Changes In Mesenchymal Cells Research Articles

Classical and Innovative Evidence for Therapeutic Strategies in Retinal Dysfunctions.

The human retina is a complex anatomical structure that has no regenerative capacity. The pathogenesis of most retinopathies can be attributed to inflammation, with the activation of the inflammasome protein platform, and to the impact of oxidative stress on the regulation of apoptosis and autophagy/mitophagy in retinal cells. In recent years, new therapeutic approaches to treat retinopathies have been investigated. Experimental data suggest that the secretome of mesenchymal cells could reduce oxidative stress, autophagy, and the apoptosis of retinal cells, and in turn, the secretome of the latter could induce changes in mesenchymal cells. Other studies have evidenced that noncoding (nc)RNAs might be new targets for retinopathy treatment and novel disease biomarkers since a correlation has been found between ncRNA levels and retinopathies. A new field to explore is the interaction observed between the ocular and intestinal microbiota; indeed, recent findings have shown that the alteration of gut microbiota seems to be linked to ocular diseases, suggesting a gut-eye axis. To explore new therapeutical strategies for retinopathies, it is important to use proper models that can mimic the complexity of the retina. In this context, retinal organoids represent a good model for the study of the pathophysiology of the retina.

Cellular mechanisms of frontal bone development in spotted gar (Lepisosteus oculatus).

The cellular and molecular mechanisms initiating vertebrate cranial dermal bone formation is a conundrum in evolutionary and developmental biology. Decades of studies have determined the developmental processes of cranial dermal bones in various vertebrates and identified possible inducers of dermal bone. However, evolutionarily derived characters of current experimental model organisms, such as non-homologous frontal bones between teleosts and sarcopterygians, hinder investigations of ancestral and conserved mechanisms of vertebrate cranial dermal bone induction. Thus, investigating such mechanisms with animals diverging at evolutionarily informative phylogenetic nodes is imperative. We investigated the cellular foundations of skull frontal bone formation in the spotted gar Lepisosteus oculatus, a basally branching non-teleost actinopterygian. Whole-mount bone and cartilage staining and hematoxylin-eosin section staining revealed that mesenchymal cell condensations in the frontal bone of spotted gar develop in close association with the underlying cartilage. We also identified novel aspects of frontal bone formation: enrichment of F-actin, cellular membranes, and E-cadherin in condensing cells, and extension of podia-like structures from osteoblasts to the frontal bone, which may be responsible for bone mineral transport. This study highlights the process of frontal bone formation with dynamic architectural changes of mesenchymal cells in spotted gar, an emerging non-teleost fish model system, illuminating supposedly ancestral and likely conserved developmental mechanisms of skull bone formation among vertebrates.

ANALYSIS OF THE EFFECTS OF RADIOTHERAPY ON DIFFERENTIATION AND VIABILITY OF MESENCHYMAL CELLS

Objective: In this project the objective was to verify the effects of radiotherapy on viability, differentiation, cellular activity, and the damage caused to the DNA of mesenchymal cells of irradiated animals. Material and methods: Forty mice submitted to radiotherapy and implant placement in the femurs were divided: control (C): no irradiation, only implants; late irradiation (IrT): implants and irradiation; early irradiation (IrP): irradiation and later implants; and immediate irradiation (IrI): implants and irradiation on the same day. At the time of euthanasia, mesenchymal cells were isolated from the femurs, which were analyzed after 7 days of culture for morphology, viability, and cellular activity as well as cellular damage to DNA. The data were statistically analyzed (P< .05). Results: Cellular viability, IrP, and IrI groups presented a lower number than the control (P< .05). In the production of alkaline phosphatase and total protein, IrI showed a lower value, and IrP showed a higher value. There was greater formation of mineralized matrix in IrP in relation to the control, (P< .05). The formation of micronuclei was not observed in the control, but in the irradiated ones, it was not possible to perform its count due to the presence of nuclear aberrations. Conclusion: All irradiated groups presented changes in mesenchymal cells, however, IrP exhibited higher osteoblastic activity than the other irradiated groups. Promotion Agency: FAPESP (2016/25246-0). Objective: In this project the objective was to verify the effects of radiotherapy on viability, differentiation, cellular activity, and the damage caused to the DNA of mesenchymal cells of irradiated animals. Material and methods: Forty mice submitted to radiotherapy and implant placement in the femurs were divided: control (C): no irradiation, only implants; late irradiation (IrT): implants and irradiation; early irradiation (IrP): irradiation and later implants; and immediate irradiation (IrI): implants and irradiation on the same day. At the time of euthanasia, mesenchymal cells were isolated from the femurs, which were analyzed after 7 days of culture for morphology, viability, and cellular activity as well as cellular damage to DNA. The data were statistically analyzed (P< .05). Results: Cellular viability, IrP, and IrI groups presented a lower number than the control (P< .05). In the production of alkaline phosphatase and total protein, IrI showed a lower value, and IrP showed a higher value. There was greater formation of mineralized matrix in IrP in relation to the control, (P< .05). The formation of micronuclei was not observed in the control, but in the irradiated ones, it was not possible to perform its count due to the presence of nuclear aberrations. Conclusion: All irradiated groups presented changes in mesenchymal cells, however, IrP exhibited higher osteoblastic activity than the other irradiated groups. Promotion Agency: FAPESP (2016/25246-0).

Modification of Gene Expression in Mesenchymal Stromal Cells of the Leukemia Patients during Chemotherapy

BackgroundIn patients with acute leukemia the stromal microenvironment is deeply modified. Disturbances in signaling pathways, genetic abnormalities and functional changes in mesenchymal cells of these patients have been previously described. Chemotherapy also affect stromal progenitor cells. A damaged microenvironment might impair hematopoiesis in acute leukemia patients.AimsTo investigate the relative expression level in MMSCs and CFU-Fs, derived from the bone marrow (BM) of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients before and over the course of chemotherapy.Methods54 newly diagnosed cases (33 AML, 21 ALL) were involved in the study after informed consent. BM was aspirated prior to any treatment (time-point 0) and at days 37, 100 and 180 since the beginning of treatment of acute leukemia. MMSCs were cultured in aMEM with 10% fetal calf serum, CFU-Fs, in aMEM with 20% fetal calf serum. The relative expression level (REL) of different genes was measured by TaqMan RQ-PCR. As a control MMSCs and CFU-Fs from 88 healthy donors were used.ResultsAt the time of the disease manifestation the analysis of gene expression in MMSCs from acute leukemia patients revealed a significant increase in the REL of genes which regulate immune system responses and thereby can influence on the leukemic cell proliferation and migration (IL-6, IL-8, IL-1b and IL-1R1) (Pic.1). Also at the time of the diagnosis an increase in REL of genes, that are responsible for hematopoiesis regulation, was observed. For example, the REL of CSF1 that can influence on leukemic cells proliferation was increased at the disease manifestation and became normal during the treatment. The same dynamics was observed in the REL of JAG1 that has an antiapoptotic effect on leukemic cells. The REL of LIF had been also significantly increased at the disease manifestation, reflecting the efforts of MMSCs to inhibit leukemic proliferation. Chemotherapy affected REL of the studied genes differently. The treatment lead to the downregulation of IGF, TGFB1 and TGFB2 (Pic.2). As far asTGFB1 and 2 inhibit the differentiation of mesenchymal stem cells, and IGF is associated with myelodysplastic changes in elderly bone marrow, so their downregulation may refer to the effectiveness of therapy. The REL of genes regulating MMSC proliferation (PDGFRa and PDGFRb, FGF2, FGFR1 and 2) increased during chemotherapy. Exploring cell adhesion molecules, the decrease in the REL of their encoding genes was observed. As far as VCAM facilitate the leukemic cell extravasation and ICAM was shown to depress the Th17 cell differentiation, the down-regulation of their genes may reflect the microenvironment restoration. The influence of chemotherapy lead to decrease in REL of genes, associated with MMSCs differentiation (BGLAP and SOX9 (Pic.3)), reflecting the mechanism of the blocking of MMSCs migration and differentiation under the stress conditions.The alterations of bone marrow stroma were more pronounced in patients who didn't achieve remission.The REL of 9 genes was studied in CFU-F colonies. There were no differences in gene expression in CFU-Fs before the treatment, except for an increase in the REL of PPARg in acute leukemia CFU-Fs. During the treatment, a decrease in the REL of SPP1 and an increase in the REL of FGFR1 and 2 were observed.ConclusionTherefore, chemotherapy used does not impair the functional ability of MMSCs and CFU-Fs, but influence on their gene expression profile. The two types of precursors are affected differently, indicating their different differentiation level and functions. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Changes in Dermal Fibroblasts from Abcc6−/− Mice Are Present before and after the Onset of Ectopic Tissue Mineralization

Pseudoxanthoma elasticum (PXE), a rare genetic disease caused by mutations in the ABCC6 gene, is characterized by progressive calcification of elastic fibers in the skin, eyes and the cardiovascular system. The pathomechanisms of the mineralization is still obscure. Several hypotheses have been proposed, one of them suggesting a role for fibroblasts in controlling the amount and the quality of the calcified extracellular matrix. This hypothesis raises the question whether changes in mesenchymal cells are the cause and/or the consequences of the calcification process. In this study, fibroblasts were isolated and cultured from Abcc6+/+ and Abcc6−/− mice of different ages in order to investigate parameters known to be associated with the phenotype of fibroblasts from PXE patients. Results demonstrate few changes (Ank and Opn down-regulation) are already present before the occurrence of calcification. By contrast, a modification of other parameters (intracellular O2− content, Tnap activity and Bmp2 up-regulation) can be observed in Abcc6−/− mice after the onset of tissue mineralization. These data suggest that in the Abcc6−/− genotype, dermal fibroblasts actively contribute to changes that promote matrix calcification and that these cells can be further modulated with time by the calcified environment, thus contributing to the age-dependent progression of the disease.

Introduction: ‘Why is there persistent disease despite aggressive therapy of rheumatoid arthritis?’.

During the last 20 years, obvious progress has been made in the care of patients with rheumatoid arthritis (RA). This results from the combination of better diagnosis, earlier use of classic drugs, and the introduction of new targeted therapies. Despite these positive results, RA remains a chronic disease and induction of remission is still rarely obtained [1]. It is as if RA were a series of lost battles. More recently, although RA care improved, chronic inflammation was shown to have an effect on mortality, specifically from cardiovascular events. The goal of this series of short articles on RA chronicity is to give readers of Arthritis Research & Therapy the views of leading experts in the field. This combines lessons coming from daily clinical observations to a better understanding of basic molecular changes in mesenchymal cells. A question of definition RA is a heterogeneous disease, as reflected by the various degrees of joint destruction and disability between patients. The complexity of such heterogeneity has been increasing over time with more markers, from autoantibodies and human leukocyte antigen (HLA) subtypes to extensive genome-wide polymorphisms and serological markers. Use of these markers has been helpful at a statistical level to compare populations but much less so to predict severity for a given patient. If it is not possible to have a simple definition of a disease, we could mix different entities under the same definition. This is strongly stated by Gary Firestein, who proposes that we now need to consider RA a syndrome in order to include the different etiologies, clinical presentation and severity, and response rates to treatment [2]. If RA is now a syndrome, how do we use diagnostic criteria? Here again these criteria are indeed useful to compare populations but, even updated, they have limitations at the level of an individual patient. As a consequence of their (too) strict use, the associated risk is then the delay needed to fulfill enough of those criteria so that the diagnosis can be made.

Mesenchymal cells infiltrating a bladder acellular matrix gradually lose smooth muscle characteristics in intraperitoneally regenerated urothelial lining tissue in rats

To characterize serial long-term histological changes in mesenchymal cells infiltrating a collagen-based matrix, as in a hollow organ with differentiated urothelial lining created intraperitoneally by grafting cultured urothelial cells, mesenchymal cells with smooth-muscle immunohistochemical characteristics infiltrated into the scaffold, despite no mesenchymal cells being seeded into the scaffold before grafting. To regenerate a urothelial lining tissue intraperitoneally, rat urothelial cells were cultured and seeded with the feeder-layer technique onto bladder acellular matrix (BAM). After 7 days of cultivation to attach urothelial cells on the BAM, the matrix was folded with the urothelial cells inside and grafted onto the mesentery of the previously partially cystectomized rat. The grafted urothelial cells on the BAM, which formed a monolayer before grafting, stratified into three to four layers as early as 4 days after grafting. Although the regenerated urothelium became thinner with time, there was urothelial stratification and a peculiar angular appearance on the apical surface of the regenerated urothelium even after 56 days. The mesenchymal cells infiltrating the BAM showed positive immunohistochemical staining to alpha-smooth muscle actin or desmin at 7 days. Subsequently, the number of actin- or desmin-positive cells gradually decreased with time. On transmission electron microscopy, the infiltrating mesenchymal cells were characterized as myofibroblasts at 7 days. Smooth muscle-like cells were identified at 14 and 28 days, and fibrocytes were the main population at 56 days. Although epithelial-mesenchymal interactions have been assumed to be one of the most critical factors in smooth-muscle development, mesenchymal cells infiltrating the scaffold in this intraperitoneal regeneration model gradually lost smooth muscle characteristics with time. These results suggest that interactions between cultured urothelial cells and infiltrating mesenchymal cells alone could not maintain the smooth muscle character of infiltrating mesenchymal cells.

Expression of platelet-derived growth factor proteins and their receptor alpha and beta mRNAs during fracture healing in the normal mouse.

Platelet-derived growth factor (PDGF), abundant in bone tissue, has been reported to stimulate mesenchymal cell proliferation and migration. To elucidate the functional roles of PDGF during fracture healing, we investigated the expression of PDGF-A and -B chain proteins and receptor alpha and beta mRNAs in fractured mouse tibiae. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibia. On days 2, 4, 7, 10, 14, 21, and 28 after the operation, the fractured tibiae were excised, fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin to prepare 7-microm sections. Immunohistochemistry using polyclonal antibodies against human PDGF-A and -B chains was carried out by the avidin-biotin-peroxidase method. For in situ hybridization, we used digoxigenin-labeled single-stranded DNA probes specific for mouse PDGF receptors alpha and beta generated by unidirectional polymerase chain reaction. In the inflammatory phase on days 2-4 after the fracture, mesenchymal cells gathering at the fracture site expressed the PDGF-B chain and beta receptor mRNA. At the stage of cartilaginous callus formation on day 7, the immunoreactivity for PDGF-A and -B chains on proliferating and hypertrophic chondrocytes and the signals of alpha and beta receptor mRNAs on proliferating chondrocytes became manifest. At the stage of bony callus and bone remodeling on days 14-21, the predominant expression of the PDGF-B chain and beta receptor was observed on both osteoclasts and osteoblasts. On day 28, signals for PDGF ligand proteins and receptor mRNAs diminished. The coincidental localization of PDGF ligands and their receptors implies a paracrine and autocrine mechanism. Our data suggested that PDGF contributed in part to the promotion of the chondrogenic and osteogenic changes of mesenchymal cells from the early to the midphase of fracture healing; the functions mediated by the beta receptor, including cell migration, might be prerequisites to the recruitment of mesenchymal cells in the initial step and to the interaction between osteoclasts and osteoblasts in the bone remodeling phase.

Ultrastructural Alteration of Trypsin- and Pancreatin-separated Embryonic Rabbit Palate Epithelium and Mesenchyme

The effects of 30- and 45-minute trypsin- and pancreatin-separation on embryonic rabbit palate epithelium and mesenchyme were studied with the electron microscope. Changes in epithelium included fragmentation of the basal lamina and formation of cytoplasmic blebs associated with the basal cell layer. Changes in mesenchymal cells were first evident at 45 min of incubation and included cell fragmentation, nuclear pyknosis, and dilation of extracellular space. Results indicate a differential susceptibility of mesenchyme to the separating agent.

Electron microscopic study of alcoholic liver disease with special attention to the changes of mesenchymal cells of the liver

The authors investigated electron microscopically the biopsy material of 48 chronic alcoholic liver patients. On the basis of clinical and histological criteria 5 stages were differentiated: fatty liver, fatty liver with increased mesenchymal activity, acute alcoholic hepatitis, chronic alcoholic hepatitis, alcoholic cirrhosis. In each group changes of liver cell organelles and mesenchymal cells were compared. In this investigation it was demonstrated that liver cell damage--except for acute alcoholic hepatitis was not parallel with the severity of the clinical picture. On the contrary, the proliveration of mesenchymal cells, signs of its secretory activity and fibre forming, were in correlation with the progression of liver disease. The authors suggest that an intermediate stage must be taken into account between acute alcoholic hepatitis and alcoholic cirrhosis: the chronic progressive alcoholic hepatitis. It reminds morphologically to a great extent of the active chronic hepatitis and it is characterized not by severity of the injury of liver cell organelles, but by great proliferation of mesenchymal cells and lymphoid infiltration.

Observations on cultures of rabbit retina infected with vaccinia and herpes simplex virus.

SUMMARY Retinal cultures from newborn rabbits were infected with herpes simplex virus and neuro- and non-neuroadapted strains of vaccinia virus. Herpes simplex virus gave rise to characteristic nuclear changes of mesenchymal cells, and rod cells and a rapid destruction of neurons. The vaccinia virus strains all caused development of cytoplasmic inclusion bodies in the mesenchymal cells, which then degenerated. Changes in neuroectodermal cells were observed relatively late and might be secondary to the deterioration of mesenchymal cells. The implication of these findings for the study of the problem of selective cellular susceptility of the central nervous system to viruses is discussed.