary procedures. In results, we observed significant up-regulation of SCARB1, PTGS2, DUSP5, ITGB3, PLK2, CCL2, TGFB1, CCL8, RFC4, LYN, ETS1, REL, LIF, SPP1, and FGER1G genes, belonging to two ontological groups, namely “positive regulation of metabolic process”, and “regulation of homeostatic process” at 7 day of IVC as compared to down-regulation at days 15 and 30. These findings suggest that the metabolic processes and homeostatic regulations are much more intense in porcine mucosal cells at day 7 of IVC. Moreover, the increased expression of marker genes, for both of these ontological groups, may suggest the existence of not only “morphological lifespan” during tissue keratinization, but also “physiological checkpoint” dedicated to metabolic processes in oral mucosa. This knowledge may be useful for preclinical experiments with drugs delivery and metabolism in both animals and humans

Significant Down-Regulation of “Biological Adhesion” Genes in Porcine Oocytes after IVM

Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes’ maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB+ oocytes were directly exposed to microarray assays and RT-qPCR (“before IVM” group), or first in vitro matured and then if classified as BCB+ passed to molecular analyses (“after IVM” group). As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes’ successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte’s achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes

Transcriptomic and morphological analysis of cells derived from porcine buccal mucosa—studies on an in vitro model

Transcriptional analysis and live-cell imaging are a powerful tool to investigate the dynamics of complex biological systems. In vitro expanded porcine oral mucosal cells, consisting of populations of epithelial and connective lineages, are interesting and complex systems for study via microarray transcriptomic assays to analyze gene expression profile. The transcriptomic analysis included 56 ontological groups with particular focus on 7 gene ontology groups that are related to the processes of differentiation and development. Most analyzed genes were upregulated after 7 days and downregulated after 15 and 30 days of in vitro culture. The performed transcriptomic analysis was then extended to include automated analysis of differential interference contrast microscopy (DIC) images obtained during in vitro culture. The analysis of DIC imaging allowed to identify the different populations of keratinocytes and fibroblasts during seven days of in vitro culture, and it was possible to evaluate the proportion of these two populations of cells. Porcine mucosa may be a suitable model for reference research on human tissues. In addition, it can provide a reference point for research on the use of cells, scaffolds, or tissues derived from transgenic animals for applications in human tissues reconstruction

Photobiomodulation—Underlying Mechanism and Clinical Applications

The purpose of this study is to explore the possibilities for the application of laser therapy in medicine and dentistry by analyzing lasers’ underlying mechanism of action on different cells, with a special focus on stem cells and mechanisms of repair. The interest in the application of laser therapy in medicine and dentistry has remarkably increased in the last decade. There are different types of lasers available and their usage is well defined by different parameters, such as: wavelength, energy density, power output, and duration of radiation. Laser irradiation can induce a photobiomodulatory (PBM) effect on cells and tissues, contributing to a directed modulation of cell behaviors, enhancing the processes of tissue repair. Photobiomodulation (PBM), also known as low-level laser therapy (LLLT), can induce cell proliferation and enhance stem cell differentiation. Laser therapy is a non-invasive method that contributes to pain relief and reduces inflammation, parallel to the enhanced healing and tissue repair processes. The application of these properties was employed and observed in the treatment of various diseases and conditions, such as diabetes, brain injury, spinal cord damage, dermatological conditions, oral irritation, and in different areas of dentistry

Time- and Dose-Dependent Effects of 17 Beta-Estradiol on Short-Term, Real-Time Proliferation and Gene Expression in Porcine Granulosa Cells

The key mechanisms responsible for achievement of full reproductive and developmental capability in mammals are the differentiation and transformation of granulosa cells (GCs) during folliculogenesis, oogenesis, and oocyte maturation. Although the role of 17 beta-estradiol (E2) in ovarian activity is widely known, its effect on proliferative capacity, gap junction connection (GJC) formation, and GCs-luteal cells transformation requires further research. Therefore, the goal of this study was to assess the real-time proliferative activity of porcine GCs in vitro in relation to connexin (Cx), luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), and aromatase (CYP19A1) expression during short-term (168 h) primary culture. The cultured GCs were exposed to acute (at 96 h of culture) and/or prolonged (between 0 and 168 h of culture) administration of 1.8 and 3.6 μM E2. The relative abundance of Cx36, Cx37, Cx40, Cx43, LHR, FSHR, and CYP19A1 mRNA was measured. We conclude that the proliferation capability of GCs in vitro is substantially associated with expression of Cxs, LHR, FSHR, and CYP19A1. Furthermore, the GC-luteal cell transformation in vitro may be significantly accompanied by the proliferative activity of GCs in pigs

Influence of Estradiol-17beta on Progesterone and Estrogen Receptor mRNA Expression in Porcine Follicular Granulosa Cells during Short-Term, In Vitro Real-Time Cell Proliferation

Progesterone (P4) and estradiol (E2) play a significant role in mammalian reproduction. Our study demonstrated that separated porcine cumulus cells (CCs) and/or granulosa cells (GCs) might proliferate in vitro during short-term, real-time primary culture. The GCs were analyzed according to gene expression of the progesterone receptor (nuclear form) (pgr), progesterone receptor membrane component 1 (pgrmc1), and estrogen-related receptor beta 3 (esrrb3) in relation to two housekeeping genes: actb and pbgd. GCs were cultivated in medium with the E2. Both pgr/actb and pgr/pbgd revealed higher expression between 24 and 168 h of IVC of prolonged E2 treatment and at 48 h of IVC after acute E2 administration. The pgrmc1/actb and pgrmc1/pbgd displayed increased expression after prolonged E2 treatment between 24 and 120 h of IVC. The highest level of esrrb3/actb at 120 and 144 h, as well as esrrb3/pbgd at 120 h, in untreated controls as compared to the hormone-stimulated group, was observed. We suggest that E2 significantly influences the upregulation of pgr, pgrmc1, and esrrb3 expression in porcine GCs during real-time cell proliferation. Since esrrb3 expression is stimulated by E2 in both an acute and prolonged manner, estradiol may be recognized as a potential estrogen receptor agonist in GCs

Expression of genes associated with BMP signaling pathway in porcine oocytes before and after IVM – a microarray approach

Abstract Background The full maturational capability of mammalian oocytes is accompanied by nuclear and cytoplasmic modifications, which are associated with proliferation and differentiation of surrounding cumulus cells. These events are regulated on molecular level by the expression of target genes involved in signal transduction pathways crucial for folliculogenesis and oogenesis. Transforming growth factor beta signaling includes several molecules that are involved in the regulation of oogenesis and embryo growth, including bone morphogenetic protein (BMP). However, the BMP-related gene expression profile in oocytes at different maturational stages requires further investigation. Methods Oocytes were isolated from pubertal crossbred Landrace gilts follicles, selected with a use of BCB staining test and analyzed before and after in vitro maturation. Gene expression profiles were examined using an Affymetrix microarray approach and validated by RT-qPCR. Database for Annotation, Visualization, and Integrated Discovery (DAVID) software was used for the extraction of the genes belonging to a BMP-signaling pathway ontology group. Results The assay revealed 12,258 different transcripts in porcine oocytes, among which 379 genes were down-regulated and 40 were up-regulated. The DAVID database indicated a “BMP signaling pathway” ontology group, which was significantly regulated in both groups of oocytes. We discovered five up-regulated genes in oocytes before versus after in vitro maturation (IVM): chordin-like 1 (CHRDL1), follistatin (FST), transforming growth factor-beta receptor-type III (TGFβR3), decapentaplegic homolog 4 (SMAD4), and inhibitor of DNA binding 1 (ID1). Conclusions Increased expression of CHRDL1, FST, TGFβR3, SMAD4, and ID1 transcripts before IVM suggested a subordinate role of the BMP signaling pathway in porcine oocyte maturational competence. Conversely, it is postulated that these genes are involved in early stages of folliculogenesis and oogenesis regulation in pigs, since in oocytes before IVM increased expression was observed