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Type 2 diabetes mellitus is a leading health issue worldwide. Among cases of diabetes mellitus nephropathy (DN), the major complication of type 2 diabetes mellitus, the nephrotic phenotype is often intractable to clinical intervention and demonstrates the rapid decline of renal function to end-stage renal disease. We recently identified the gene for glypican-5 (GPC5), a cell-surface heparan sulfate proteoglycan, as conferring susceptibility for acquired nephrotic syndrome and additionally identified an association through a genome-wide association study between a variant in GPC5 and DN of type 2 diabetes mellitus. In vivo and in vitro data showed a progressive increase of GPC5 in type 2 DN along with severity; the excess was derived from glomerular mesangial cells. In this study, diabetic kidney showed that accumulation of fibroblast growth factor (Fgf)2 strikingly induced progressive proteinuria that was avoided in Gpc5 knockdown mice. The efficacy of Gpc5 inhibition was exerted through expression of the Fgf receptors 3 and 4 provoked in the diabetic kidney attributively. Extraglomerular Fgf2 was pathogenic in DN, and the deterrence of Gpc5 effectively inhibited the glomerular accumulation of Fgf2, the subsequent increase of mesangial extracellular matrix, and the podocytes' small GTPase activity. These findings elucidate the pivotal role of GPC5, identified as a susceptible gene in the genome-wide association study, in hyperglycemia-induced glomerulopathy.
Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
The hippocampus is a brain area characterized by its high plasticity, observed at all levels of organization: molecular, synaptic, and cellular, the latter referring to the capacity of neural precursors within the hippocampus to give rise to new neurons throughout life. Recent findings suggest that promoter methylation is a plastic process subjected to regulation, and this plasticity seems to be particularly important for hippocampal neurogenesis. We have detected the enzyme GNMT (a liver metabolic enzyme) in the hippocampus. GNMT regulates intracellular levels of SAMe, which is a universal methyl donor implied in almost all methylation reactions and, thus, of prime importance for DNA methylation. In addition, we show that deficiency of this enzyme in mice (Gnmt-/-) results in high SAMe levels within the hippocampus, reduced neurogenic capacity, and spatial learning and memory impairment. In vitro, SAMe inhibited neural precursor cell division in a concentration-dependent manner, but only when proliferation signals were triggered by bFGF. Indeed, SAMe inhibited the bFGF-stimulated MAP kinase signaling cascade, resulting in decreased cyclin E expression. These results suggest that alterations in the concentration of SAMe impair neurogenesis and contribute to cognitive decline.
© 2014 Wiley Periodicals, Inc.
Glypican-1 is a cell membrane heparan sulfate proteoglycan. It is composed of a core protein with covalently attached glycosaminoglycan, and N-linked glycosylated (N-glycosylated) chains, and is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage. Glypican-1 plays a key role in the growth and development of muscle by regulating fibroblast growth factor 2 (FGF2). The GPI anchor of glypican-1 can be cleaved, resulting in glypican-1 being secreted or shed into the extracellular matrix environment. The objective of the current study was to investigate the role of glypican-1 shedding and the glycosaminoglycan and N-glycosylated chains in regulating the differentiation of turkey myogenic satellite cells. A glypican-1 construct without the GPI anchor was cloned into the mammalian expression vector pCMS-EGFP, and glypican-1 without the GPI anchor and glycosaminoglycan and N-glycosylated chains were also cloned. These constructs were co-transfected into turkey myogenic satellite cells with a small interference RNA targeting the GPI anchor of endogenous glypican-1. The soluble glypican-1 mutants were not detected in the satellite cells but in the cell medium, suggesting the secretion of the soluble glypican-1 mutants. Soluble glypican-1 increased satellite cell differentiation and enhanced myotube formation in the presence of exogenous FGF2. The increase in differentiation was supported by the elevated expression of myogenin. In conclusion, the shedding of glypican-1 from the satellite cell surface acts as a positive regulator of satellite cell differentiation and sequesters FGF2, permitting further differentiation.
Copyright © 2012 Elsevier Inc. All rights reserved.
During fetal lung development, cells within the mesenchyme differentiate into vascular endothelia. This process of vasculogenesis gives rise to the cells that will eventually form the alveolar capillary bed. The cellular mechanisms regulating lung vasculogenesis are poorly understood, partly due to the lack of experimental systems that model this process. Here, we have developed and characterized a novel fetal mouse lung cell model of mesenchymal to endothelial differentiation. Using mesenchymal cells from the lungs of embryonal day 15 Immortomice, we show that endothelial growth media containing fibroblast growth factor-2 and vascular endothelial growth factor can stimulate formation of vascular endothelial cells in culture. These newly formed endothelial cells retain plasticity, as removing endothelial growth media causes loss of vascular markers and renewed formation of α-smooth muscle actin positive stress fibers. Cells with the highest Flk-1 expression differentiated into endothelia more efficiently. Individual mesenchymal cell clones had varied ability to acquire an endothelial phenotype. These fetal lung mesenchymal cells were multipotent, capable of differentiating into not only vascular endothelia, but also osteogenic and chondrongenic cell lineages. Our data establish a cell culture model for mesenchymal to endothelial differentiation that could prove useful for future mechanistic studies in the process of vasculogenesis both during normal development and in the pathogenesis of pulmonary vascular disease.
OBJECTIVES - The overarching goal of this line of research is to translate basic fibroblast growth factor (bFGF) treatment for vocal fold scarring into practical clinical use. In a previous canine investigation, we demonstrated that bFGF improves phonation threshold pressure, mucosal wave amplitude, and histologic measures in vocal folds treated after injury. In the present study, we studied the effects of bFGF on gene expression of the extracellular matrix and growth factors in rat vocal fold fibroblasts.
METHODS - Fibroblasts harvested from the vocal folds of 5 rats were treated with 3 concentrations of bFGF (0, 10, and 100 ng/mL). The fibroblasts were collected at 24 hours and 72 hours after bFGF administration. Quantitative polymerase chain reaction was then used to investigate the gene expression of the investigated growth factors and extracellular matrices.
RESULTS - The results revealed significantly down-regulated expression of procollagen I and significantly up-regulated expression of hyaluronic acid synthase (HAS) 2 and fibronectin in fibroblasts treated with bFGF. The administration of bFGF also resulted in the up-regulation of bFGF and hepatocyte growth factor (HGF). No changes in the expression of HAS-1, tropoelastin, or procollagen III were observed between the treatment and control conditions.
CONCLUSIONS - Treatment with bFGF induces the down-regulation of procollagen I and the up-regulation of HAS-2 in vocal fold fibroblast cell cultures. These gene expression alterations to key mediators of the wound healing process may translate into potential benefits in the remediation of vocal fold injury. The up-regulation of HGF, an antifibrotic effector molecule, may demonstrate additional benefits by optimizing the wound healing environment and by accelerating the wound repair cascade. These findings may provide fuel for additional discoveries into the development of growth factor therapy for the treatment of vocal fold scar.
CONCLUSIONS - Results of the current study revealed improved phonation threshold pressure (PTP), normalized mucosal wave amplitude (NMWA), and less contraction of the lamina propria in injured larynges treated with basic fibroblast growth factor (bFGF).
OBJECTIVES - We investigated the effects of local injection of bFGF for treatment of acute vocal fold injury in a canine model.
METHODS - Vocal folds of eight beagles were unilaterally injured by removal of the mucosa under direct laryngoscopy. Four beagles received local injections of bFGF delivered to the scarred vocal fold at 1 month after injury. The remaining four beagles received local injections of saline and served as a sham-treatment group. Larynges were harvested 5 months after treatment and excised larynx experiments were performed to measure PTP, NMWA, and normalized glottal gap (NGG). Histologic staining was performed to evaluate structural changes of the extracellular matrix.
RESULTS - Excised larynx measurements revealed significantly lower PTP and increased NMWA in bFGF-treated vocal fold. Elastica Van Gieson staining revealed less contraction of the bFGF-treated vocal fold. Histologic measurements revealed that the thickness of the lamina propria was significantly greater in the bFGF-treated vocal fold. Alcian blue staining revealed improved restoration of hyaluronic acid in the bFGF-treated vocal fold.
The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.
2010 Elsevier Inc. All rights reserved.
OBJECTIVES - We investigated acute changes in extracellular matrix (ECM) gene expression and histologic changes in the deposition of collagen and hyaluronan (hyaluronic acid; HA) after basic fibroblast growth factor (bFGF) treatment of the aged rat vocal fold.
METHODS - For the polymerase chain reaction (PCR) experiments, we divided ten 18-month-old Sprague-Dawley rats into two groups that received serial injections of sham (saline solution) or bFGF (2 ng/microL) and euthanized them 2 weeks after the initial injection to investigate acute changes in ECM gene expression. We treated a separate group of 5 animals unilaterally and sacrificed them 4 weeks after the initial injection to investigate histologic changes in the deposition of collagen and HA.
RESULTS - Real-time PCR revealed significantly up-regulated HA synthase (HAS)-2, HAS-3, matrix metalloproteinase (MMP)-2, and procollagen type I gene expression in the bFGF treatment group as compared to the sham treatment group. Histologic staining revealed significantly increased deposition of HA in the bFGF-treated vocal fold as compared to the sham-treated vocal fold. No differences in ECM collagen levels were observed between treatment sides.
CONCLUSIONS - Basic fibroblast growth factor induced the up-regulation of HAS-2, HAS-3, MMP-2, and procollagen type I. Histologically, aged vocal folds treated with bFGF revealed increased deposition of HA as compared to sham-treated vocal folds.
Successful approaches to tissue engineering smooth muscle tissues utilize biodegradable scaffolds seeded with autologous cells. One common problem in using biological scaffolds specifically is the difficulty of inducing cellular penetration and controlling de novo extracellular matrix deposition/remodeling in vitro. Our hypothesis was that small intestinal submucosa (SIS) exposed to specific mechanical stimulation regimes would modulate the synthesis of de novo collagen and elastin by bladder smooth muscle cells (BSMC) within the SIS matrix. We further hypothesized that the cytokines vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), two key growth factors involved in epithelial mesenchymal signaling, will promote the cellular penetration into SIS necessary for mechanical stimulation. BSMC were seeded at 0.5 x 10(6) cells/cm(2) onto the luminal side of SIS specimens. VEGF (10 ng/mL) and FGF-2 (5 ng/mL) were added to each insert in the media every other day for up to 7 days in static culture. Following static culture, specimens were stretched strip-biaxially under 15% peak strain at either 0.5 or 0.1 Hz for an additional 7 days. Following the culture period, specimens were assayed histologically and biochemically for cellular penetration, proliferation, elastin, collagen, and protease activity. Histological analyses demonstrated that in standard culture media, BSMC remained on the surface of the SIS while both FGF-2 and VEGF profoundly promoted ingrowth of the BSMC into the SIS. The penetration of the cells in response to these cytokines was confirmed using a Transwell assay. Following cellular penetration, BSMC produced significant amounts of elastic fibers under cyclic mechanical stretching at 0.1 Hz under 15% stretch, as evidenced by colorimetric assay and histology using a Verhoeff-Van Gieson stain. Protease activity was assessed in the media and found to be statistically increased in static culture following FGF-2 treatment. These findings demonstrate, for the first time, the capability of BSMC to produce histologically apparent elastin fibers in vitro. Moreover, our results suggest that a strategy involving growth factors and controlled mechanical stimulation may be used to engineer functional, elastin-rich tissue replacements using decellularized biologically derived scaffolds.
OBJECTIVES - Our objective in this study was to apply an elastic, biodegradable polyester urethane urea (PEUU) cardiac patch onto subacute infarcts and to examine the resulting cardiac ventricular remodeling and performance.
BACKGROUND - Myocardial infarction induces loss of contractile mass and scar formation resulting in adverse left ventricular (LV) remodeling and subsequent severe dysfunction.
METHODS - Lewis rats underwent proximal left coronary ligation. Two weeks after coronary ligation, a 6-mm diameter microporous PEUU patch was implanted directly on the infarcted LV wall surface (PEUU patch group, n = 14). Sham surgery was performed as an infarction control (n = 12). The LV contractile function, regional myocardial wall compliance, and tissue histology were assessed 8 weeks after patch implantation.
RESULTS - The end-diastolic LV cavity area (EDA) did not change, and the fractional area change (FAC) increased in the PEUU patch group (p < 0.05 vs. week 0), while EDA increased and FAC decreased in the infarction control group (p < 0.05). The PEUU patch was largely resorbed 8 weeks after implantation and the LV wall was thicker than infarction control (p < 0.05 vs. control group). Abundant smooth muscle bundles with mature contractile phenotype were found in the infarcted myocardium of the PEUU group. The myocardial compliance of the PEUU group was distributed between normal myocardium and infarction control (p < 0.001).
CONCLUSIONS - Implantation of a novel biodegradable PEUU patch onto a subacute myocardial infarction promoted contractile phenotype smooth muscle tissue formation and improved cardiac remodeling and contractile function at the chronic stage. Our findings suggest a new therapeutic option against post-infarct cardiac failure.