Ionizing irradiation-induced Fgr in senescent cells mediates fibrosis.

The role of cellular senescence in radiation-induced pulmonary fibrosis (RIPF) and the underlying mechanisms are unknown. We isolated radiation-induced senescent tdTOMp16 positive mesenchymal stem cells, established their absence of cell division, then measured levels of irradiation-induced expression of biomarkers of senescence by RNA-seq analysis. We identified a Log2 6.17-fold upregulation of tyrosine kinase Fgr, which was a potent inducer of biomarkers of fibrosis in target cells in non-contact co-cultures. Inhibition of Fgr by shRNA knockdown did not block radiation-induced senescence in vitro; however, both shRNA knockdown, or addition of a specific small-molecule inhibitor of Fgr, TL02-59, abrogated senescent cell induction of profibrotic genes in transwell-separated target cells. Single-cell RNA-seq (scRNAseq) analysis of mouse lungs at day 150 after 20 Gy thoracic irradiation revealed upregulation of Fgr in senescent neutrophils, and macrophages before detection of lung fibrosis. Thus, upregulated Fgr in radiation-induced senescent cells mediates RIPF and is a potential therapeutic target for the prevention of this radiation late effect.

Myofibroblast transcriptome indicates SFRP2hi fibroblast progenitors in systemic sclerosis skin.

Skin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells. The number of myofibroblasts in SSc skin correlates with the modified Rodnan skin score, the most widely used clinical measure of skin disease severity. Murine fibrosis models indicate that myofibroblasts can arise from a variety of different cell types, but their origin in SSc skin has remained uncertain. Utilizing single cell RNA-sequencing, we define different dermal fibroblast populations and transcriptome changes, comparing SSc to healthy dermal fibroblasts. Here, we show that SSc dermal myofibroblasts arise in two steps from an SFRP2hi/DPP4-expressing progenitor fibroblast population. In the first step, SSc fibroblasts show globally upregulated expression of transcriptome markers, such as PRSS23 and THBS1. A subset of these cells shows markers indicating that they are proliferating. Only a fraction of SFRP2hi SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. Bioinformatics analysis of the SSc fibroblast transcriptomes implicated upstream transcription factors, including FOSL2, RUNX1, STAT1, FOXP1, IRF7 and CREB3L1, as well as SMAD3, driving SSc myofibroblast differentiation.

Trps1 transcription factor represses phosphate-induced expression of SerpinB2 in osteogenic cells.

Serine protease inhibitor SerpinB2 is one of the most upregulated proteins following cellular stress. This multifunctional serpin has been attributed a number of pleiotropic activities, including roles in cell survival, proliferation, differentiation, immunity and extracellular matrix (ECM) remodeling. Studies of cancer cells demonstrated that expression of SerpinB2 is directly repressed by the Trps1 transcription factor, which is a regulator of skeletal and dental tissues mineralization. In our previous studies, we identified SerpinB2 as one of the novel genes highly upregulated by phosphate (Pi) at the initiation of the mineralization process, however SerpinB2 has never been implicated in formation nor homeostasis of mineralized tissues. The aim of this study was to establish, if SerpinB2 is involved in function of cells producing mineralized ECM and to determine the interplay between Pi signaling and Trps1 in the regulation of SerpinB2 expression specifically in cells producing mineralized ECM. Analyses of the SerpinB2 expression pattern in mouse skeletal and dental tissues detected high SerpinB2 protein levels specifically in cells producing mineralized ECM. qRT-PCR and Western blot analyses demonstrated that SerpinB2 expression is activated by elevated Pi specifically in osteogenic cells. However, the Pi-induced SerpinB2 expression was diminished by overexpression of Trps1. Decreased SerpinB2 levels were also detected in osteoblasts and odontoblasts of 2.3Col1a1-Trps1 transgenic mice. Chromatin immunoprecipitation assay (ChIP) revealed that the occupancy of Trps1 on regulatory elements in the SerpinB2 gene changes in response to Pi. In vitro functional assessment of the consequences of SerpinB2 deficiency in cells producing mineralized ECM detected impaired mineralization in SerpinB2-deficient cells in comparison with controls. In conclusion, high and specific expression of SerpinB2 in cells producing mineralized ECM, the impaired mineralization of SerpinB2-deficient cells and regulation of SerpinB2 expression by two molecules regulating formation of mineralized tissues suggest involvement of SerpinB2 in physiological mineralization.

Reactive oxygen species (ROS) generation as an underlying mechanism of inorganic phosphate (Pi)-induced mineralization of osteogenic cells.

Reactive Oxygen Species (ROS) are a natural byproduct of oxygen metabolism. At physiological levels, ROS regulate multiple cellular processes like proliferation, migration, and differentiation. Increased levels of ROS are associated with pathological conditions, such as inflammation and vascular calcification, where they elicit cytotoxic effects. These contrasting outcomes of ROS have also been reported in osteogenic precursor cells. However, the role of ROS in committed osteogenic cells has not been investigated. Cytotoxic and physiologic effects have also been demonstrated for extracellular phosphate (Pi). Specifically, in committed osteogenic cells Pi stimulates their major function (mineralization), however in osteogenic precursors and endothelial cells Pi cytotoxicity has been reported. Interestingly, Pi cytotoxic effects have been associated with ROS production in the pathological vascular mineralization. In this study, we investigated a molecular mechanistic link between elevated Pi and ROS production in the context of the mineralization function of committed osteogenic cells. Using committed osteogenic cells, 17IIA11 odontoblast-like cell and MLO-A5 osteoblast cell lines, we have unveil that Pi enhances intracellular ROS production. Furthermore, using a combination of mineralization assays and gene expression analyses, we determined that Pi-induced intracellular ROS supports the physiological mineralization process. In contrast, the exogenous ROS, provided in a form of H2O2, was detrimental for osteogenic cells. By comparing molecular signaling cascades induced by extracellular ROS and Pi, we identified differences in signaling routes that determine physiologic versus toxic effect of ROS on osteogenic cells. Specifically, while both extracellular and Pi-induced intracellular ROS utilize Erk1/2 signaling mediator, only extracellular ROS induces stress-activated mitogen-activated protein kinases P38 and JNK that are associated with cell death. In summary, our results uncovered a physiological role of ROS in the Pi-induced mineralization through the molecular pathway that is distinct from ROS-induced cytotoxic effects.

Trps1 Regulates Development of Craniofacial Skeleton and Is Required for the Initiation of Palatal Shelves Fusion.

Trichorhinophalangeal syndrome (TRPS) is an autosomal dominant disorder resulting from heterozygous mutations of the TRPS1 gene. Common craniofacial abnormalities in TRPS patients include micrognathia, hypoplastic zygomatic arch, high-arched palate, and, occasionally, cleft palate. Studies have demonstrated that mice with a heterozygous Trps1 mutation (Trps1+/- mice) have similar features to patients with TRPS, including high-arched palates. However, mice with a homozygous Trps1 mutation (Trps1-/- mice) exhibit similar but more severe abnormalities, including cleft palate. Our study aimed to characterize the craniofacial phenotype to understand the role of Trps1 in craniofacial development and gain insight on the cleft palate pathogenesis in Trps1 deficiency. Whole-mount skeletal staining revealed hypoplastic skeletal and cartilaginous elements, steep nasal slope, and missing presphenoid in Trps1-/- mice. Although several craniofacial skeleton elements were abnormal in Trps1-/- mice, the Trps1 deficiency did not appear to disrupt cranial vault development. All Trps1-/- mice presented with cleft palate. Analyses of Trps1 expression during palatogenesis detected Trps1 mRNA and protein in palatal mesenchyme and in specific regions of palatal epithelium, which suggested that Trps1 is involved in palatal fusion. Ex vivo culture experiments demonstrated that Trps1-/- palatal shelves were unable to initiate the fusion process. On the molecular level, Trps1 deficiency resulted in decreased epithelial expression of proteins involved in palatal fusion, including chondroitin sulfate proteoglycan, transforming growth factor-beta 3, Twist1, and beta-catenin. Mesenchymal expression of chondroitin sulfate proteoglycan expression was unaffected, indicating a cell type-specific mechanism of Trps1 regulation on chondroitin sulfate proteoglycan. In conclusion, we demonstrated that Trps1 is involved in the development of craniofacial skeletal elements and in the initiation of the palatal shelves fusion. Furthermore, our studies uncovered that Trps1 is required for epithelial expression of several proteins involved in the palatal shelves fusion.

Trps1 transcription factor regulates mineralization of dental tissues and proliferation of tooth organ cells.

Mutations of the TRPS1 gene cause trichorhinophalangeal syndrome (TRPS), a skeletal dysplasia with dental abnormalities. TRPS dental phenotypes suggest that TRPS1 regulates multiple aspects of odontogenesis, including the tooth number and size. Previous studies delineating Trps1 expression throughout embryonic tooth development in mice detected strong Trps1 expression in dental mesenchyme, preodontoblasts, and dental follicles, suggesting that TRPS dental phenotypes result from abnormalities in early developmental processes. In this study, Trps1+/- and Trps1-/- mice were analyzed to determine consequences of Trps1 deficiency on odontogenesis. We focused on the aspects of tooth formation that are disturbed in TRPS and on potential molecular abnormalities underlying TRPS dental phenotypes. Microcomputed tomography analyses of molars were used to determine tooth size, crown shape, and mineralization of dental tissues. These analyses uncovered that disruption of one Trps1 allele is sufficient to impair mineralization of dentin in both male and female mice. Enamel mineral density was decreased only in males, while mineralization of the root dental tissues was decreased only in females. In addition, significantly smaller teeth were detected in Trps1+/- females. Histomorphometric analyses of tooth organs showed reduced anterior-posterior diameter in Trps1-/- mice. BrdU-incorporation assay detected reduced proliferation of mesenchymal and epithelial cells in Trps1-/- tooth organs. Immunohistochemistry for Runx2 and Osx osteogenic transcription factors revealed changes in their spatial distribution in Trps1-/- tooth organs and uncovered cell-type specific requirements of Trps1 for Osx expression. In conclusion, this study has demonstrated that Trps1 is a positive regulator of cell proliferation in both dental mesenchyme and epithelium, suggesting that the microdontia in TRPS is likely due to decreased cell proliferation in developing tooth organs. Furthermore, the reduced mineralization observed in Trps1+/- mice may provide some explanation for the extensive dental caries reported in TRPS patients.

Proteomic profiling of extracellular vesicles released from vascular smooth muscle cells during initiation of phosphate-induced mineralization.

Purpose/Aim: Elevated serum phosphate is one of the major factors contributing to vascular calcification. Studies suggested that extracellular vesicles released from vascular smooth muscle cells significantly contribute to the initiation and progression of this pathology. Recently, we have demonstrated that elevated phosphate stimulates release of extracellular vesicles from osteogenic cells at the initiation of the mineralization process. Here, we used MOVAS cell line as an in vitro model of vascular calcification to examine whether vascular smooth muscle cells respond to high phosphate levels in a similar way and increase formation of extracellular vesicles. MATERIALS AND METHODS: Vesicles residing in extracellular matrix as well as vesicles released to culture medium were evaluated by nanoparticle tracking analyses. In addition, using mass spectrometry and protein profiling, protein composition of extracellular vesicles released by MOVAS cells under standard growth conditions and upon exposure to high phosphate was compared. RESULTS: Significant increase of the number of extracellular vesicles was detected after 72 h of exposure of cells to high phosphate. Elevated phosphate levels also affected protein composition of extracellular vesicles released from MOVAS cells. Finally, the comparative analyses of proteins in extracellular vesicles isolated from extracellular matrix and from conditioned medium identified significant differences in protein composition in these two groups of extracellular vesicles. CONCLUSIONS: Results of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.

Condiloma vaginal en niña: presentación de un caso / Vaginal condyloma in a baby girl: report of a case

Se presenta el caso de una niña de 9 años, de raza negra con condilomas acuminados de vulva, cuyas lesiones se extendían desde el clítoris a los labios y región perineal. Se han registrado pocos casos de esta enfermedad en niños en la literatura mundial. Como antecedentes patológicos de importancia fue diagnosticada infección por VIH sin tratamiento antirretroviral. El examen físico reveló múltiples condilomas y pápulas en ambos labios y clítoris. El examen ginecológico no mostró evidencia de abuso sexual. Las lesiones fueron extirpadas por tratamiento quirúrgico seguido de curas con solución antiséptica de betadine (yodo) con resultados satisfactorios. La familia recibió apoyo psicológico según fue necesario. Se concluye que los condilomas en niños son un fuerte índice de abuso sexual, pero no exclusivo(AU)

For a 9 year old girl, black with vulvar condyloma, whose lesions extended from the clitoris and perineum lip are presented. There have been few cases of this disease in children in other literatures. As significant medical history, she was diagnosed with HIV without antiretroviral treatment. Physical examination revealed multiple condylomas and bumps on both lips and clitoris. The gynecological examination showed no evidence of sexual abuse. The lesions were excised by surgery followed by healing with antiseptic Betadine (iodine) with satisfactory results. The family received counseling as necessary. As a conclusion, the condyloma in children is a strong rate of sexual abuse, but not exclusive