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Critical Reviews in Eukaryotic Gene... 2023Endometriosis is a pathological condition defined by the occurrence of endometrial glandular and stromal structures in anatomical compartments different from the uterine...
Endometriosis is a pathological condition defined by the occurrence of endometrial glandular and stromal structures in anatomical compartments different from the uterine cavity. Endometriosis is a genetic polymorphism, estrogen-dependent inflammatory disease. This very common pathological entity causes a high level of morbidity in patients; it is also considered one of the most important causes of infertility. We and others have proposed as a pathogenetic mechanism of endometriosis a modification in the fine tuning of the processes of organogenesis of the uterus. We have correlated the immunohistochemical expression in deep endometriotic lesions and in normal endometrial tissue of several molecular factors that are implicated in the embryonic development of the uterine glands. We noticed a significant higher expression both for epithelium and stroma in the controls respect to the endometriosis samples for FGF7, FGF-10 and HGF. Interestingly, regarding FGF-23 and IFN-τ, we observed a significant higher expression in the ectopic endometrial stroma compared to the eutopic endometrium, while thepithetlium expression did not display a significant differential expression in endometriosis tissues respect to normal endometrium. The data generated support the fact that endometriosis tissues, both the epithelial and stromal component, have a different phenotype respect to the eutopic endometrium and sustain the hypothesis that alterations in the molecular mechanisms in control for adenogenesis and survival of endometrial structures are linked to the genesis and survival of endometriosis lesions outside of the uterus.
Topics: Humans; Female; Endometriosis; Respect; Endometrium; Epithelium; Fibroblast Growth Factor 10; Hepatocyte Growth Factor; Fibroblast Growth Factor 7
PubMed: 37183948
DOI: 10.1615/CritRevEukaryotGeneExpr.2023047178 -
Production of a 135-residue long N-truncated human keratinocyte growth factor 1 in Escherichia coli.Microbial Cell Factories May 2023Palifermin (trade name Kepivance®) is an amino-terminally truncated recombinant human keratinocyte growth factor 1 (KGF-1) with 140 residues that has been produced...
BACKGROUND
Palifermin (trade name Kepivance®) is an amino-terminally truncated recombinant human keratinocyte growth factor 1 (KGF-1) with 140 residues that has been produced using Escherichia coli to prevent and treat oral mucositis following radiation or chemotherapy. In this study, an amino-terminally shortened KGF-1 variant with 135 residues was produced and purified in E. coli, and its cell proliferation activity was evaluated.
RESULTS
We expressed soluble KGF-1 fused to thioredoxin (TRX) in the cytoplasmic fraction of E. coli to improve its production yield. However, three N-truncated forms (KGF-1 with 140, 138, and 135 residues) were observed after the removal of the TRX protein from the fusion form by cleavage of the human enterokinase light chain C112S (hEK C112S). The shortest KGF-1 variant, with 135 residues, was expressed by fusion with TRX via the hEK cleavage site in E. coli and purified at high purity (> 99%). Circular dichroism spectroscopy shows that purified KGF-1 had a structure similar to that of the KGF-1 as a random coiled form, and MCF-7 cell proliferation assays demonstrate its biological activity.
CONCLUSIONS
We identified variations in N-terminus-truncated KGF-1 and selected the most stable form. Furthermore, by a simple two-step purification, highly purified KGF-1 was obtained that showed biological activity. These results demonstrate that KGF-1 may be considered an alternative protein to KGF-1.
Topics: Humans; Fibroblast Growth Factor 7; Escherichia coli
PubMed: 37170276
DOI: 10.1186/s12934-023-02097-z -
Scientific Reports May 2023Intrahepatic nerves are involved in the regulation of metabolic reactions and hepatocyte-based regeneration after surgical resection, although their contribution to...
Intrahepatic nerves are involved in the regulation of metabolic reactions and hepatocyte-based regeneration after surgical resection, although their contribution to chronic liver injury remains unknown. Given that intrahepatic nerves are abundant in the periportal tissue, they may be correlated also with cholangiocyte-based regeneration. Here we demonstrate that isoproterenol (ISO), a β-adrenergic receptor agonist, promoted ductular expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in vivo. Immunofluorescence analysis shows that nerve fibers positive for tyrosine hydroxylase form synaptophysin-positive nerve endings on epithelial cell adhesion molecule-positive (EpCAM) cholangiocytes as well as on Thy1 periportal mesenchymal cells (PMCs) that surround bile ducts, suggesting that the intrahepatic biliary tissue are targeted by sympathetic nerves. In vitro analyses indicate that ISO directly increases cAMP levels in cholangiocytes and PMCs. Mechanistically, ISO expands the lumen of cholangiocyte organoids, resulting in promotion of cholangiocyte proliferation, whereas it increases expression of fibroblast growth factor 7, a growth factor for cholangiocytes, in PMCs. Taken together, the results indicate that intrahepatic sympathetic nerves regulate remodeling of bile ducts during DDC-injury by the activation of β-adrenergic receptors on cholangiocytes and PMCs.
Topics: Liver; Pyridines; Bile Ducts; Adrenergic beta-Agonists
PubMed: 37127664
DOI: 10.1038/s41598-023-33882-w -
Biomedicine & Pharmacotherapy =... Mar 2023Human embryonic stem cell (hES)-derived mesenchymal stem cells (-MSCs) are an unlimited source of MSCs. The hair growth-promoting effects of diverse MSCs have been...
Human embryonic stem cell (hES)-derived mesenchymal stem cells (-MSCs) are an unlimited source of MSCs. The hair growth-promoting effects of diverse MSCs have been reported, but not that of hES-MSCs. In the present study, we investigated the hair growth-promoting effects of hES-MSCs and their underlying mechanisms. hES-MSCs or conditioned medium of hES-MSCs exhibited hair-growth effects, which increased the length of mouse vibrissae and human hair follicles. hES-MSCs accelerated the telogen-to-anagen transition in C3H mice and were more effective than adipose-derived stem cells. We further examined whether hypoxia could enhance the hair-growth promoting effects of hES-MSCs. The injection of hES-MSCs or conditioned medium (Hyp-CM) cultured under hypoxia (2% O) enhanced the telogen-to-anagen transition in C3H mice. Additionally, Hyp-CM increased the length of mouse vibrissae, human hair follicles, and the proliferation of human dermal papilla and outer root sheath cells. Moreover, fibroblast growth factor 7, interleukin 12B, and teratocarcinoma-derived growth factor 1 were upregulated under hypoxia, and the co-treatment with these three proteins increased the hair length and induced telogen-to-anagen transition. Hypoxia increased reactive oxygen species (ROS) production, and ROS scavenging attenuated the secretion of growth factors. NADPH oxidase 4 was primarily expressed in hES-MSCs and generated ROS under hypoxia. Collectively, our results suggest that hES-MSCs exhibit hair-growth effects, which is enhanced by hypoxia.
Topics: Humans; Animals; Mice; NADPH Oxidase 4; Reactive Oxygen Species; Culture Media, Conditioned; Mice, Inbred C3H; Cell Proliferation; Hair Follicle; Mesenchymal Stem Cells; Embryonic Stem Cells; Intercellular Signaling Peptides and Proteins; Hypoxia; Cells, Cultured
PubMed: 36706635
DOI: 10.1016/j.biopha.2023.114303 -
Cell Biology International May 2023Ovarian granulosa cells (OGCs) play an essential role in the regulation of follicular growth and development. However, previous studies of OGCs have concentrated on...
Ovarian granulosa cells (OGCs) play an essential role in the regulation of follicular growth and development. However, previous studies of OGCs have concentrated on traditional 2D cultures. In the present study, we used the hanging drop culture method to culture rat OGCs (rOGCs) and assessed the effects of 3D conditions on their proliferation and gene expression profiles. Compared with those grown in 2D conditions, rOGCs grown in 3D cultures showed a significantly different spatial cell distribution and cell alignment under electron microscopy. In particular, rOGCs in 3D cultures showed abundant rough and microvilli-like structures on their cell surface. Here, we showed that these cells grew slowly following 3D culture; the G0/G1-phase increased and the S- and G2/M-phases decreased. Using whole-transcriptome sequencing analysis, 501 genes were shown to have been significantly upregulated and 502 were shown to have been downregulated. Differentially expressed genes were most enriched in pathways involved in focal adhesion, MAPK, and PI3K/Akt signaling according to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Western blotting revealed that SPP1 and FGF7 in the PI3K/Akt pathway were significantly upregulated following 3D culture. These findings improve our understanding of OGCs in real 3D environments in vivo and provide possible avenues for future research on OGCs.
Topics: Female; Rats; Animals; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Granulosa Cells; Signal Transduction; Transcriptome; Fibroblast Growth Factor 7
PubMed: 36701359
DOI: 10.1002/cbin.11998 -
Gerontology 2023Intravesical prostatic protrusion (IPP) has been reported to be associated with bladder outlet obstruction and is the main cause of lower urinary tract symptoms (LUTS)...
INTRODUCTION
Intravesical prostatic protrusion (IPP) has been reported to be associated with bladder outlet obstruction and is the main cause of lower urinary tract symptoms (LUTS) during the development of benign prostatic hyperplasia (BPH). However, the molecular mechanism of IPP remains unclear.
METHODS
Clinical data analysis was performed to analyze the association between IPP and long-term complications in patients with BPH. RNA sequencing was performed on prostate tissues (IPP or not). Stromal cells were obtained from IPP-derived primary cultures to explore the molecular mechanism of IPP formation. Cell proliferation was evaluated by a CCK-8 assay. Multiple proteins in the signaling pathway were assessed using Western blot.
RESULTS
First, we confirmed that IPP is a prognostic factor for long-term complications in patients with BPH. Then, we observed that FGF7 was upregulated in both IPP tissues and IPP primary stromal cells through immunohistochemistry, Western blot, and quantitative real-time PCR. Furthermore, FGF7 was significantly upregulated in high IPP-grade prostate tissues. The coculture experiments showed that the downregulation of FGF7 in IPP-derived stromal cells inhibited the proliferation and migration of the prostate epithelial cells. Additionally, FGF7 was bound to FGFR2 to induce the epithelial-mesenchymal transition process through binding to FGFR2. RNA sequencing analysis also revealed the activation of the MAPK/ERK1/2 signaling pathway. The MAPK/ERK1/2 was downregulated by a specific inhibitor affecting the FGF7 stimulation in vitro.
CONCLUSIONS
Our data reveal a novel amplification effect, i.e., stromal cell-derived FGF7 promotes epithelial cell proliferation and stromal cell phenotype, ultimately inducing IPP formation. Targeting FGF7 can significantly reduce epithelial to stromal transition and provide a potential therapeutic target for BPH progression.
Topics: Humans; Male; Prostatic Hyperplasia; Prostate; Up-Regulation; MAP Kinase Signaling System; Urinary Bladder Neck Obstruction; Fibroblast Growth Factor 7
PubMed: 36693332
DOI: 10.1159/000527929 -
The Protein Journal Feb 2023Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF...
Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF by engineering the aggregation hotspots. Initially, 21 mutants were designed based on the previously-identified aggregation-prone regions (APRs) and then four of them including mutants No. 4 (L91K, I119K), 7 (V13S, L91K), 14 (L91D, I119D), and 21 (A51E) were selected based on molecular dynamics (MD) simulations for further experimental studies. The recombinantly produced rhKGF and mutants were analyzed regarding secondary structure, thermal stability, aggregation propensity, and biological activity. Far-UV CD spectroscopy showed that the mutants have similar secondary structure with rhKGF. A51E mutant showed enhanced stability and decreased monomer loss under heat stress suggesting its reduced aggregation propensity compared to rhKGF. Mutant No. 14 showed higher stability and less aggregation tendency than mutant No. 4 indicating that only mutations decreasing pI of rhKGF are effective in reducing its aggregation tendency. All of the mutants were at least as potent as rhKGF in stimulating proliferation of MCF-7 epithelial cells. Our results identified A51E as an equally potent, more stable, and less aggregation-prone analog of rhKGF which could be a promising alternative drug candidate for the commercially available rhKGF (Palifermin).
Topics: Humans; Fibroblast Growth Factor 7; Molecular Dynamics Simulation
PubMed: 36683078
DOI: 10.1007/s10930-023-10089-6 -
International Journal of Biological... Mar 2023Ulcerative colitis (UC) is a chronic recurrent disease affecting the gastrointestinal tract especially colorectum. Keratinocyte growth factor (KGF) plays the vital roles...
Ulcerative colitis (UC) is a chronic recurrent disease affecting the gastrointestinal tract especially colorectum. Keratinocyte growth factor (KGF) plays the vital roles in maintaining the colonic mucosal barrier. The poor stability and off-target of KGF were two hindering factors for its clinical application. Herein, in situ hydrogel (PE) with mucoadhesive ability was constructed by using temperature-sensitive poloxamer and EGCG as hydrogel-forming material and adhesive enhancer, respectively. Incorporation of EGCG led to the slight decrease of the gelled temperature and shortened the gelled time of PE hydrogel. When the concentration of EGCG is 0.1 %, PE hydrogel exhibits the suitable viscosity of 280 ± 20 Pa·s and the strong adhesive force of 725 ± 25 mN. KGF was soluble in cold PE solution to obtain KGF-loaded PE hydrogel (KGF@PE). PE hydrogel could improve the stability of KGF in vitro. KGF@PE not only could recover greatly the body weight of TNBS-induced rats but also repair their colonic morphology and goblet cell function. Moreover, the potential of repairing the epithelial barrier was indicated by upregulating tight junction proteins. Importantly, the safety of KGF@PE hydrogel for colitis was also confirmed on AOM/DSS-induced mice models. Conclusively, KGF@PE may be a promising therapeutic platform without obvious side effect for ulcerative colitis.
Topics: Rats; Mice; Animals; Colitis, Ulcerative; Hydrogels; Fibroblast Growth Factor 7; Adhesives; Colon; Disease Models, Animal; Dextran Sulfate; Intestinal Mucosa; Colitis
PubMed: 36669631
DOI: 10.1016/j.ijbiomac.2023.123323 -
Current Oncology (Toronto, Ont.) Jan 2023Oral mucositis is a common and most debilitating complication associated with cancer therapy. Despite the significant clinical and economic impact of this condition,... (Review)
Review
Oral mucositis is a common and most debilitating complication associated with cancer therapy. Despite the significant clinical and economic impact of this condition, there is little to offer to patients with oral mucositis, and the medications used in its management are generally only palliative. Given that mucositis is ultimately a predictable and, therefore, potentially preventable condition, in this study we appraised the scientific literature to evaluate effective methods of prevention that have been tested in randomised controlled trials (RCTs). Published high-level evidence shows that multiple preventative methods are potentially effective in the prevention of oral mucositis induced by radiotherapy, chemotherapy, or both. Anti-inflammatory medications (including benzydamine), growth factors and cytokines (including palifermin), cryotherapy, laser-and-light therapy, herbal medicines and supplements, and mucoprotective agents (including oral pilocarpine) showed some degree of efficacy in preventing/reducing the severity of mucositis with most anticancer treatments. Allopurinol was potentially effective in the prevention of radiotherapy-induced oral mucositis; antimicrobial mouthwash and erythropoietin mouthwash were associated with a lower risk of development of severe oral mucositis induced by chemotherapy. The results of our review may assist in highlighting the efficacy and testing the effectiveness of low-cost, safe preventative measures for oral mucositis in cancer patients.
Topics: Humans; Mucositis; Mouthwashes; Stomatitis; Neoplasms; Anti-Inflammatory Agents; Randomized Controlled Trials as Topic
PubMed: 36661723
DOI: 10.3390/curroncol30010074 -
Otology & Neurotology : Official... Mar 2023To evaluate the effectiveness of the menin-MLL inhibitor, MI503, as a conservative treatment of middle ear cholesteatoma (cholesteatoma) in a mouse model and to confirm...
HYPOTHESIS
To evaluate the effectiveness of the menin-MLL inhibitor, MI503, as a conservative treatment of middle ear cholesteatoma (cholesteatoma) in a mouse model and to confirm its safety profile regarding auditory function in vivo.
BACKGROUND
Cholesteatoma is a mass formed by the keratinizing squamous epithelium in the tympanic cavity and/or mastoid and subepithelial connective tissue and by the progressive accumulation of keratin debris with/without a surrounding inflammatory reaction. Although the main treatment is surgical therapy, the techniques to prevent recurrence remain a critical area of research. Recently, the use of MI503 in experiments resulted in the inhibition of the growth of cholesteatoma in vivo under histone modification.
METHODS
After cholesteatoma was induced in ICR mice (n = 7) by keratinocyte growth factor expression vector transfection, MI503 (50 μM) or phosphate-buffered saline was topically injected for 14 days. The effects of MI503 against cholesteatoma were analyzed by micro-computed tomography images. For the in vivo ototoxicity study, a single intratympanic injection of MI503 (50 or 500 μM) or phosphate-buffered saline (n = 4 each) was done in the ICR mice. An auditory brainstem response was performed at days 0, 1, and 14. For morphological analysis, immunostaining for Phalloidin/F-actin and Myo7a was performed.
RESULTS
MI503 reduced keratinocyte growth factor-induced cholesteatoma in vivo (4 of 4 [100%]). No difference was found in the mean variation of the average of the auditory brainstem response thresholds between the three groups in the in vivo ototoxicity study, thus confirming its safety profile regarding auditory function. MI503 does not demonstrate any deleterious effects on murine hair cells when assessed by immunostaining.
CONCLUSION
These findings demonstrate an encouraging safety profile for the use of menin-MLL inhibitor for the conservative treatment of cholesteatoma.
Topics: Animals; Mice; Cholesteatoma, Middle Ear; Fibroblast Growth Factor 7; Ototoxicity; Epigenesis, Genetic; Mice, Inbred ICR; X-Ray Microtomography; Cholesteatoma; Ear, Middle; Phosphates
PubMed: 36593557
DOI: 10.1097/MAO.0000000000003795