KGF-2 Regulates STAP-2-Mediated Signal Transducer and Activator of Transcription 3 Signaling and Reduces Skin Scar Formation.

Hypertrophic scar is a common complication of burns, skin trauma, and postoperative trauma, which involves excessive proliferation of fibroblasts and accumulation of a large amount of disorganized collagen fibers and extracellular matrix. KGF-2 plays important roles in the regulation of cellular homeostasis and wound healing. In this study, we investigated the effect and underlying mechanism of KGF-2 on scar formation after wound healing both in vitro and in vivo. We show that KGF-2 attenuates mechanical stress-induced scar formation while promoting wound healing. Mechanistically, KGF-2 inhibits STAP-2 expression and signal transducer and activator of transcription 3 activation, leading to significantly reduced collagen I and collagen III levels. Our results provide an insight into the role of KGF-2 in wound healing and scar formation and the therapeutic potential for reducing scarring while promoting wound healing.

Toxicity Evaluation of Long-Term Topical Application of Recombinant Human Keratinocyte Growth Factor-2 Eye Drops on Macaca Fascicularis.

Recombinant human keratinocyte growth factor-2 (rhKGF-2), an effective agent for the regeneration of epithelial tissue, was found to have great potential for use in treatments of corneal diseases that involve corneal epithelial defects. Furthermore, the safety of long-term and high-dose external use of KGF-2 eye drops in rabbits has been well established previously. The aim of this study is to determine the safe dose range and target organs for toxicity of rhKGF-2 eye drops in Macaca fascicularis (M. fascicularis). The M. fascicularis animals were administered with different doses of rhKGF-2 eye drops (125, 500, and 2000 µg/ml) for four consecutive weeks, followed by a 2 week recovery period. No significant differences in weight, electrocardiogram characteristics, blood and urine indexes, pathology, and bone marrow cells were detected among the animals in different groups. The corneas of some animals in the middle- and high-dose groups showed fluorescence when stained with sodium fluorescein, and then the staining disappeared on days 28 and 42. Anti-rhKGF-2 antibodies were detected in a small number of animals in the high-dose group, and their level decreased after rhKGF-2 withdrawal. No neutralizing antibodies were detected. The result demonstrated that there was no obvious adverse reaction when topical application of rhKGF-2 eye drops at the dosage of 125 or 500 µg/ml on the M. fascicularis. This study is of great significance for the future clinical transformation of rhKGF-2 eye drops.

Controlled release of KGF-2 for regulation of wound healing by KGF-2 complexed with "lotus seedpod surface-like" porous microspheres.

Keratinocyte growth factor-2 (KGF-2) can regulate the proliferation and differentiation of keratinocyte, which plays a remarkable role in maintaining normal tissue structure and promoting wound healing. As an effective strategy, KGF-2 solution is widely used in the treatment of wounds in clinical applications. However, KGF-2 in solution cannot achieve sustained release, which results in drug loss and unnecessary waste. Polysaccharide hemostasis microspheres (PHMs) are an ideal drug loading platform due to their special "lotus seedpod surface-like" morphology and structure. Herein, to realize the controllable release of KGF-2, PHMs loaded with KGF-2 (KGF-2@PHMs) were prepared. It was found that the bioavailability of KGF-2 was improved greatly. Most importantly, KGF-2@PHMs can reduce inflammation and accelerate the wound healing process due to the controlled release of KGF-2. KGF-2@PHMs might be a potential alternative strategy for wound healing in future clinical applications.

Hypoglycemic Efficacy of Rh-aFGF Variants in Treatment of Diabetes in ZDF Rats.

Acidic fibroblast growth factor (aFGF) is a promising regulator of glucose with no adverse effects of hypoglycemia. Previous researches revealed that aFGF mediated adipose tissue remodeling and insulin sensitivity. These findings supported rh-aFGF135 would be used as a new candidate for the treatment of insulin resistance and type 2 diabetes. In this study, we aimed to investigate the hypoglycemic efficacy of recombinant human acidic fibroblast growth factor 135 (rh-aFGF135) with low mitogenic in type 2 diabetic ZDF rats. ZDF rats were treated with rh-aFGF135 at a daily dosage of 0.25 and 0.50 mg/kg by tail intravenous injection for 5 weeks. The blood glucose levels, oral glucose tolerance test, insulin tolerance test, HOMA-IR for insulin resistance, serum biochemical parameters, and the histopathological changes of adipose tissue, liver and other organs were detected at designed time point. The glucose uptake activity and anti-insulin resistance effect of rh-aFGF135 were also detected in HepG2 cells. Results revealed that rh-aFGF135 exhibited a better hypoglycemic effect compared with vehicle group and without the adverse effect of hypoglycemia in ZDF rats. Compared with vehicle group, rh-aFGF135 significantly improved the situation of hyperglycemia and insulin resistance. Rh-aFGF135 decreased ALT, AST, GSP, and FFA levels noticeably compared with vehicle control group (P < 0.01 or P < 0.001). After 5 weeks of treatment, high-dosage rh-aFGF135 could remodel adipose tissue, and has no influence on other organs. H&E staining showed that rh-aFGF135 reduced the size of adipocytes. In addition, rh-aFGF135 may improve insulin resistance partly by increasing the protein expression of p-IRS-1 (human Ser 307). As a hypoglycemic drug for long-term treatment, rh-aFGF135 would be a potentially safe candidate for the therapy of type 2 diabetes.

Preparation of a recombinant collagen-peptide (RHC)-conjugated chitosan thermosensitive hydrogel for wound healing.

Thermosensitive chitosan hydrogels have been widely used in drug delivery and tissue repair, but further applications of these hydrogels have been limited by their weak mechanical strength and poor bioactivity. A thermoresponsive hydrogel formed by conjugating recombinant human collagen-peptide (RHC) with chitosan might be better suited for cell encapsulation and wound repair. RHC-chitosan hydrogels were prepared and tested, and the results showed that moderate RHC conjugation led to hydrogels with lower gelation temperature. The prepared RHC-containing hydrogels showed superior mechanical strength to chitosan-only hydrogels. Additionally, cells exhibited superior viability when cultured with RHC-modified hydrogels compared with hydrogels that had not been conjugated with RHC. Finally, RHC-chitosan hydrogels were injected onto the backs of rats with second-degree burns and promoted cell infiltration, vessel formation, and wound healing. Overall, the use of RHC-chitosan hydrogels is a promising and effective therapeutic approach for burn wound treatment.

KGF-2 and FGF-21 poloxamer 407 hydrogel coordinates inflammation and proliferation homeostasis to enhance wound repair of scalded skin in diabetic rats.

OBJECTIVE: The present study focused on the development of a poloxamer 407 thermosensitive hydrogel loaded with keratinocyte growth factor-2 (KGF-2) and fibroblast growth factor-21 (FGF-21) as a therapeutic biomaterial in a scald-wound model of type-2 diabetes in Goto-Kakizaki (GK) rats. RESEARCH DESIGN AND METHODS: In this study, a poloxamer 407 thermosensitive hydrogel loaded with KGF-2 and/or FGF-21 was prepared and its physical and biological properties were characterized. The repairing effects of this hydrogel were investigated in a scald-wound model of type-2 diabetes in GK rats. The wound healing rate, epithelialization, and formation of granulation tissue were examined, and biomarkers reflecting regulation of proliferation and inflammation were quantified by immunostaining and Western blotting. T tests and analyses of variance were used for statistical analysis via Graphpad Prism V.6.0. RESULTS: A 17.0% (w/w) poloxamer 407 combined with 1.0% (w/w) glycerol exhibited controlled release characteristics and a three-dimensional structure. A KGF-2/FGF-21 poloxamer hydrogel promoted cellular migration without apoptosis. This KGF-2/FGF-21 poloxamer hydrogel also accelerated wound healing of scalded skin in GK rats better than that of a KGF-2 or FGF-21 hydrogel alone due to accelerated epithelialization, formation of granulation tissue, collagen synthesis, and angiogenesis via inhibition of inflammatory responses and increased expression of alpha-smooth muscle actin (α-SMA), collagen III, pan-keratin, transforming growth factor-ß (TGF-ß), vascular endothelial growth factor (VEGF), and CD31. CONCLUSIONS: A KGF-2/FGF-21 poloxamer hydrogel accelerated wound healing of scalded skin in GK rats, which was attributed to a synergistic effect of KGF-2-mediated cellular proliferation and FGF-21-mediated inhibition of inflammatory responses. Taken together, our findings provide a novel and potentially important insight into improving wound healing in patients with diabetic ulcers.

Comparative Analysis of KGF-2 and bFGF in Prevention of Excessive Wound Healing and Scar Formation in a Corneal Alkali Burn Model.

PURPOSE: Basic fibroblast growth factor (bFGF) is an effective drug for corneal injury. However, the explicit role of bFGF in corneal scar formation still remains unclear. Keratinocyte growth factor-2 (KGF-2) is associated with the treatment of wound healing. We aimed to compare the efficacy of bFGF and KGF-2 in prevention of excessive wound healing and consequent scar formation in a rat alkali burn model, which provides important clues on the significance of KGF-2 to be developed as a new drug for such injuries. METHODS: The epithelial defect area was evaluated using fluorescein sodium at a concentration of 0.5%. The therapeutic effect of KGF-2 and bFGF on proliferation of rabbit corneal fibroblasts (RCFs) was evaluated by methylthiazoletetrazolium. RCF migration assays were performed with a modified scratch method. Activation of mitogen-activated protein kinase (MAPK) was evaluated by Western blot with specific antibodies. RESULTS: All corneal wounds treated with KGF-2 were found closed within 7 days; however, the wounds treated with bFGF or phosphate buffer saline (PBS) required 14 days to close. RCFs treated with KGF-2 or bFGF showed similar dose-dependent proliferation. The KGF-2 group significantly promoted cell migration compared with the bFGF group. The KGF-2 group showed less expression of α-smooth muscle actin (SMA) and numbers of myofibroblasts compared with the bFGF group. Our findings suggested identification of cascade reaction of extracellular regulated protein kinases (ERK)1/2 and p38 signals in KGF-2- and bFGF-induced proliferation and migration of RCFs. In addition, KGF-2 showed stronger effects during ERK1/2 and p38 phosphorylation in methylthiazoletetrazolium proliferation assay and scratch migration assay. CONCLUSIONS: KGF-2 exhibited better effects than bFGF in reepithelialization, acceleration of migration, and reduction of scar formation, which has potential to become a new drug to cure corneal injury.

Two-hundred-liter scale fermentation, purification of recombinant human fibroblast growth factor-21, and its anti-diabetic effects on ob/ob mice.

Fibroblast growth factor-21 (FGF-21) is a potential cytokine for type II diabetes mellitus. This study aimed to optimize recombinant human FGF-21 (rhFGF-21) production in Escherichia coli BL21 (DE3) employing high cell density fermentation at a 200-L scale and pilot-scale purification. FGF-21 was eventually expressed in E. coli BL21 (DE3) using human FGF-21 synthetic DNA sequence via the introduction of vector pET-3c; the product is used as seed strain during the fermentation of rhFGF-21. Fermentation of rhFGF-21 was performed in a 30-L and 200-L fermenters. rhFGF-21 was primarily expressed in the form of inclusion bodies after IPTG induction. At the 200-L scale, the bacterial production and expression levels of rhFGF-21 were 38.8 ± 0.6 g/L and 30.9 ± 0.7%, respectively. Additionally, the high purification (98%) of rhFGF-21 was tested with HPLC analysis and reducing & non-reducing SDS-PAGE analysis. The final yield of purified rhFGF-21 was 71.1 ± 13.9 mg/L. The activity of rhFGF-21 stock solution reached at 68.67 ± 8.74 IU/mg. Blood glucose controlling and insulin sensitization were improved with treatment of rhFGF-21 in type II diabetic ob/ob mice. Our results showed that the relatively stable and time-saving pilot-scale production process was successfully established, providing an efficient and cost-effective strategy for large-scale and industrial production of rhFGF-21.

Higher Biostability of rh-aFGF-Carbomer 940 Hydrogel and Its Effect on Wound Healing in a Diabetic Rat Model.

Hydrogels are excellent drug delivery carriers with excellent ductility. Here, we report the design of a higher biostability of a recombinant human acidic fibroblast growth factor (rh-aFGF) carbomer hydrogel formulation. To verify the optimality of this formula, we prepared various prescriptions and tested the resulting physical properties including micromorphology, long-term stability, accelerated stability, and destructive test. Furthermore, the efficacy for promoting wound healing in full-thickness injury and scald wound diabetic rat models was explored. We found that rh-aFGF-carbomer hydrogel had good physical properties. It was stable for 24 months at 5 ± 3 °C, and for 6 months at 25 ± 3 °C. In vivo, the rh-aFGF-carbomer 940 hydrogel achieved a remarkable promotion of skin wound healing in diabetic rats with full-thickness injuries or scald wounds. Our data suggest that rh-aFGF-carbomer hydrogel may have applications for the treatment of diabetic ulcers combined with other wounds.

Design and Evaluation of Lyophilized Fibroblast Growth Factor 21 and Its Protection against Ischemia Cerebral Injury.

This study investigated the effect of the excipients, including glycine, mannitol, arginine, trehalose, sorbitol, and poloxamer188, on the stability of recombinant human fibroblast growth factor 21(FGF21) during the process of lyophilization and storage. The glass transition temperature (Tg), protein secondary structure, aggregation ratio, and the bioactivity of lyophilized FGF21 were measured. We furthermore investigated the effect of FGF21 against ischemia cerebral injury using the middle cerebral artery occlusion (MCAO) model in rats. The ischemia cerebral injury of MCAO rats was analyzed via 2,3,5-triphenyltetrazolium chloride and Nissl-staining. Endoplasmic reticulum (ER) stress related proteins were detected via Western blot. In this study, we found that aggregation was the primary mode of deterioration of lyophilized FGF21under accelerated storage conditions. Mannitol combined with trehalose and glycine formulations offers the most effective protein protection to reduce the aggregation. Administration of FGF21 protected cerebral ischemia and decreased ER stress related proteins in MCAO rats and PC12 cells.