KGF-2 ameliorates UVB-triggered skin photodamage in mice by attenuating DNA damage and inflammatory response and mitochondrial dysfunction.

BACKGROUND: Long-term exposure to UVB induces DNA damage, inflammatory response, mitochondrial dysfunction, and apoptosis in skin cells, thus causing skin photodamage. Research has demonstrated the noteworthy antioxidant, anti-inflammatory, DNA repair, and mitochondrial protective properties of keratinocyte growth factor-2 (KGF-2). METHODS: To examine the impact of KGF-2 on UVB-triggered skin photodamage in mice, hair-removed mice were initially exposed under UVB radiation and subsequently treated with KGF-2 hydrogel and repeated for 6 days. On day 7, the assessment of histopathological alterations, inflammation, DNA damage, mitochondrial function, and apoptosis in mouse skin was assessed. RESULTS: It was found that KGF-2 could effectively relieve cutaneous photodamage symptoms and inhibit epidermal proliferation in mice. Meanwhile, KGF-2 was found to significantly reduce DNA damage, attenuate the inflammatory response, and inhibit the mitochondria-mediated intrinsic apoptotic pathway in the UVB-exposed mouse skin photodamage model. CONCLUSION: To summarize, our results indicated that KGF-2 reduces the severity of mouse skin photodamage caused by UVB rays by attenuating DNA damage and the inflammatory response, besides inhibiting the mitochondria-mediated intrinsic apoptosis pathway.

High-level production of keratinocyte growth factor 2 in Escherichia coli.

Recombinant human keratinocyte growth factor 2 (KGF-2), also known as repifermin, is used in various therapeutic applications. However, KGF-2 production has not been optimized for facilitating large-scale production. Therefore, we attempted to attain high-level production of bioactive KGF-2. KGF-2 was fused with 6HFh8 (6HFh8-KGF-2) at the tobacco etch virus protease cleavage site. The 6HFh8-KGF-2 was expressed in Escherichia coli with high expression levels of approximately 33% and 20% of soluble protein in flask culture and 5 L fermentation, respectively. 6HFh8-KGF-2 was purified via nickel affinity chromatography. To maintain a stable form of KGF-2, the conditions of the cleavage reaction were optimized based on the isoelectric point. KGF-2 was purified via ion-exchange chromatography to high purity (>99%) with an optimal purification yield (91%). Circular dichroism spectroscopy demonstrated that purified KGF-2 had a secondary structure and thermal stability similar to that of commercial KGF-2. Bioactivity assays indicated that purified KGF-2 could induce MCF-7 cell proliferation in the same manner as commercial KGF-2. These results demonstrate that bioactive KGF-2 was overexpressed in E. coli and purified to high quality. Our findings indicated that bioactive KGF-2 can be produced in large quantities in E. coli.

Evaluation of clinical and histological effects of KGF-2 and NGF on corneal wound healing in an experimental alkali burn rabbit model.

Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-ß (TGF-ß) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-ß was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.

Toxicology study of long-term administration of rhKGF-2 eye drops on rabbit corneas.

Keratinocyte growth factor -2 promotes corneal repair. Its mechanism of action involves regulating regeneration and migration of corneal cells, as well as activating corneal limbal stem cells. However, KGF-2 being a carcinogenic growth factor and its potential adverse effect in over dosage long-term treatment had not yet been reported. In this study, we used New Zealand white rabbits to study possible toxic effects of ocular administration of recombinant human keratinocyte growth factor-2 eye drops. Animals in the medium- and high-dose groups had some ocular irritant reactions during the course of drug administration; however this reaction was harmless to the cornea and it ended up when administration was stopped. Serum biochemistries were largely unaffected by treatment. Pathological examinations were unremarkable. We found that over-dosed administration of these eye drops caused some ocular irritation, but this irritant reaction was harmless to the eye, and it reversed after the drug was stopped. There were no apparent systemic effects of the drug.

Keratinocyte growth factor-2 inhibits bacterial infection with Pseudomonas aeruginosa pneumonia in a mouse model.

To determine protective effects of concurrent administration of Keratinocyte growth factor-2 (KGF-2) with Pseudomonas aeruginosa (P. aeruginosa) inoculation on the induced pneumonia. KGF-2 (5 mg/kg) was concurrently administered into the left lobe of 55 mice with P. aeruginosa PAO1 (5 × 10(6) CFU, half-lethal dose); 55 mice in the control group were concurrently administered PBS with the PAO1. We detected and analyzed: body temperature; amount of P. aeruginosa in homogenates; count of total number of nucleated cells and of mononuclear macrophages; protein concentration in bronchoalveolar lavage fluid (BALF); lung wet-to-dry weight ratio; cytokines in BALF and blood; and lung morphology. To study survival rate, concurrent administration of KGF-2 (experimental group) versus PBS (control) with a lethal dose of PAO1 (1 × 10(7) CFU was performed, and survivorship was documented for 7 days post-inoculation. The bacterial CFU in lung homogenates was significantly decreased in the KGF-2 group compared to the control group. There were significantly more mononuclear macrophages in the BALF from the KGF-2 group than from the control group (p < 0.05). KGF-2 increased the surfactant protein and GM-CSF mRNA in lung at 6 h and 72 h after inoculation. Significant reduction of lung injury scores, protein concentrations, lung wet-to-dry weight ratio, and IL-6 and TNF-α levels was noted in the KGF-2 treated rats at 72 h after inoculation (p < 0.05). The 7-day survival rate of the KGF-2 group was significantly higher than that of the control group (p < 0.05). Concurrent administration of KGF-2 facilitates the clearance of P. aeruginosa from the lungs, attenuates P. aeruginosa-induced lung injury, and extends the 7-day survival rate in mice model with P. aeruginosa pneumonia.

Application of oleosin-flanked keratinocyte growth factor-2 expressed from Arabidopsis thaliana promotes hair follicle growth in mice.

OBJECTIVE: To determine an efficient expression strategy in Arabidopsis thaliana and to determine whether a dimeric oleosin fusion approach could achieve keratinocyte growth factor-2 (KGF2) expression and bioactivity. RESULTS: Higher recombinant protein accumulation was observed in the two dimeric oleosin constructs than in a single-oleosin-fused protein (O::KGR2) or KGF2 control. Highest expression was in O-O::KGF2-transgenic seeds. MTT assay in FGFR2 III b-BaF3 cells revealed comparable levels of bioactivity due to O-O::KGF2 and O::KGR2, whereas O::KGF2-O had no effect on FGFR2 III b-BaF3 opithelial cell growth. The transgenic proteins had a pronounced stimulatory effect on hair follicle proliferation in C57BL/6 mice. CONCLUSION: A dimeric oleosin approach can be used to express KGF2 with the non-symmetrical O-O::KGF2 construct showing the highest expression and bioactivity.

Pharmacokinetics of topically applied recombinant human keratinocyte growth factor-2 in alkali-burned and intact rabbit eye.

Keratinocyte growth factor-2 (KGF-2), an effective agent in the development of epithelial tissue and regeneration during corneal wound healing, is a potential therapeutic option to treat the corneal diseases with corneal epithelial defects. However the tissue distribution and pharmacokinetics of KGF-2 have not been explored yet in eye upon topical application. Using (125)I-labeled recombinant human KGF-2 ((125)I-rhKGF-2), tissue distribution of rhKGF-2 in alkali-burned and control rabbit eyes was studied. Our results revealed that (125)I-rhKGF-2 was distributed to all eye tissues examined. The highest radioactivity level was found in the cornea, followed by iris, sclera, ciliary body, lens, aqueous humor, vitreous body, and serum in a greatest to least order. The levels of (125)I-rhKGF-2 were higher in corneas of alkali-burned eyes than those in control eyes though without statistical significance. Calculated pharmacokinetic parameters of t1/2, Cmax, and Tmax of rhKGF-2 in the rabbit corneas were 3.4 h, 135.2 ng/ml, and 0.5 h, respectively. In iris, lens, aqueous humor, and tear, t1/2, Cmax, and Tmax values were 6.2, 6.5, 5.2, and 2.5 h; 23.2, 4.5, 24.1, and 29,498.9 ng/ml; and 1.0, 0.5, 0.5, and 1.0 h, respectively. Predominant and rapid accumulation of rhKGF-2 in corneas suggests that therapeutic doses of rhKGF-2 could be delivered by topical application for treatment of corneal diseases.

Keratinocyte growth factor-2 intratracheal instillation significantly attenuates ventilator-induced lung injury in rats.

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. Keratinocyte growth factor-2 (KGF-2), a potent epithelial cell mitogen, may have a role in preventing ventilator-induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF-2 in VILI in rats. Forty healthy adult male Sprague-Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high-volume zero positive end-expiratory pressure) and HVZP+KGF-2 were given intratracheally equal PBS and 5 mg/kg KGF-2 72 hrs before 4 hrs HVZP ventilation (20 ml/kg), respectively, while PBS and KGF-2 were administered in the same manner in Groups Control and KGF-2, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis factor-α, macrophage inflammatory protein 2), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre-treatment with KGF-2 showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF-2 might be considered a promising prevention for human VILI or other acute lung injury diseases.

Keratinocyte Growth Factor 2 Ameliorates UVB-Induced Skin Damage via Activating the AhR/Nrf2 Signaling Pathway.

Objective: Exposure to ultraviolet B (UVB) can cause skin damage through oxidative stress, DNA damage, and apoptosis. Keratinocyte growth factor (KGF) has been shown to reduce the content of intracellular reactive oxygen species (ROS) following UVB exposure, a role that is crucial for the efficient photoprotection of skin. The present study evaluated the photoprotective effect of KGF-2 on UVB-induced skin damage and explored its potential molecular mechanism. Methods: To evaluate the effect of KGF-2 on UVB-induced damage ex vivo, a human epidermal full-thickness skin equivalent was pretreated without or with KGF-2 and then exposed to UVB and the levels of histopathological changes, DNA damage, inflammation, and apoptosis were then evaluated. The ability of KGF-2 to protect the cells against UVB-inflicted damage and its effect on ROS production, apoptosis, and mitochondrial dysfunction were determined in HaCaT cells. Results: Pretreatment of the epidermis with KGF-2 ameliorated the extent of photodamage. At the cellular level, KGF-2 could attenuate ROS production, apoptosis, DNA damage, and mitochondrial dysfunction caused by UVB exposure. KGF-2 could also activate the aryl hydrocarbon receptor (AhR) to trigger the Nrf2 signaling pathway. Conclusion: Taken together, our findings suggested that KGF-2 could ameliorate UVB-induced skin damage through inhibiting apoptosis, reducing oxidative stress, and preventing DNA damage and mitochondrial dysfunction via regulating AhR/Nrf2 signaling pathway.

[Effects of aerosol inhalation of recombinant human keratinocyte growth factor 2 on the lung tissue of rabbits with severe smoke inhalation injury].

Objective: To investigate the effect of recombinant human keratinocyte growth factor 2 (rhKGF-2) on lung tissue of rabbits with severe smoke inhalation injury. Methods: A total of 120 New Zealand rabbits were divided into 5 groups by random number table after being inflicted with severe smoke inhalation injury, with 24 rats in each group. Rabbits in the simple injury group inhaled air, while rabbits in the injury+phosphate buffer solution (PBS) group inhaled 5 mL PBS once daily for 7 d. Rabbits in injury+1 mg/kg rhKGF-2 group, injury+2 mg/kg rhKGF-2 group, and injury+5 mg/kg rhKGF-2 group received aerosol inhalation of 1 mg/kg, 2 mg/kg, and 5 mg/kg rhKGF-2 (all dissolved in 5 mL PBS) once daily for 7 d, respectively. On treatment day 1, 3, 5, and 7, blood samples were taken from the ear central artery of 6 rabbits in each group. After the blood was taken, the rabbits were sacrificed, and the tracheal carina tissue and lung were collected. Blood pH value, arterial oxygen partial pressure (PaO(2)), arterial blood carbon dioxide pressure (PaCO(2)), and bicarbonate ion were detected by handheld blood analyzer. The expressions of pulmonary surfactant-associated protein A (SP-A) and vascular endothelial growth factor (VEGF) in lung tissue were detected by Western blotting. Pathomorphology of lung tissue and trachea was observed by hematoxylin-eosin staining. Data were processed with analysis of variance of two-way factorial design and Tukey test. Results: (1) Compared with those in simple injury group, the blood pH values of rabbits in the latter groups on treatment day 1-7 had no obvious change (P>0.05). The PaO(2) of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 5 and 7 were (75.0±2.4) and (71.0±4.5) mmHg (1 mmHg=0.133 kPa), respectively, which were significantly higher than (62.0±6.8) and (63.0±3.0) mmHg in simple injury group (q=4.265, 8.202, P<0.05 or P<0.01). The PaO(2) of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 7 was (82.0±4.9) mmHg, which was significantly higher than that in simple injury group (q=6.234, P<0.01). Compared with that in simple injury group, the PaCO(2) of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 3 was significantly decreased (q=4.876, P<0.01) and significantly increased on treatment day 5 (q=5.562, P<0.01); the PaCO(2) of rabbits in injury+5 mg/kg rhKGF-2 group was significantly increased on treatment day 5 and 7 (q=5.013, 4.601, P<0.05 or P<0.01). Compared with that in simple injury group, the serum bicarbonate ion of rabbits in injury+1 mg/kg rhKGF-2 group on treatment day 7 was significantly increased (q=5.142, P<0.01); the serum bicarbonate ion of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 5 and 7 were significantly increased (q=4.830, 6.934, P<0.01); the serum bicarbonate ion of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 5 were significantly increased (q=3.973, P<0.05). (2) The expressions of SP-A in lung tissue of rabbits in simple injury group and injury+PBS group in each treatment time point were close (P>0.05). The expressions of SP-A in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group on treatment day 3 were 0.091±0.007 and 0.101±0.009, respectively, significantly higher than 0.069±0.009 in simple injury group (q=10.800, 13.580, P<0.01). The expressions of SP-A in lung tissue of rabbits in injury+1 mg/kg rhKGF-2 group, injury+2 mg/kg rhKGF-2 group, and injury+5 mg/kg rhKGF-2 group on treatment day 5 and 7 were 0.127±0.008, 0.132±0.006, 0.194±0.006, 0.152±0.017, 0.166±0.004, 0.240±0.008, significantly higher than 0.092±0.003 and 0.108±0.005 in simple injury group (q=6.789, 12.340, 17.900, 9.875, 31.480, 40.740, P<0.01). (3) On treatment day 1 and 5, there was no significant difference in the expression of VEGF in lung tissue of rabbits among the 5 groups (P>0.05). Compared with those in simple injury group, the expressions of VEGF in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 3 and 7 were significantly increased (q=4.243, 8.000, P<0.05 or P<0.01), and the expression of VEGF in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 7 was significantly increased (q=20.720, P<0.01). (4) On treatment day 1, the injury of rabbits in each group was similar, with a large number of neutrophils infiltrated and abscess formed in the alveolar and interstitial tissue, thickened alveolar septum, some collapsed alveolar and atelectasis; large area of tracheal mucosa was degenerated and necrotic, with a large amount of inflammatory exudates blocking in the cavity. On treatment day 3, the inflammation of lung tissue and trachea in each group were improved, but the inflammation in simple injury group and injury+PBS group was also serious. On treatment day 5, the inflammation in lung tissue and trachea of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group were improved much obviously than those in the other groups. On treatment day 7, the inflammation in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group alleviated obviously than those in the other groups, most alveoli had no obvious exudative fluid, the alveolar cavity was intact and clear, the local alveolar dilated like a cyst, and the alveolar septum thinning; the improvement of inflammation of trachea was more obvious than the other groups, the tracheal mucosa tended to be more complete, and few neutrophils were infiltrated in the endotracheal cavity. Conclusions: Atomization inhalation of rhKGF-2 can improve the PaO(2) level of rabbits with severe smoke inhalation injury, reduce airway inflammation, increase the expression of SP-A and VEGF in lung tissue, thus promoting the repair of lung tissue.

Therapeutic efficacy of a mutant of keratinocyte growth factor-2 on trinitrobenzene sulfonic acid-induced rat model of Crohn's disease.

BACKGROUND: Keratinocyte growth factor-2 (KGF-2) has been testified to be a multifunctional growth factor, which can stimulate the regeneration and reconstruction of epidermis, corium and mucosa. Its effect on Crohn's disease has hitherto not been evaluated. Here, we investigated the preventive and therapeutic actions of STEA, a mutant of human KGF-2 with high activity, on trinitrobenzene sulfonic acid (TNBS)-induced rat model of Crohn's disease. METHODS: Rats with TNBS-induced colitis were treated with STEA and clinical scores were evaluated. Body weight, mortality, macroscopic and microscopic damage of the colonic tissue were examined. The levels of inflammatory cytokines in serum were detected by ELISA, the T cell subpopulations and the cell cycle of intestinal epithelial cells were analyzed by flow cytometry. RESULTS: Both preventive and therapeutic administration of STEA significantly ameliorated body weight loss, diarrhea, and intestinal inflammation, reduced the high mortality and histopathologic damage of rats with TNBS-induced colitis. The serum level of inflammatory cytokines, such as TNF-α, IL-1ß, IFN-γ and IL-6 were markedly decreased in colitis rats treated with STEA. The CD4+ and CD8+ T lymphocytes in peripheral blood were reduced with STEA administration at early stage of colitis. In addition, STEA treatment could promote the growth of intestinal epithelial cells by increasing the cell proportion in S phase of cell cycle and inhibiting cell apoptosis. CONCLUSIONS: Both preventive and therapeutic administration of STEA could ameliorate the colonic damages in rats with TNBS-induced colitis. STEA might be a promising option for the treatment of Crohn's disease.

Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats.

Keratinocyte growth factor-2 (KGF-2) plays a key role in lung development, but its role in acute lung injury has not been well characterized. Lipopolysaccharide instillation caused acute lung injury, which significantly elevated lung wet-to-dry weight ratio, protein and neutrophils in bronchoalveolar lavage fluid (BALF), inhibited surfactant protein A and C expression in lung tissue, and increased pathological injury. Pretreatment with KGF-2 improved the above lung injury parameters, partially restored surfactant protein A and C expression, and KGF-2 given 2-3 days before LPS challenge showed maximum lung injury improvement. Pretreatment with KGF-2 also markedly reduced the levels of TNF-α, MIP-2, IL-1ß and IL-6 in BALF and the levels of IL-1ß and IL-6 in lung tissue. Histological analysis showed there was increased proliferation of alveolar type II epithelial cells in lung parenchyma, which reached maximal 2 days after KGF-2 instillation. Intratracheal administration of KGF-2 attenuates lung injury induced by LPS, suggesting KGF-2 may be potent in the intervention of acute lung injury.

Keratinocyte growth factor-2 and autologous serum potentiate the regenerative effect of mesenchymal stem cells in cornea damage in rats.

AIM: To investigate the healing process after severe corneal epithelial damage in rats treated with mesenchymal stem cells (MSCs) cultured with or without keratinocyte growth factor (KGF-2) and autologous serum (AS) on amniotic membrane (AM). Many patients are blind and devastated by severe ocular surface diseases due to limbal stem cell deficiency. Bone marrow-derived MSCs are potential sources for cell-based tissue engineering to repair or replace the corneal tissue, having the potential to differentiate to epithelial cells. METHODS: The study included 5 groups each including 10 female "Sprague Dawley" rats in addition to 20 male rats used as bone marrow donors. Group I rats received AM+MSCs, Group II rats AM+MSCs cultured with KGF-2, Group III rats AM+MSCs cultured with KGF-2+AS, Group IV rats only AM and Group V rats, none. AS was derived from blood drawn from male rats and bone marrow was obtained from the femur and tibia bones of the same animals. Therapeutic effect was evaluated with clinical, histopathological and immunohistochemical assessment. MSC engraftment was demonstrated via detection of donor genotype (Y+) in the recipient tissue (X) with polymerase chain reaction. RESULTS: Corneal healing was significantly better in Groups I-III rats treated with MSC transplantation compared to Group IV and Group V rats with supportive treatment only. The best results were obtained in Group III rats with 90% transparency, 70% lack of neovascularization, and 100% epithelium damage limited to less than 1/4 of cornea. CONCLUSION: We suggest that culture of MSCs with KGF-2 and AS on AM is effective in corneal repair in case of irreversible damage to limbal stem cells.