FGF-2 mRNA Levels Research Articles

Curcumin Reduces Hypoxia/Reperfusion Injury of Cardiomyocytes byStimulating Vascular Endothelial Cells to Secrete FGF2.

Endothelial cells (ECs) can provide cell protection for cardiomyocytes (CMs) under hypoxia-reoxygenation (HR) conditions by secreting derived factors. This study aimed to explore the role of curcumin (CUR) in ECs for protecting CMs from HR injury. A co-culture system for ECs and CMs was set up, and subjected to HR. The transcription, expression, and secretion of FGF2 were detected by RT-qPCR, western blot, and ELISA, respectively. siRNAs specifically targeting FGF2 were transfected into ECs. FGF2 receptor- specific inhibitors (AZD4547) were used to treat CMs. The co-culture with ECs did not affect the proliferation of CMs, while CUR and ECs co-culture had a synergistic effect on promoting the proliferation of CMs in HR. Furthermore, the co-culture with ECs did not affect the apoptosis and autophagy of CMs in HR. However, the co-culture of ECs after CUR treatment inhibited the apoptosis and autophagy of CMs in HR. CUR treatment significantly enhanced FGF2 mRNA, protein, and secretion levels of ECs in HR. In addition, CUR treatment increased FGF2 levels in the CMs medium in the ECs and CMs co-culture system. The reduction of FGF2 levels in the medium and the inhibition of FGF2 receptors significantly inhibited the proliferation of CMs and significantly promoted the apoptosis and autophagy of CMs in HR. Focusing on the protective effects of CUR and ECs on cardiomyocytes is of great significance for the treatment of clinical myocardial HR injury.

Gα11 deficiency increases fibroblast growth factor 23 levels in a mouse model of familial hypocalciuric hypercalcemia.

Fibroblast growth factor 23 (FGF23) production has recently been shown to increase downstream of Gαq/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding Gα11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the effect of Gα11 deficiency on FGF23 production in mice with heterozygous (Gna11+/-) or homozygous (Gna11-/-) ablation of Gna11. Both Gna11+/- and Gna11-/- mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11-/- mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and also showed evidence of systemic inflammation with elevated serum IL-1β levels. Furin gene expression was significantly increased in the Gna11-/- liver, suggesting enhanced FGF23 cleavage despite the observed rise in circulating intact FGF23 levels. Gna11-/- mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, Gα11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that patients with FHH - at least those with GNA11 mutations - may be at risk for these complications.

The XPO1 inhibitor selinexor ameliorates bleomycin-induced pulmonary fibrosis in mice via GBP5/NLRP3 inflammasome signaling

Pulmonary fibrosis is an irreversible and progressive lung disease with limited treatments available. Selinexor (Sel), an orally available, small-molecule, selective inhibitor of XPO1, exhibits notable antitumor, anti-inflammatory and antiviral activities. However, its potential role in treating pulmonary fibrosis is unknown. C57BL/6J mice were used to establish a pulmonary fibrosis model by intratracheal administration of bleomycin (BLM). Subsequently, Sel was administered intraperitoneally. Our data demonstrated that Sel administration ameliorated BLM-induced pulmonary fibrosis by increasing mouse body weights; reducing H&E staining, Masson staining scores, and shadows in mouse lung computed tomography (CT) images, decreasing the total cell and neutrophil counts in the lung and bronchoalveolar lavage fluid (BALF); and decreasing the levels of TGF-β1. We next confirmed that Sel reduced the deposition of extracellular matrix (ECM) components in the lungs of BLM-induced pulmonary fibrosis mice. We showed that collagen I, alpha-smooth muscle actin (α-SMA), and hydroxyproline levels and the mRNA levels of Col1a1, Eln, Fn1, Ctgf, and Fgf2 were reduced. Mechanistically, tandem mass tags (TMT)- based quantitative proteomics analysis revealed a significant increase in GBP5 in the lungs of BLM mice but a decrease in that of BLM + Sel mice; this phenomenon was confirmed by western blotting and RT-qPCR. NLRP3 inflammasome signaling was significantly enriched in both the BLM group and BLM + Sel group based on GO and KEGG analyses of differentially expressed proteins between the groups. Furthermore, Sel reduced the expression of NLRP3, cleaved caspase 1, and ASC in vivo and in vitro, and decreased the levels of IL-1β, IL-18, and IFN-r in lung tissue and BALF. SiRNA-GBP5 inhibited NLRP3 signaling in vitro, and overexpression of GBP5 inhibited the protective effect of Sel against BLM-induced cellular injury. Taken together, our findings indicate that Sel ameliorates BLM-induced pulmonary fibrosis by targeting GBP5 via NLRP3 inflammasome signaling. Thus, the XPO1 inhibitor – Sel might be a potential therapeutic agent for pulmonary fibrosis.

Effect of collagen endometrial patch loaded with adipose-derived mesenchymal stem cells on endometrial regeneration in rats with a thin endometrium.

This study aimed to investigate the effects of a collagen endometrial patch (EM patch) loaded with adipose-derived mesenchymal stem cells (ADSCs) on endometrial regeneration in a rat model with thin endometrium. Thin endometrium was induced in female rats and divided into treatment groups as outlined: control, group 1(G1), local injection of ADSCs into the uterus, group 2 (G2), an EM patch without ADSCs, group 3 (G3), and an EM patch loaded with ADSCs, group 4 (G4). The rats were euthanized at either two weeks or four weeks after modeling and treatment followed by histological and biochemical analyses to examine the regenerative effects on the injured endometrium. Transplantation of the ADSC-loaded EM patch significantly promoted endometrial proliferation and increased the luminal epithelial area. Two weeks after treatment, the mean number of von Villebrand factor (vWF)+ or cluster of differentiation (CD) 31+-stained blood vessels was significantly higher in G4 than in G1 and G2. The mRNA and protein expression levels of TGF-β and FGF2 were significantly upregulated in G4 compared to those in the control. G4 exhibited significantly increased LIF mRNA levels and immunoreactivity compared with the other groups at both two weeks and four weeks after treatment. Cell tracking after ADSCs treatment revealed the presence of a substantial number of ADSCs grafted in the uterine tissues of G4, whereas a low number of ADSCs that were focally clustered were present in G2. Transplantation of EM patches loaded with ADSCs resulted in the histological and biochemical restoration of an injured endometrium. The strategic integration of EM patches and ADSCs holds significant promise as an innovative therapeutic approach for effectively treating impaired endometrial conditions.

Kidney Damage in Long COVID: Studies in Experimental Mice.

Signs and symptoms involving multiple organ systems which persist for weeks or months to years after the initial SARS-CoV-2 infection (also known as PASC or long COVID) are common complications of individuals with COVID-19. We recently reported pathophysiological changes in various organs post-acute infection of mice with mouse hepatitis virus-1 (MHV-1, a coronavirus) (7 days) and after long-term post-infection (12 months). One of the organs severely affected in this animal model is the kidney, which correlated well with human studies showing kidney injury post-SARS-CoV-2 infection. Our long-term post-infection pathological observation in kidneys includes the development of edema and inflammation of the renal parenchyma, severe acute tubular necrosis, and infiltration of macrophages and lymphocytes, in addition to changes observed in both acute and long-term post-infection, which include tubular epithelial cell degenerative changes, peritubular vessel congestion, proximal and distal tubular necrosis, hemorrhage in the interstitial tissue, and vacuolation of renal tubules. These findings strongly suggest the possible development of renal fibrosis, in particular in the long-term post-infection. Accordingly, we investigated whether the signaling system that is known to initiate the above-mentioned changes in kidneys in other conditions is also activated in long-term post-MHV-1 infection. We found increased TGF-β1, FGF23, NGAL, IL-18, HIF1-α, TLR2, YKL-40, and B2M mRNA levels in long-term post-MHV-1 infection, but not EGFR, TNFR1, BCL3, and WFDC2. However, only neutrophil gelatinase-associated lipocalin (NGAL) increased in acute infection (7 days). Immunoblot studies showed an elevation in protein levels of HIF1-α, TLR-2, and EGFR in long-term post-MHV-1 infection, while KIM-1 and MMP-7 protein levels are increased in acute infection. Treatment with a synthetic peptide, SPIKENET (SPK), which inhibits spike protein binding, reduced NGAL mRNA in acute infection, and decreased TGF-β1, BCL3 mRNA, EGFR, HIF1-α, and TLR-2 protein levels long-term post-MHV-1 infection. These findings suggest that fibrotic events may initiate early in SARS-CoV-2 infection, leading to pronounced kidney fibrosis in long COVID. Targeting these factors therapeutically may prevent acute or long-COVID-associated kidney complications.

Modulation of fibroblast growth factor-23 expression and transepithelial calcium absorption in Caco-2 monolayer by calcium-sensing receptor and calcineurin under calcium hyperabsorptive state

Fibroblast growth factor (FGF)-23 and calcium-sensing receptor (CaSR) have previously been postulated to be parts of a negative feedback regulation of the intestinal calcium absorption to prevent excessive calcium uptake and its toxicity. However, the underlying mechanism of this feedback regulation remained elusive, especially whether it required transcription of FGF-23. Herein, we induced calcium hyperabsorptive state (CHS) by exposing intestinal epithelium-like Caco-2 monolayer to 30 mM CaCl2 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after which FGF-23 mRNA levels and transepithelial calcium flux were determined. We found that CHS upregulated FGF-23 transcription, which was reverted by CaSR inhibitors (Calhex-231 and NPS2143) but without effect on CaSR transcription. Although 10 nM 1,25(OH)2D3 was capable of enhancing transepithelial calcium flux, the higher-than-normal calcium inundation as in CHS led to a decrease in calcium flux, consistent with an increase in FGF-23 protein expression. Administration of inhibitors (≤10 μM CN585 and cyclosporin A) of calcineurin, a mediator of CaSR action to control transcription and production of its target proteins, was found to partially prevent FGF-23 protein production and the negative effect of CHS on calcium transport, while having no effect on FGF-23 mRNA expression. Direct exposure to FGF-23, but not FGF-23 + PD173074 (FGFR1/3 inhibitor), also completely abolished the 1,25(OH)2D3-enhanced calcium transport in Caco-2 monolayer. Nevertheless, CHS and CaSR inhibitors had no effect on the mRNA levels of calcineurin (PPP3CB) or its targets (i.e., NFATc1–4). In conclusion, exposure to CHS induced by high apical calcium and 1,25(OH)2D3 triggered a negative feedback mechanism to prevent further calcium uptake. CaSR and its downstream mediator, calcineurin, possibly contributed to the regulatory process, in part by enhancing FGF-23 production to inhibit calcium transport. Our study, therefore, corroborated the physiological significance of CaSR-autocrine FGF-23 axis as a local feedback loop for prevention of excessive calcium uptake.

Effectiveness of topical administration of platelet-rich plasma on the healing of methicillin-resistant Staphylococcus aureus-infected full-thickness wound model

This study aimed to investigate the wound-healing activity of animal platelet-rich plasma (PRP) in wounds infected with methicillin-resistant Staphylococcus aureus (MRSA) in rats. After wound induction, the rats were divided into three groups: noninfected animals treated with PRP (PRP group), MRSA-infected animals treated with mupirocin (standard control group), and MRSA-infected animals treated with PRP (MRSA+PRP group). Scratch assays, MTT test, and live/dead cells were also investigated. Total bacterial count, parameters of wound area, histopathological assessment, and expressions of IL-1β, TNF-α, iNOS, PDGF, FGF-2, and TGF-β mRNA levels and immunofluorescent staining of CD31 and collagen type 1 were assessed. The results showed that culture with PRP increased migration. PRP only showed cytotoxicity in a concentration of 100%. Topical application of PRP (50 µL) reduced the wound area and total bacterial count compared with the control group (P<0.05). The mRNA levels of IL-1β, TNF-α, and iNOS expression on days 7 and 14 (P<0.05) decreased in the treated groups compared with control rats. The mRNA levels of PDGF and TGF-β expression (P<0.05) increased in the treatment groups compared with control rats on days 3 and 7 (P<0.05). FGF-2 expression was significantly higher in the treated groups compared with the control group on days 7 and 14 (P<0.05). Moreover, positive expressions of macrophage colony-stimulating factor (M-CSF), CD31, collagen type 1 and cytokeratin proteins keratinocyte proliferation, and re-epithelization were significantly (P<0.05) increased in both PRP and MRSA+PRP-treated groups compared with the control groups on days 7 and 14. Topical administration of PRP accelerated the wound healing in MRSA-infected wound by decreasing the inflammation and improving the proliferative phase.

Role of the Phosphate Transport Facilitator XPR1 in Bone Homeostasis and Phosphate‐Dependent FGF23 Secretion

BackgroundEvidence point at Xenotropic and Polytropic retrovirus Receptor 1 (XPR1) as a protein involved in the cellular phosphate export pathway and phosphate homeostasis: systemic constitutive deletion of Xpr1 in mice is lethal and kidney‐specific deletion of Xpr1 induces hypophosphatemic rickets with hypercalciuria. XPR1 is expressed in the bone, but its function remains elusive. We hypothesized that XPR1 is crucial for bone homeostasis and that is involved in the sensing mechanism of phosphate in the bone.MethodsWe established an inducible whole body Xpr1 KO mouse model. The mice were placed in metabolic cages for urine and blood analysis, followed by femur uCT and histomorphometry. The Fgf23 mRNA and protein (c‐terminal and intact FGF23) levels were assessed from tibia and femur incubated ex vivo for 24h with increasing phosphate concentrations. Primary osteoblast cells were isolated from control and Xpr1 KO mouse calvaria.ResultsConditional total body Xpr1KO mice are constantly decreasing their body weight upon Xpr1 deletion, and develop profound hypophosphatemia, compared to their control littermates. Histomorphometry analysis and uCT on femurs of Xpr1 KO mice show high trabecular bone density, despite low bone formation rate and low number of osteoclasts. Increased intact FGF23 secretion by tibia and femur was observed in control mice in response to increased phosphate concentration in the media. However, the bone samples from Xpr1KO mice are resistant to phosphate induced FGF23 secretion. No changes were observed for the c‐terminal FGF23 in response to increased phosphate concentration, for both the control and Xpr1 KO mice.Osteoblasts were successfully prepared from control and Xpr1 KO mouse calvarie and Xpr1 expression was inactivated by tamoxifen treatment.ConclusionXPR1 is essential for phosphate homeostasis and FGF23 secretion upon phosphate exposure. Osteoblasts will now be used to further study phosphate dependent FGF23 secretion.

Lactic acid induces fibroblast growth factor 23 (FGF23) production in UMR106 osteoblast-like cells

Endocrine and paracrine fibroblast growth factor 23 (FGF23) is a protein predominantly produced by bone cells with strong impact on phosphate and vitamin D metabolism by targeting the kidney. Plasma FGF23 concentration early rises in kidney and cardiovascular diseases correlating with progression and outcome. Lactic acid is generated in anaerobic glycolysis. Lactic acidosis is the consequence of various physiological and pathological conditions and may be fatal. Since FGF23 production is stimulated by inflammation and lactic acid induces pro-inflammatory signaling, we investigated whether and how lactic acid influences FGF23. Experiments were performed in UMR106 osteoblast-like cells, Fgf23 mRNA levels estimated from quantitative real-time polymerase chain reaction, and FGF23 protein determined by enzyme-linked immunosorbent assay. Lactic acid dose-dependently induced Fgf23 gene expression and up-regulated FGF23 synthesis. Also, Na+-lactate as well as formic acid and acetic acid up-regulated Fgf23. The lactic acid effect was significantly attenuated by nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB) inhibitors wogonin and withaferin A. Lactic acid induces FGF23 production, an effect at least in part mediated by NFκB. Lactic acidosis may, therefore, be paralleled by a surge in plasma FGF23.

Suppression of autophagy promotes fibroblast activation in p53-deficient colorectal cancer cells

Deficiency of p53 in cancer cells activates the transformation of normal tissue fibroblasts into carcinoma-associated fibroblasts; this promotes tumor progression through a variety of mechanisms in the tumor microenvironment. The role of autophagy in carcinoma-associated fibroblasts in tumor progression has not been elucidated. We aimed to clarify the significance of autophagy in fibroblasts, focusing on the TP53 status in co-cultured human colorectal cancer cell lines (TP53-wild-type colon cancer, HCT116; TP53-mutant colon cancer, HT29; fibroblast, CCD-18Co) in vitro. Autophagy in fibroblasts was significantly suppressed in association with ACTA2, CXCL12, TGFβ1, VEGFA, FGF2, and PDGFRA mRNA levels, when co-cultured with p53-deficient HCT116sh p53 cells. Exosomes isolated from the culture media of HCT116sh p53 cells significantly suppressed autophagy in fibroblasts via inhibition of ATG2B. Exosomes derived from TP53-mutant HT29 cells also suppressed autophagy in fibroblasts. miR-4534, extracted from the exosomes of HCT116sh p53 cells, suppressed ATG2B in fibroblasts. In conclusion, a loss of p53 function in colon cancer cells promotes the activation of surrounding fibroblasts through the suppression of autophagy. Exosomal miRNAs derived from cancer cells may play a pivotal role in the suppression of autophagy.

The Tmprss6-/- Mouse Model of Iron Refractory Iron Deficiency Anemia (IRIDA) Exhibits Disrupted Phosphate Homeostasis, Elevated Circulating FGF23 Levels, and Increased Fgf23 Expression in Bone Marrow

While the effects of chronic iron deficiency on erythropoiesis are well documented, the effects of iron deficiency on metabolism remain poorly understood. Recent studies suggest that iron deficiency anemia may impact systemic phosphate regulation (Farrow et al ., PNAS, 2011; Clinkenbeard et al ., J Bone Miner Res, 2014; David et al ., Kidney Int, 2016; Hanudel et al ., Am J Physiol Renal Physiol, 2016). Here we used Tmprss6-/- mice, a model of iron refractory iron deficiency anemia (IRIDA) characterized by hepcidin elevation, to explore the relationship between systemic iron deficiency and phosphate homeostasis. By intercrossing Tmprss6+/- mice, we generated Tmprss6-/- mice and littermate controls, which were raised on a standard rodent diet (Teklad 2018S containing 200 ppm iron and 0.7% phosphorus) prior to study at 8 weeks of age. Tmprss6-/- mice displayed a trend toward lower serum phosphate concentration, as well as a significant elevation in the urinary phosphate/creatinine ratio. Because of the role of phosphate in bone mineralization, we also analyzed the tibiae of Tmprss6-/- mice and found that they showed alterations in bone histology and biomechanical properties.To determine if fibroblast growth factor 23 (FGF23), a key phosphate-regulating hormone, may contribute to these changes in phosphate balance, we compared plasma FGF23 levels of 8-week-old Tmprss6-/- mice to littermate controls using enzyme-linked immunosorbant assays (ELISAs). Tmprss6-/- mice showed significantly higher levels of the biologically-active (“intact”) form of FGF23 as well as “total” FGF23 (a measure of both the intact form and the C-terminal cleavage product of FGF23). This FGF23 elevation was also observed in 3-week-old Tmprss6-/- pups (i.e. prior to weaning). Because osteocytes in bone have been described as a major site of FGF23 production, we measured Fgf23 mRNA levels by qRT-PCR in RNA that was prepared from femur bones of Tmprss6-/- mice or littermate controls after marrow removal by flushing. We observed a four-fold increase in Fgf23 expression in the femurs of Tmprss6-/- mice as compared to littermate controls. Because Fgf23 expression has previously been reported in Ter119+ erythroid cells from wild type mice (Coe et al ., J Biol Chem, 2014), and because our RNA preparations from femur bones showed evidence of residual bone marrow contamination (as assessed by alpha hemoglobin expression), we also measured Fgf23 mRNA expression in RNA prepared from total bone marrow. Tmprss6-/- mice exhibited significantly higher bone marrow Fgf23 mRNA levels than littermate controls. Notably, when RNA was prepared from bones that had been more effectively depleted of marrow by mechanical disruption and hypotonic lysis, Tmprss6-/- mice no longer showed an increase in Fgf23 mRNA levels in the femur RNA as compared to littermate controls. Collectively, these results suggest that increased Fgf23 production by one or more bone marrow cell populations contributes to elevated circulating FGF23 in Tmprss6-/- mice.FGF23 has been proposed as the basis for a bone-kidney axis that coordinates bone health with renal handling of phosphate. Our findings suggest that altered phosphate homeostasis in Tmprss6-/- mice may result from increased Fgf23 expression in the bone marrow and a corresponding elevation in circulating FGF23. These results may have relevance to the skeletal health of patients with IRIDA due to TMPRSS6 mutation and those with other forms of iron-restricted anemia resulting from hepcidin elevation. Additionally, as Tmprss6-/- mice are known to exhibit growth retardation, our results raise the possibility that disrupted phosphate homeostasis may contribute to the commonly observed association of childhood iron deficiency with growth impairment. Future studies will characterize the source of Fgf23 production in the bone marrow of Tmprss6-/- mice as well as the mechanism by which iron deficiency elevates Fgf23 expression. DisclosuresNo relevant conflicts of interest to declare.

FC 018HIGH DIETARY PHOSPHATE INTAKE AND INTRA-CARDIAC SYNTHESIS OF FIBROBLAST GROWTH FACTOR 23 SYNERGISTICALLY WORSEN CARDIAC FUNCTION

Abstract Background and Aims Left ventricular hypertrophy (LVH) is a major complication of CKD and associates with increased levels of the phosphaturic hormone fibroblast growth factor (FGF) 23. FGF23 induces hypertrophic growth of cardiac myocytes in vitro and LVH in rodents, suggesting that FGF23 can directly affect the heart. Besides the bone, cardiac myocytes express FGF23, too, and recent studies demonstrate that its expression is increased in cardiac and kidney injury, suggesting that cardiotoxicity of FGF23 may be at least partly due to the paracrine effects of heart-derived FGF23. However, it is still questioned whether elevated FGF23 per se is able to induce pathologic alterations in the heart or whether additional factors in CKD, such as Klotho deficiency or hyperphosphatemia are required for FGF23 to tackle the heart. By generating a mouse model with cardiac-specific overexpression of FGF23 via myocardial gene transfer using adeno-associated virus (AAV), we elucidated the cardiotoxic properties of elevated FGF23 in (1) unchallenged mice, unbiased of alterations usually associated with CKD, and (2) in the presence of high dietary phosphate intake, mimicking the exposure of enhanced serum phosphate. Method First, an adeno-associated virus that expresses murine Fgf23 (AAV-Fgf23) under the control of the cardiac troponin T promotor was injected subcutaneously into eight-week-old male C57BL/6 wildtype mice. After four months, cardiac function and geometry was assessed by cardiac magnetic resonance imaging (MRI) and echocardiography and heart tissue was analysed by qPCR, immunoblot and histological analyses. The biological activity of AAV-Fgf23-derived cardiac Fgf23 was determined using isolated neonatal rat ventricular myocytes (NRVM) in vitro. Second, AAV-Fgf23 and control mice were fed a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD) and cardiac phenotype was investigated after six months. Results AAV-Fgf23 mice showed increased cardiac-specific Fgf23 expression and synthesis of intact Fgf23 (iFgf23) protein in the heart resulting in enhanced circulating iFgf23 compared to control. Serum of AAV-Fgf23 mice stimulated hypertrophic growth of isolated NRVM and induced pro-hypertrophic gene expression in vitro, indicating that cardiac iFgf23 is biologically active. Likewise, AAV-Fgf23 mice revealed an activation of renal FGFR1/Klotho/MAPK signalling and subsequent down-regulation of renal sodium phosphate transporters NaPi-2a and NaPi-2c, causing reduced tubular phosphate reabsorption. Nevertheless, in unchallenged AAV-Fgf23 mice, impaired cardiac function, LVH and LV fibrosis were lacking. In contrast, HPD stimulated the bone expression of Fgf23 in both AAV-Fgf23 and Ctrl mice, while intra-cardiac Fgf23 mRNA levels were only increased in both AAV-Fgf23 groups irrespective of NPD or HPD. However, HPD in AAV-Fgf23 mice promoted O-glycosylation of cardiac iFgf23, suggesting stabilization of biologically active Fgf23 protein. Echocardiography showed impaired cardiac function in AAV-Fgf23 on HPD compared to its NPD group, demonstrated by enhanced end-systolic and end-diastolic volumes, increased systolic and diastolic LV diameters as well as enlarged LV inner diameters, respectively. Pressure-volume analysis using Millar catheter showed higher end-systolic and end-diastolic blood pressure (ESP, EDP) in AAV-Fgf23 mice on HPD compared to NPD. HPD in Ctrl only enhanced EDP, although this did not reach the level of statistical significance. Conclusion Chronic exposure to biologically active cardiac iFgf23 per se does not tackle the heart, while high intra-cardiac Fgf23 synthesis in the presence of high dietary phosphate promotes cardiotoxicity of Fgf23, which could pose a significant health risk to the general population.

The role of sperm in inducing genomic changes in the implantation: An experimental study.

Endometrial receptivity and implantation are important topics in reproductive sciences. No evidence was found to support sperm involvement in endometrial receptivity and its associated factors. This study aimed to explore the effect of the normal human spermatozoa-endometrium cell interaction in regulating genes in the endometrial receptivity pathway. Semen samples were collected from a healthy and fertile man; then, they were incubated with endometrial cells for 24hr and considered as the sperm group. A group was cultured without spermatozoa and considered as a control group. About 24hr later, cells were collected from the bottom of the culture dish. The expressions of the VEGF, FGF2, HBEGF, LIFR, EGF, LIF, MUC1, HOXA10, CSF and PGR genes were evaluated in the two groups. Statistical analysis was performed using an independent sample test. Compared with the control group, in the sperm group, the mRNA levels of PGR (p=.0451), VEGF (p=.0101), HBEGF (p=.0163), EFG (p=.0339), FGF2 (p=.012), LIF (p=.0324), LIFR (p=.0321) and HOXA10 (p=.0098) were significantly upregulated. The results showed that there is a need for the interaction between spermatozoa and endometrium for implantation and can be used for preparing uterine in in vitro fertilisation cycles.

Effects of a Catechol-Functionalized Hyaluronic Acid Patch Combined with Human Adipose-Derived Stem Cells in Diabetic Wound Healing.

Introduction: Chronic inflammation and impaired neovascularization play critical roles in delayed wound healing in diabetic patients. To overcome the limitations of current diabetic wound (DBW) management interventions, we investigated the effects of a catechol-functionalized hyaluronic acid (HA-CA) patch combined with adipose-derived mesenchymal stem cells (ADSCs) in DBW mouse models. Methods: Diabetes in mice (C57BL/6, male) was induced by streptozotocin (50 mg/kg, >250 mg/dL). Mice were divided into four groups: control (DBW) group, ADSCs group, HA-CA group, and HA-CA + ADSCs group (n = 10 per group). Fluorescently labeled ADSCs (5 × 105 cells/100 µL) were transplanted into healthy tissues at the wound boundary or deposited at the HA-CA patch at the wound site. The wound area was visually examined. Collagen content, granulation tissue thickness and vascularity, cell apoptosis, and re-epithelialization were assessed. Angiogenesis was evaluated by immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blot. Results: DBW size was significantly smaller in the HA-CA + ADSCs group (8% ± 2%) compared with the control (16% ± 5%, p < 0.01) and ADSCs (24% ± 17%, p < 0.05) groups. In mice treated with HA-CA + ADSCs, the epidermis was regenerated, and skin thickness was restored. CD31 and von Willebrand factor-positive vessels were detected in mice treated with HA-CA + ADSCs. The mRNA and protein levels of VEGF, IGF-1, FGF-2, ANG-1, PIK, and AKT in the HA-CA + ADSCs group were the highest among all groups, although the Spred1 and ERK expression levels remained unchanged. Conclusions: The combination of HA-CA and ADSCs provided synergistic wound healing effects by maximizing paracrine signaling and angiogenesis via the PI3K/AKT pathway. Therefore, ADSC-loaded HA-CA might represent a novel strategy for the treatment of DBW.

Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro.

ObjectiveTo investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro.Materials and MethodsRecombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis.ResultsThe prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis.ConclusionsCo-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

Accelerative effect of topical Zataria multiflora essential oil against infected wound model by modulating inflammation, angiogenesis, and collagen biosynthesis

Context Zataria multiflora Boiss (Lamiaceae) essential oil (ZME) is believed to be a bactericide herbal medicine and might alleviate negative effects of infection. Objective This study evaluates the effects of an ointment prepared from ZME (ZMEO) on infected wounds. Materials and methods A full-thickness excisional skin wound was surgically created in each mouse and inoculated with 5 × 107 suspension containing Pseudomonas aeruginosa and Staphylococcus aureus. The BALB/c mice (n = 72) were divided into four groups: (1) negative control that received base ointment (NCG), (2) positive control that daily received Mupirocin® (MG), (3) therapeutic ointment containing 2% ZMEO and (4) therapeutic ointment containing 4% ZMEO, for 21 days. Wound contraction, total bacterial count, histopathological parameters, antioxidant activity, qRT-PCR analysis for expression of IL-1β, TNF-α, VEGF, IGF-1, TGF-β, IL-10, and FGF-2 mRNA levels were assessed on days 3, 7, and 14 following the wounding. Results Topical administration of ZMEO significantly decreased the total bacterial count and wound area and also expression of IL-1β and TNF-α compared to the control groups (p < 0.05) in all days. This could also increase significantly the expression of TGF-β, IL-10 IGF-1, FGF-2, and VEGF, and also angiogenesis, fibroblasts, fibrocytes, epithelialization ratio, and collagen deposition and improve antioxidant status compared to the control group (p < 0.05). Discussion and conclusion ZMEO accelerated the healing process of infected wounds by shortening the inflammatory factors and increasing proliferative phase. Applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested.

Interaction of sperm with endometrium can regulate genes involved in endometrial receptivity pathway in mice: An experimental study.

BackgroundMany researchers consider implantation and endometrial receptivity as pertinent issues in reproductive science. Although, several experiments have been performed and their results evaluated, yet there is no confirmed evidence about the related factors and the role of sperm in endometrial receptivity.ObjectiveTo investigate the effect of the sperm-endometrium interaction in regulating genes involved in the endometrial receptivity pathway.Materials and Methods In this experimental study, 10 male and 30 female NMRI mice were included, and half of the male cases were vasectomized. The subjects were divided into two groups as follows; group 1 (case) comprised of 15 females mated with 5 non-vasectomized male mice, while group 2 (control) consisted of 15 females mated with 5 vasectomized males. Cases were sacrificed and assessed after 36 hr and the endometrial tissue was extracted and kept at -80°C until the next use. The expression of the endometrial receptivity pathway genes, including VEGF, HBEGF, FGF2, EGF, LIF, LIFR, HOXA10, MUC1, PGR, and CSF, was examined in both groups. For statistical analysis, an independent samples test (Mean SD) was used.ResultsThe mRNA levels of LIF (p = 0.045), LIFR (p = 0.040), MUC1 (p = 0.032), VEGF (p = 0.022), EFG (p = 0.035), and FGF2 (p = 0.040) were significantly upregulated in the case group compared with the control group.ConclusionFinally, seminal plasma was observed to be effective in expressing the involved genes in the successful implantation pathway, including LIF, LIFR, MUC1, VEGF, EGF, and FGF2.

Mo1290 THE EFFECT OF PIRFENIDONE FOR ESOPHAGEAL STRICTURE AFTER CIRCUMFEREENTIAL ESOPHAGEAL ENDOSCOPIC SUBMUCOSAL DISSECTION IN A PORCINE MODEL

Esophageal stricture inevitably comes up after endoscopic submucosal dissection (ESD) for circumferential esophageal cancer. The effect of current preventive treatments has not been sufficient, although several studies have been reported in multifaceted methods. We focused on Pirfenidone, which is an oral antifibrotic drug commonly used for idiopathic pulmonary fibrosis. Our aim was to investigate the effect of Pirfenidone for preventing esophageal stricture after circumferential esophageal ESD in a porcine model. Eight four-months-old female three-way crossed (LWD) pigs were used for this study. Eight pigs underwent a 5cm-long circumferential ESD under general anesthesia on day 1. Two pigs were used as a control group. Pirfenidone was orally administrated (800mg/day) to 2 pigs and (1200mg/day) to 4 pigs for fourteen consecutive days. Biological tissues were sampled by using forceps on day 1, day 8 and day 15. The pigs were euthanized on day 15, followed by evaluations on esophageal stricture macroscopically and microscopically, and the expression of genes closely associated with the development of fibrosis. The esophageal contraction rate (%) was calculated in extracted specimens macroscopically. The contraction rate (%) was defined as 100 (A - B)/A, where A is the mean value of oral diameter and anal diameter each 2cm from the edge after ESD and B is the narrowest diameter of esophagus. Esophageal fibrosis was identified by Masson’s Trichrome staining microscopically. The expression of genes was evaluated on the messenger RNA (mRNA) levels of IL-6, IL-1β, TNFα, TGF-β1, CTGF, FGF2, EGF, collagen1 and αSMA by Real-Time RT-PCR method. The contraction rate was 61.1% in the control group, 67.8% in Pirfenidone (800mg/day) and 58.1% in Pirfenidone (1200mg/day) groups. Pirfenidone didn’t reduce the stricture rate. The pathological findings or the expression of genes showed no significant differences. In the control group, the expression levels of TGF-β1 reached the maximum value on day 8 and decreased on day15. Whereas the expression levels of CTGF, FGF2, EGF, collagen1 and αSMA escalated stepwise. We couldn’t demonstrate the effect of Pirfenidone for esophageal stricture after circumferential esophageal ESD in a porcine model. However, results of this study might include some beneficial information for our further study.

Endothelin receptor B controls the production of fibroblast growth factor 23.

Endothelin-1 (ET-1) is a member of the endothelin family of peptide hormones first discovered as endothelium-derived mediators regulating vascular tone. ET-1 also regulates the proliferation and differentiation of bone cells that synthesize fibroblast growth factor 23 (FGF23). FGF23 is a hormone controlling renal phosphate and vitamin D metabolism. Here, we studied the role of ET-1 and endothelin receptor B (ETB) for FGF23 production. Fgf23 gene expression was studied in IDG-SW3 bone cells by quantitative RT-PCR. ETB-expressing (etb+/+ ) and rescued ETB-deficient mice (etb-/- ) were studied in metabolic cages. Their serum FGF23, PTH, and 1,25(OH)2 D3 concentrations were determined by ELISA, serum and urinary phosphate and Ca2+ by photometric methods. ET-1 and ETB agonist sarafotoxin 6c suppressed Fgf23 mRNA in IDG-SW3 cells. Serum C-terminal and intact FGF23 as well as bone Fgf23 mRNA levels were significantly higher in etb-/- mice than in etb+/+ mice. Renal phosphate excretion was significantly higher in etb-/- mice despite lower phosphate levels. In addition, etb-/- animals exhibited calciuria and a significantly higher serum 1,25(OH)2 D3 concentration compared to etb+/+ mice. In conclusion, ETB-dependent ET-1 signaling is a potent suppressor of FGF23 formation. This effect is likely to be of clinical relevance given the use of endothelin receptor antagonists in various diseases.

Resistance to MET/VEGFR2 Inhibition by Cabozantinib Is Mediated by YAP/TBX5-Dependent Induction of FGFR1 in Castration-Resistant Prostate Cancer

The overall goal of this study was to elucidate the role of FGFR1 induction in acquired resistance to MET and VEGFR2 inhibition by cabozantinib in prostate cancer (PCa) and leverage this understanding to improve therapy outcomes. The response to cabozantinib was examined in mice bearing patient-derived xenografts in which FGFR1 was overexpressed. Using a variety of cell models that reflect different PCa disease states, the mechanism underpinning FGFR1 signaling activation by cabozantinib was investigated. We performed parallel investigations in specimens from cabozantinib-treated patients to confirm our in vitro and in vivo data. FGFR1 overexpression was sufficient to confer resistance to cabozantinib. Our results demonstrate transcriptional activation of FGF/FGFR1 expression in cabozantinib-resistant models. Further analysis of molecular pathways identified a YAP/TBX5-driven mechanism of FGFR1 and FGF overexpression induced by MET inhibition. Importantly, knockdown of YAP and TBX5 led to decreased FGFR1 protein expression and decreased mRNA levels of FGFR1, FGF1, and FGF2. This association was confirmed in a cohort of hormone-naïve patients with PCa receiving androgen deprivation therapy and cabozantinib, further validating our findings. These findings reveal that the molecular basis of resistance to MET inhibition in PCa is FGFR1 activation through a YAP/TBX5-dependent mechanism. YAP and its downstream target TBX5 represent a crucial mediator in acquired resistance to MET inhibitors. Thus, our studies provide insight into the mechanism of acquired resistance and will guide future development of clinical trials with MET inhibitors.