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Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), plays a critical role in embryonic development, cell proliferation, and differentiation. However, efficient production of recombinant KGF remains a challenge due to its low expression levels and high tendency for aggregation in Escherichia coli. This study aimed to enhance the expression and solubility of KGF by employing different protein tags—PDIb'a', MBP, and His—fused to the N-terminus of KGF. Among these, H-PDIb'a'-KGF demonstrated superior stability and was selected for large-scale production and purification. The purified KGF was confirmed through liquid chromatography with tandem mass spectrometry analysis, which showed an 81% fragment mass identification coverage. Biological activity assessments using human breast cancer MCF-7 cells indicated that purified KGF significantly increased cell proliferation, with an EC 50 of 6.4 ± 0.5 pM. Interestingly, PDIb'a' alone also exhibited a stimulatory effect on MCF-7 cells. Furthermore, the purified KGF enhanced the wound healing of HaCaT keratinocytes in a dose-dependent manner. These findings provide valuable insights into the efficient production and functional characterization of recombinant KGF for potential applications in therapeutic interventions. • PDIb'a' fusion tag enhanced soluble expression of KGF in E. coli, enabling efficient purification. • Purified KGF potently stimulated MCF-7 cell proliferation with EC50 of 6.4 pM. • KGF promoted wound healing of HaCaT keratinocytes in dose-dependent manner. • PDIb'a' tag alone exhibited mitogenic activity on MCF-7 cells. • Approach enables cost-effective production of bioactive KGF for therapeutic use. [ABSTRACT FROM AUTHOR]

NKX2.1 is a master regulator of lung morphogenesis and cell specification; however, interactions of NKX2.1 with various transcription factors to regulate cell-specific gene expression and cell fate in the distal lung remain incompletely understood. FOXO1 is a key regulator of stem/progenitor cell maintenance/differentiation in several tissues but its role in the regulation of lung alveolar epithelial progenitor homeostasis has not been evaluated. We identified a novel role for FOXO1 in alveolar epithelial cell (AEC) differentiation that results in the removal of NKX2.1 from surfactant gene promoters and the subsequent loss of surfactant expression in alveolar epithelial type I-like (AT1-like) cells. We found that the FOXO1 forkhead domain potentiates a loss of surfactant gene expression through an interaction with the NKX2.1 homeodomain, disrupting NKX2.1 binding to the SFTPC promoter. In addition, blocking PI-3K/AKT signaling reduces phosphorylated FOXO-1 (p-FOXO1), allowing accumulated nuclear FOXO1 to interact with NKX2.1 in differentiating AEC. Inhibiting AEC differentiation in vitro with keratinocyte growth factor (KGF) maintained an AT2 cell phenotype through increased PI3K/AKT-mediated FOXO1 phosphorylation, resulting in higher levels of surfactant expression. Together these results indicate that FOXO1 plays a central role in AEC differentiation by directly binding NKX2.1 and suggests an essential role for FOXO1 in mediating AEC homeostasis.

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway.

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Reindeer is the only deer species in both sexes grow antlers. Many apparent differences exist in antlers of female and male reindeer and sika deer. Insulin like growth factor 1 (IGF1), Keratinocyte growth factor (KGF) and Nerve Growth Factor (NGF) are essential for antler growth. To investigate whether epigenetic regulation of the growth factors is important in growth of sika deer, female and male reindeer antlers, methylation status were evaluated using bisulfite sequencing PCR (BSP). The 5' flanking regions of IGF1, KGF and NGF were cloned from reindeer and sika deer antlers, and the lengths were 2089bp/2107bp, 1474bp/1474bp and 865bp/865bp, respectively. Based on bioinformatics analysis, the regions of IGF1 (-50bp ~ +615bp), KGF (-285bp ~ +207bp) and NGF (-388bp ~ +109bp) were selected for studying the methylation status. The antler mesenchyme of male reindeer was the only methylation sample in IGF1 (2.23 ± 0.92%), significantly higher in methylation level than that of female reindeer and sika deer (0.01

Objective: To investigate the protective effect and mechanism of KGF combined with HIF-1α on IEC-6 cells knocked out by BECN1 under hypoxia stress. Methods: The stable BECN1 knockout cell line IEC-6B-cells were constructed and divided into blank control group (BC), negative control group (NC) and HIF-1 α Group, KGF joint HIF-1 α Group (KH). Observed the changes of cell morphology in each group. Cell survival rate, ATP content, cell cycle and apoptosis rate, autophagy related gene expression, apoptosis and autophagy related protein expression were detected in each group. Results: After 24 hours of hypoxia treatment, spindle, star and other abnormal morphological changes were observed in cells of each group. Electron microscopy showed that a large number of lysosomal vesicles with different sizes were found in the cytoplasm of NC cells. Other groups of cells showed morphological changes of early apoptosis, and autophagic vesicles were found in the cytoplasm of cells in BC group and KH group. Compared with BC group, the cell survival rate of each knockout group was significantly lower (P＜0.01), and the cell survival rate of KH group was higher than NC group,KGF group and HIF-1α group (P＜0.05). The content of ATP in BC group was significantly higher than that in other groups (P＜0.05).Compared with BC group and KH group, the percentage of G0/G1 phase and apoptosis rate of other three groups were significantly increased (P＜0.05). There was no significant difference between KH group and BC group (P＞0.05). Compared with BC group, the expression of BECN1, SQSTM1 and LC3 genes in NC group, KGF group, HIF-1α group and KH group decreased significantly (P＜0.05). Compared with BC group, the expression of Beclin1 protein and LC3 II/LC3 I in NC group, KGF group, HIF-1 α group and KH group decreased significantly (P＜0.05), and the expression of p62 protein increased significantly (P＜0.05). The ratio of Bax/Bcl-2 and the expression of Caspase3 protein in KH group were lower than those in NC group (P＜0.05). Conclusion: KGF combined with HIF-1α can protect IEC-6B-cells from hypoxia stress by promoting cell proliferation, enhancing cell energy metabolism, reducing cell arrest rate in G0/G1phase and inhibiting apoptosis. [ABSTRACT FROM AUTHOR]

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose‐derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen‐binding domain from Vibrio mimicus metalloprotease (vibrioCBD‐KGF). KGF and vibrioCBD‐KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK‐293 and MCF‐7 cells. vibrioCBD‐KGF demonstrated enhanced collagen‐binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD‐KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen‐coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin‐10 and Involucrin), and stem cell markers (Collagen‐I and Vimentin) by real‐time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD‐KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down‐regulation of Collagen‐I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD‐KGF. The present study showed that vibrioCBD‐KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen‐based biomaterials. [ABSTRACT FROM AUTHOR]

Disseminated superficial actinic porokeratosis (DSAP) is an inherited dermatosis triggered by chronic ultraviolet light exposure. Cosmetic improvement has been noted with topical therapies or laser treatment. Lesions of DSAP are precancerous, having been reported to develop into squamous cell carcinomas (SCCs) or basal cell carcinomas (BCCs) occasionally. We describe a patient with longstanding DSAP who developed a primary CNS lymphoma. Palifermin (a keratinocyte growth factor analogue) was used as an adjuvant for his chemotherapy to minimize mucositis. Our patient noted marked clinical improvement of his DSAP following his chemotherapy for lymphoma. Follow-up has shown a sustained improvement of his DSAP with no development of any SCCs. Palifermin may be safe for use in individuals at high risk of developing SCCs and further research into keratinocyte growth factors and risk of development of SCC is warranted. Systemic chemotherapy appears to improve DSAP lesions.

This study strategically incorporates epidermal growth factor (EGF) and keratinocyte growth factor (KGF) within a hyaluronic acid (HA) hydrogel to enhance corneal wound healing. The controlled release of EGF and KGF from the HA hydrogel is engineered to promote the regeneration of both the epithelial and stromal layers. Specifically, EGF plays a pivotal role in the regeneration of the epithelial layer, while KGF exhibits efficacy in the regeneration of the stromal layer. The combination of these growth factors facilitates efficient regeneration of each layer and demonstrates the capability to modulate each other's regenerative effects. The interplay between EGF and KGF provides an understanding of their cooperative influence on the dynamics of corneal wound healing. The results of this study contribute to the development of advanced strategies for corneal wound management and offer insights into the complex process of corneal regeneration. [ABSTRACT FROM AUTHOR]

Postoperative abdominal adhesion (PAA) is one of the more universal complications of abdominal surgery with a frequent incidence. Currently available keratinocyte growth factor (KGF)-based glues for the prevention of adhesions remain a great bottleneck since their long-term biological activity in vivo is insufficient. In this study, we fabricated hybrid polydopamine (PDA)-KGF nanoparticles (PDA-KGF NPs) by using an in situ self-assembly and polymerization method. The physicochemical properties of the PDA-KGF nanoparticles were systematically characterized. The effect of preventing PAA in rats was evaluated by using hybrid PDA-KGF NPs combined with hyaluronate (Ha). The expression levels of inflammatory factors and the degree of inflammatory cell infiltration in the injured peritoneum were evaluated by enzyme-linked immunosorbent assays and hematoxylin-eosin staining, respectively. The levels of phospho-Src expression were revealed by Western blotting. The degree of fibrosis and the density of deposited collagen fibers were measured with real-time reverse-transcription polymerase chain reaction and picrosirius red staining. The results indicated that the PDA-KGF NPs combined with Ha greatly prevented the incidence of abdominal adhesion s and promoted the repair of mesothelial cells in injured peritoneum. More importantly, the PDA-KGF NPs combined with Ha obviously reduced collagen deposition and fibrosis and inhibited the inflammatory response. Our results suggest that PDA-KGF NPs combined with Ha are promising barrier-like biomaterials for the effective prevention of postoperative tissue adhesion. Postoperative abdominal adhesion (PAA) as an inevitable postoperative complication affected the quality of life of patients. Currently available methods for preventing adhesions mainly employ degradable biomaterials. Previous research demonstrated that a hybrid keratinocyte growth factor (KGF)-sodium hyaluronate (Ha) gel could prevent the formation of PAAs. However, its clinical outcomes are not satisfactory since their bioactivity in vivo is too short. In this article, we fabricated hybrid polydopamine (PDA)-KGF nanoparticles (PDA-KGF NPs), which extend KGF bioactivity, effectively prevent PAA. Moreover, PDA-KGF NPs could remarkably reduce both collagen deposition and fibrosis, inhibit the inflammatory response, and promote mesothelial regeneration. Overall, the PDA-KGF NPs combined with Ha exhibit efficient antiadhesion properties, may provide a promising clinical protocol for the prevention of PAA. [Display omitted] [ABSTRACT FROM AUTHOR]

Kai Jia,* Yan Wang,* Xin Tong, Rong Wang Department of Nutrition, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Kai JiaDepartment of Nutrition, Beijing Chao-Yang Hospital, No. 8 Gongti South Road, Chaoyang District, Beijing 100020, People’s Republic of ChinaTel +86-85231275Fax +86-10-83911276Email kaijiamyemail@sina.comIntroduction: KGF-modified MSCs can promote the repair of spinal cord injury and pulmonary fibrosis injury in rats. However, the effect of KGF-modified MSCs on UC rats is unclear. We aimed to explore the therapeutic effect and possible mechanism of KGF gene-modified MSCs on trinitrobenzene sulfonic acid (TNBS)-induced UC rats.Methods: The lentivirus-mediated KGF gene was introduced into bone marrow MSCs of male rats. Female SD rats were induced to establish a UC model by TNBS. Untreated MSCs, MSCs carrying empty vectors (MSCs-vec) or MSCs carrying KGF gene (MSCs-KGF) were transplanted into UC rats by tail vein injection.Results: Significantly high expression of KGF was observed in the intestinal tissues of the MSCs-KGF group. Compared with the challenged control group, the DAI score, CMDI score and TDI score of the MSCs group, MSCs-vec group and MSCs-KGF group were markedly lower. Treatment with MSCs obviously promoted the expression of claudin-1 and PCNA in intestinal tissues of UC rats. Simultaneously, compared with the challenged control group, the levels of TNF-α, IL-6 and IL-8 in the intestinal tissues of the MSCs groups were significantly decreased, while the levels of IL-10 were significantly increased. Most importantly, we found that MSCs-KGF significantly improved colonic morphology and tissue damage and inflammation in UC rats compared with MSCs and MSCs-vec. Further analysis showed that MSCs-KGF clearly promoted phosphorylation of PI3K and Akt and inhibited nuclear translocation of NF-κB in intestinal tissues of UC rats.Discussion: MSCs, especially KGF-modified MSCs, can improve colonic tissue damage in UC rats by promoting intestinal epithelial cell proliferation and reducing colonic inflammatory response, which may be related to activation of PI3K/Akt pathway and inhibition of NF-κB activation.Keywords: ulcerative colitis, bone marrow mesenchymal stem cells, keratinocyte growth factor, gene modification, PI3K/Akt pathway, NF-κB

The authors have previously demonstrated that one phage-displayed keratinocyte growth factor (KGF) model peptide that can bind to epidermal cells and facilitate cellular proliferation. In this study, the authors investigated the role of phage-displayed KGF model peptide on wound healing in diabetic rats. Full-thickness excisional dorsal wounds were created in the diabetic rats, after which the rats were randomly divided into five groups: negative control group [normal saline (NS)], two KGF control groups which were respectively treated with low-dose KGF (5 ng/mL, K-LD) and high-dose KGF (50 ng/mL, K-HD), and two KGF model-peptide-treated groups which were respectively treated with low-dose model peptide (5 ng/mL, M-LD) and high-dose model peptide (50 ng/mL, M-HD). On day 14 post-injury, wound closure was followed by digital planimetry and wound tissues were harvested for histologic assay and real-time polymerase chain reaction. Wounds treated with model peptide closed markedly faster than negative control wounds and were comparable to KGF treated wounds. Histology and immunohistology results demonstrated significantly higher levels of re-epithelization, granulation tissue formation and vascularization in the model peptide groups. Furthermore, real-time polymerase chain reaction expression of KGFR, collagen I and transforming growth factor-β1 in model peptide groups, were also generally higher than that in negative control group. Phage-displayed KGF peptide promotes wound healing through accelerating re-epithelialization, enhancing dermal regeneration, and inducing angiogenesis. Model peptide possesses the potential to be a promising therapeutic option for the treatment of diabetic ulcers. [ABSTRACT FROM AUTHOR]

Objectives: To investigate whether serum amylase can predict the recovery of salivary volume and determine the correlation of the level of cytokines, including epidermal growth factor, hepatocyte growth factor and keratinocyte growth factor, with oral mucositis during chemoradiotherapy for oral cancer. Subjects and Methods: This study included 84 patients treated with preoperative chemoradiotherapy followed by curative surgery, following a phase II study protocol. We measured and analysed the correlation of the stimulated saliva volume, serum amylase and cytokines in resting saliva at baseline and 1 month after chemoradiotherapy with oral mucositis levels. Results: We observed a negative correlation between the serum amylase level at the beginning of chemoradiotherapy and the stimulated saliva volume at 1 month after chemoradiotherapy (p =.03). Epidermal growth factor in resting saliva was significantly reduced after chemoradiotherapy (p <.01). The incidence of severe oral mucositis during chemoradiotherapy was significantly higher and negatively associated with the epidermal growth factor and keratinocyte growth factor levels (p =.04, p =.05). Conclusions: The serum amylase level at the beginning of chemoradiotherapy may be a predictor of the recovery of the saliva volume. Furthermore, cytokines such as epidermal growth factor and keratinocyte growth factor in resting saliva affect the development of oral mucositis during chemoradiotherapy. [ABSTRACT FROM AUTHOR]

Bronchopulmonary dysplasia (BPD) is a devastating disease of prematurity that is associated with mechanical ventilation and hyperoxia. We used preterm pigs delivered at gestational day 102 as a translational model for 26–28-week infants to test the hypothesis administering recombinant human keratinocyte growth factor (rhKGF) at initiation of mechanical ventilation will stimulate type II cell proliferation and surfactant production, mitigate ventilator induced lung injury, and reduce epithelial to mesenchymal transition considered as a precursor to BPD. Newborn preterm pigs were intubated and randomized to receive intratracheal rhKGF (20 μg/kg; n = 6) or saline (0.5 ml 0.9% saline; control; n = 6) before initiating 24 h of ventilation followed by extubation to nasal oxygen for 12 h before euthanasia and collection of lungs for histopathology and immunohistochemistry to assess expression of surfactant protein B and markers of epithelial to mesenchymal transition. rhKGF pigs required less oxygen during mechanical ventilation, had higher tidal volumes at similar peak pressures indicative of improved lung compliance, and survival was higher after extubation (83% vs. 16%). rhKGF increased surfactant protein B expression (p < 0.05) and reduced TGF-1β (p < 0.05), that inhibits surfactant production and is a prominent marker for epithelial to mesenchymal transition. Our findings suggest intratracheal administration of rhKGF at initiation of mechanical ventilation enhances surfactant production, reduces ventilator induced lung injury, and attenuates epithelial-mesenchymal transition while improving pulmonary functions. rhKGF is a potential therapeutic strategy to mitigate pulmonary responses of preterm infants that require mechanical ventilation and thereby reduce the incidence and severity of bronchopulmonary dysplasia.

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA's globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique "fingerprint" or "biomarker" vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease. [ABSTRACT FROM AUTHOR]

RationaleIncreasing epithelial repair and regeneration may hasten resolution of lung injury in patients with the acute respiratory distress syndrome (ARDS). In animal models of ARDS, keratinocyte growth factor (KGF) reduces injury and increases epithelial proliferation and repair. The effect of KGF in the human alveolus is unknown.ObjectivesTo test whether KGF can attenuate alveolar injury in a human model of ARDS.MethodsVolunteers were randomized to intravenous KGF (60 μg/kg) or placebo for 3 days, before inhaling 50 μg LPS. Six hours later, subjects underwent bronchoalveolar lavage (BAL) to quantify markers of alveolar inflammation and cell-specific injury.Measurements and main resultsKGF did not alter leukocyte infiltration or markers of permeability in response to LPS. KGF increased BAL concentrations of surfactant protein D, matrix metalloproteinase (MMP)-9, IL-1Ra, granulocyte-macrophage colony-stimulating factor (GM-CSF), and C-reactive protein. In vitro, BAL fluid from KGF-treated subjects inhibited pulmonary fibroblast proliferation, but increased alveolar epithelial proliferation. Active MMP-9 increased alveolar epithelial wound repair. Finally, BAL from the KGF-pretreated group enhanced macrophage phagocytic uptake of apoptotic epithelial cells and bacteria compared with BAL from the placebo-treated group. This effect was blocked by inhibiting activation of the GM-CSF receptor.ConclusionsKGF treatment increases BAL surfactant protein D, a marker of type II alveolar epithelial cell proliferation in a human model of acute lung injury. Additionally, KGF increases alveolar concentrations of the antiinflammatory cytokine IL-1Ra, and mediators that drive epithelial repair (MMP-9) and enhance macrophage clearance of dead cells and bacteria (GM-CSF). Clinical trial registered with ISRCTN 98813895.

We previously found that human mesenchymal stem cells (MSC) or its conditioned medium restored lung protein permeability and reduced alveolar inflammation following Escherichia coli endotoxin-induced acute lung injury (ALI) in an ex vivo perfused human lung in part through the secretion of soluble factors such as keratinocyte growth factor (KGF). Recently, MSC were found to release microvesicles (MVs) that were biologically active because of the presence of mRNA or miRNA with reparative properties. MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes. These studies were designed to determine if MVs released by human bone marrow derived MSCs would be effective in restoring lung protein permeability and reducing inflammation in E. coli endotoxin-induced ALI in C57BL/6 mice. The intratracheal instillation of MVs improved several indices of ALI at 48 hours. Compared to endotoxin-injured mice, MVs reduced extravascular lung water by 43% and reduced total protein levels in the bronchoalveolar lavage (BAL) fluid by 35%, demonstrating a reduction in pulmonary edema and lung protein permeability. MVs also reduced the influx of neutrophils and macrophage inflammatory protein-2 levels in the BAL fluid by 73% and 49%, respectively, demonstrating a reduction in inflammation. KGF siRNA-pretreatment of MSC partially eliminated the therapeutic effects of MVs released by MSCs, suggesting that KGF protein expression was important for the underlying mechanism. In summary, human MSC-derived MVs were therapeutically effective following E. coli endotoxin-induced ALI in mice in part through the expression of KGF mRNA in the injured alveolus.

Extracellular basic pH regulates cellular processes in wounds, and consequently influenced wound healing. Oxidative defence system modulation in the skin helps heal wounds, inhibits skin ageing and improves the skin condition. Moreover, the role of keratinocyte growth factor (KGF) and nuclear factor erythroid 2‐related factor 2 (Nrf2) in antioxidant systems has been reported in various skin models. However, the effects of extracellular basic pH on wound‐ or skin ageing‐related skin damage have not been examined. Thus, we investigated the antioxidant systems affected by extracellular basic pH in a 3D human skin equivalent system (3HSE). Extracellular basic pH decreased KGF expression and enhanced the oxidative defence system, and thus activated Nrf2 in the 3HSE. Additionally, extracellular basic pH and KGF treatment up‐regulated Nrf2 activation and its regulation of the oxidative defence system in the 3HSE. This indicates that Nrf2 up‐regulation is enhanced by reactive oxygen species production, rather than KGF, and by extracellular basic pH of the skin. The inhibition of skin damage through pH imbalance and KGF regulation suggests that the development of pH‐regulating or pH‐maintaining materials may provide effective therapeutic strategies for maintaining a healthy skin. [ABSTRACT FROM AUTHOR]

Adipose‐derived stromal cells (ADSCs) showed excellent capacity in regeneration and tissue protection. Low tidal volume ventilation (LVT) strategy demonstrates a therapeutic benefit on the treatment of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). This study, therefore, aimed to undertaken determine whether the combined LVT and ADSCs treatment exerts additional protection against lipopolysaccharide (LPS)‐induced ALI in rats. The animals were randomized into seven groups: Group I (control), Group II (instillation of LPS at 10 mg/kg intratracheally), Group III (LPS+LVT 6 ml/kg), Group IV (LPS+intravenous autologous 5 × 106 ADSCs which were pretreated with a scrambled small interfering RNA [siRNA] of keratinocyte growth factor [KGF] negative control), Group V (LPS+ADSCs which were pretreated with a scrambled siRNA of KGF, Group VI (LPS+LVT and ADSCs as in the Group IV), and Group VII (LPS+LVT and ADSCs as in the Group V). We found that levels of tumor necrosis factor‐α, transforming growth factor‐β1, and interleukin (IL)‐1β and IL‐6, the proinflammatory cytokines, were remarkably increased in LPS rats. Moreover, the expressions of ENaC, activity of Na, K‐ATPase, and alveolar fluid clearance (AFC) were obviously reduced by LPS‐induced ALI. The rats treated by ADSCs showed improved effects in all these changes of ALI and further enhanced by ADSCs combined with LVT treatment. Importantly, the treatment of ADSCs with siRNA‐mediated knockdown of KGF partially eliminated the therapeutic effects. In conclusion, combined treatment with ADSCs and LVT not only is superior to either ADSCs or LVT therapy alone in the prevention of ALI. Evidence of the beneficial effect may be partly due to improving AFC by paracrine or systemic production of KGF and anti‐inflammatory properties. [ABSTRACT FROM AUTHOR]

Chronic wounds with a long healing period are considered a worldwide problem. As an important player in the wound healing process, keratinocyte growth factor (KGF) is one of the most effective methods of wound treatment. To prevent the wounds from becoming chronic, effective treatments should be applied at the first steps of wound generation. However, the low stability of KGF at acidic pH conditions at the first stages of the wound prevents its application in wound treatment. Lack of knowledge of the KGF behavior at different pHs has made it difficult to design the pH stable variants of this protein. In this study, we tried to simulate the KGF behavior at different pH conditions using molecular dynamics. To do this, the protonation and deprotonation states of KGF at different pH(s) were analyzed with PROPKA webserver. Then, the PDB files generated at different pHs were subjected to the molecular dynamic simulation at temperatures 300, 400, and 500 K using GROMACS software. The conformational stability, residue flexibility, protein compactness, and hydrogen-bond analysis were performed at the atomic level. The results showed that by decreasing the total charge of the protein from 10 at neutral pH to 7 at alkaline pH (pH: 9.0) at 500K, the KGF stability was significantly increased. Furthermore, analysis of the solvent-accessible surface area (SASA) showed that the KGF SASA is decreased with reduced protein charge. Analysis of the intra-protein and protein-solvent hydrogen bonds (H-bonds) showed that not only the number of H-bonds at alkaline pHs are higher compared to the neutral and acidic pHs, but also, they did not significantly decrease with increasing temperature at alkaline pH(s). Our results showed that the presence of more positively charged residues and their repulsion makes the KGF an unstable protein. Therefore, it seems that targeted mutations to reduce protein charge can lead to increased stability of KGF.

RationaleMesenchymal stem cells secrete paracrine factors that can regulate lung permeability and decrease inflammation, making it a potentially attractive therapy for acute lung injury. However, concerns exist whether mesenchymal stem cells' immunomodulatory properties may have detrimental effects if targeted toward infectious causes of lung injury.ObjectivesTherefore, we tested the effect of mesenchymal stem cells on lung fluid balance, acute inflammation, and bacterial clearance.MethodsWe developed an Escherichia coli pneumonia model in our ex vivo perfused human lung to test the therapeutic effects of mesenchymal stem cells on bacterial-induced acute lung injury.Measurements and main resultsClinical-grade human mesenchymal stem cells restored alveolar fluid clearance to a normal level, decreased inflammation, and were associated with increased bacterial killing and reduced bacteremia, in part through increased alveolar macrophage phagocytosis and secretion of antimicrobial factors. Keratinocyte growth factor, a soluble factor secreted by mesenchymal stem cells, duplicated most of the antimicrobial effects. In subsequent in vitro studies, we discovered that human monocytes expressed the keratinocyte growth factor receptor, and that keratinocyte growth factor decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance. Inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of mesenchymal stem cells in the ex vivo perfused human lung and monocytes grown in vitro injured with E. coli bacteria.ConclusionsIn E. coli-injured human lungs, mesenchymal stem cells restored alveolar fluid clearance, reduced inflammation, and exerted antimicrobial activity, in part through keratinocyte growth factor secretion.

• Mycophenolate mofetil (MMF) induced gastrointestinal disruption and increased intestinal mucosal permeability in a mouse model. • KGF maintained tight junction protein, reversed proliferative inhibition and suppressed the apoptosis of intestinal epithelial cells. • KGF significantly enhanced the proportion of γδ+T intraepithelial lymphocytes. Although mycophenolate mofetil-induced (MMF) effectively improves long-term graft survival, the gastrointestinal (GI) side effects due to MMF-induced GI barrier damage limit its use in clinic. Keratinocyte growth factor (KGF) plays a crucial role in the intestinal protection and repair process. This study is designed to investigate the protective effect of KGF on MMF-induced intestinal mucosal barrier disruption and the potential mechanism. Thirty adult male C57BL/6 mice were assigned to one of the following groups: the MMF group, the MMF + KGF group, and the control group (n = 10 in each group). Animals in the MMF group received MMF (500 mg/kg) by gavage once daily for 15 consecutive days; animals in the MMF + KGF group received MMF (500 mg/kg) by gavage and KGF (5 mg/kg) by intraperitoneal injection once daily for 15 consecutive days; and control mice were given an equal volume of vehicle during the 15-day experimental period. In each group, intestinal paracellular permeability, histopathological changes and shifts in tight junction (TJ) protein were evaluated; further, proliferation and apoptosis of intestinal epithelial cells (IECs) were assessed, and intraepithelial lymphocytes (IELs) were isolated and analyzed by flow cytometry. MMF caused intestinal mucosal injury, increased intestinal mucosal permeability, and altered expression of TJ protein. Moreover, MMF treatment inhibited IEC proliferation and increased apoptosis. MMF treatment resulted in a lower proportion of γ δ + T cells in IELs (γ δ + IELs). Conversely, concurrent administration of KGF with MMF effectively alleviated MMF-induced intestinal mucosal disruption, inhibited the increase in intestinal permeability, and maintained TJ protein expression. KGF also reversed the MMF-mediated inhibition of proliferation and promotion of apoptosis in IECs. In addition, KGF significantly enhanced the proportion of γ δ + IELs. Our findings suggest that MMF induces intestinal epithelial barrier disruption in mice. KGF may play a protective role to ameliorate the disruption and provide a therapeutic intervention for gastrointestinal disorders induced by MMF. [ABSTRACT FROM AUTHOR]

Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin‐like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF–ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF–ELPs. [ABSTRACT FROM AUTHOR]

Background: The skin is a dynamic body organ that can be activated by both central and local hypothalamic-pituitary-adrenal axis systems. This phenomenon might be the crucial explanation why stress can cause relapse of chronic inflammatory skin diseases, such as psoriasis. Here, we determined the effects of mast cells on keratinocyte proliferation under stress hormone stimulation. Methods: We subcutaneously injected dexamethasone on the shaved back of mice and evaluated histological changes and keratinocyte growth factor (KGF) expression on dermal mast cells. Further, human mast cell line (HMC-1) and keratinocyte cell line (HaCaT) cells were treated with dexamethasone in vitro to observe the extent of proliferation and the expression of KGF. Finally, the supernatants of HMC-1 cells treated with dexamethasone were used for the culture of HaCaT cells to investigate the effect on proliferation. Results: We observed epidermal thickening in dexamethasone-injected mice, accompanied by an increase in the number of KGF-expressing dermal mast cells. Similar to mouse dermal mast cells, KGF was highly expressed in the human mast cell line HMC-1 following stimulation with dexamethasone. Further, dexamethasone-treated mast cells promoted keratinocyte proliferation in vitro. However, the effects of mast cells on keratinocytes were significantly diminished in the presence of anti-KGF-blocking antibodies. Conclusion: Taken together, our results show that a stressful environment may disturb skin barrier homeostasis through mast cell-derived KGF expression. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway. [Display omitted] • MSC-CM and KGF had therapeutic effects on LPS-induced ALI model in mice. • KGF in MSC-CM could recover LPS-induced 3D alveolar organoid damage. • NF-κB signaling pathway may participate in the therapeutic effect of KGF in MSC-CM on ALI and the regulation of ENaC. [ABSTRACT FROM AUTHOR]

Endometrial injury usually results in intrauterine adhesion (IUA), which is an important cause of infertility and recurrent miscarriage in reproductive women. There is still lack of an effective therapeutic strategy to prevent occurrence of IUA. Keratinocyte growth factor (KGF) is a potent repair factor for epithelial tissues. Here, a temperature-sensitive heparin-modified poloxamer (HP) hydrogel with affinity to KGF (KGF-HP) was used as a support matrix to prevent IUA and deliver KGF. The rheology of KGF-HP hydrogel was carefully characterized. The cold KGF-HP solution was rapidly transited to hydrogel with suitable storage modulus (G′) and loss modulus (G″) for the applications of uterus cavity at temperature of 33 °C. In vitro release demonstrated that KGF was released from HP hydrogels in sustained release manner for a long time. In vivo bioluminescence imaging showed that KGF-HP hydrogel was able to prolong the retention of the encapsulated KGF in injured uterus of rat model. Moreover, the morphology and function of the injured uterus were significantly recovered after administration of KGF-HP hydrogel, which were evaluated by two-dimensional ultrasound imaging and receptive fertility. Not only proliferation of endometrial glandular epithelial cells and luminal epithelial cells but also angiogenesis of injured uterus were observed by Ki67 and CD31 staining after 7 d of treatment with KGF-HP hydrogel. Finally, a close relatively relationship between autophagy and proliferation of endometrial epithelial cells (EEC) and angiogenesis was firstly confirmed by detecting expression of LC3-II and P62 after KGF treatment. Overall, KGF-HP may be used as a promising candidate for IUA treatment.

Abstract Keratinocyte growth factor (KGF) was effective to treat ulcerative colitis. However, its poor stability and unspecific distribution toward inflamed bowel were two important obstacles hindering its consistent efficacy. Herein, KGF was firstly encapsulated into the liposomes (KGF-Lips) to improve its stability. Thereafter, the neutrophil membrane vesicle (NEM) was extracted from the activated neutrophil which was isolated from the healthy mice and then activated by lipopolysaccharide. Subsequently, NEM was inlaid in KGF-Lips to construct a neutrophil-like liposome (KGF-Neus). KGF was easily encapsulated into KGF-Neus with a high encapsulation efficiency of 95.3 ± 0.72%. Controlling NEM/lipid ratio at 1:50, KGF-Neus displayed the spherical morphology with D h of 154.8 ± 2.7 nm, PDI of 0.18, and zeta potential of −2.37 ± 0.14 mV. Moreover, KGF-Neus exhibited good stability of D h and significantly improved the chemical stability of KGF. Owing to NEM-associated proteins, KGF-Neus were specifically internalized by the inflammatory HUVECs. Moreover, KGF-Neus were specifically homed to the inflamed bowel in dextran sulfate sodium-induced mice after intravenous injection, resulting in the effective recovery of the morphology and function of the bowel. The therapeutic mechanisms of KGF-Neus were highly associated with alleviation of inflammation in colitis. Overall, the neutrophil-like liposome may be an excellent carrier for the colitis-targeted delivery of KGF. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is a common, costly and potentially lethal disease with characteristics of alveolar-capillary membrane disruption, pulmonary edema and impaired gas exchange due to increased apoptosis and pulmonary inflammation. There is no effective and specific therapy for ALI; however, mesenchymal stem cells (MSCs) have been demonstrated to be a potential option. Lipopolysaccharide (LPS) is a highly proinflammatory molecule that is used to mimic an in vivo inflammatory and damaged state in vitro. The present study investigated the effect of bone marrow-derived MSCs on an LPS-induced alveolar epithelial cell (A549 cell line) injury and its underlying mechanisms by a Transwell system. It was identified that a high LPS concentration caused a decrease in cell viability, increases in apoptosis, inflammatory cytokine release and NF-κB activity, disruption of the caspase-3/Bcl-2 ratio, upregulation of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and toll-interleukin-1 receptor domain-containing adaptor inducing interferon (TRIF) expression, and facilitation of TLR4/MyD88 and TLR4/TRIF complex formation in A549 cells. Coculture with MSCs attenuated all of these activities induced by LPS in A549 cells. In addition, an increased level of keratinocyte growth factor (KGF) and angiopoietin-1 (ANGPT1) secretion from MSCs was observed under inflammatory stimulation. KGF and/or ANGPT1 neutralizing antibodies diminished the beneficial effect of MSC conditioned medium. These data suggest that MSCs alleviate inflammatory damage and cellular apoptosis induced by LPS in A549 cells by modulating TLR4 signals. These changes may be partly associated with an increased secretion of KGF and ANGPT1 from MSCs under inflammatory conditions. These data provide the basis for development of MSC-based therapies for ALI. [ABSTRACT FROM AUTHOR]

Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of mesenchymal origin. Based on preclinical data, recombinant KGF plays a critical role in protecting and repairing of damaged epithelial tissues. Despite great efforts to express recombinant human KGF(rhKGF) in different organisms, attempts for finding appropriate protein expression system with the ability of producing a properly folded and processed KGF needs further investigation. Pichia pastoris has been used successfully and extensively for production of industrial enzymes and pharmaceutical proteins. Herein, we investigated the affect of pro-region-α-factor early deletion on production and secretion of rhKGF in Pichia pastoris. Initially, expression of human KGF induced in MCF-7 cell line treated with 1, 25-Dihydroxy vitamin D3. The coding sequence of full-length rhKGF194 was then cloned into the yeast integrative expression vector, downstream of α-factor and was integrated into P. pastoris genome. KGF protein was expressed in P. pastoris x33 cells, usingα-factor signal peptide for translocation of KGF to ER. An internal human signal peptide was also arranged after α-factorfor early removal of the pro-region in ER. RT-PCR results demonstrated that KGF mRNA was expressed successfully after induction by methanol. Recombinant KGF protein expression was detected by Western blotting in cell lysats, but not in conditioned media. A molecular weight of 17 kD for rhKGF194 indicates that the α-factor and internal human signal peptideshad been removed in x33 cells. The results indicate that in the absence of pro-region-α-factor, the recombinant KGF protein was not efficiently processed and transported within the biosynthesis-secretory pathway. As KGF protein is an unstable growth factor and tend to aggregate because of some native properties, It seems that presence of a chaperon molecule fusion with KGF is necessary for efficient secretion of the recombinant protein.