Keratinocyte Growth Factor Concentrations Research Articles

Manufacturing exosomes for wound healing: Comparative analysis of culture media.

Mesenchymal stem cell (MSC)-derived exosomes (EXs) have emerged as promising therapeutic agents for wound healing. However, the optimal conditions for manufacturing MSC-derived EXs that maximize their wound-healing potential have yet to be established. Hence, we compared the efficacy of five different MSC culture media, including three different serum-free, a platelet-supplemented, and a fetal bovine serum-supplemented media, in exosome manufacturing for wound healing applications. Although umbilical cord-derived MSCs (UCMSCs) cultured in these media exhibited similar proliferation, morphology, MSC surface marker expression, and stemness, EXs derived from UCMSCs cultured in different culture media displayed varying levels of growth factors and cytokines. Notably, EXs derived from platelet-supplemented media (DM-PLT_EXs) exhibited significantly higher concentrations of keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF-A), platelet-derived growth factor (PDGF-BB), interleukin 6 (IL-6), interleukin 7 (IL-7), and interleukin 8 (IL-8) than EXs from other media. These differences correlated with the superior capability of DM-PLT_EXs to promote human skin fibroblast proliferation and stimulate angiogenesis of human umbilical vein endothelial cells, making them a more suitable choice for wound healing applications. Our findings emphasize the significance of the culture medium selection in tailoring the therapeutic potential of UCMSC-derived EXs for wound healing.

Analysis of different conditioned media secreted by limbal progenitor cells in the modulation of corneal healing

Ex vivo cultivation and transplantation of limbal epithelial cells has been reported as an alternative source for ocular surface reconstruction. However, until now, the functional improvement of these patients is limited due to the low survival rate of the transplanted cells. Consequently, the clinical benefits of this therapeutic strategy are only temporary and can assign them to paracrine effects associated with the transplanted cells. With this background in mind, we aimed to analyze the effect of different conditioned media containing growth factors secreted by limbal progenitor cells on corneal epithelial healing, both in vitro and in vivo. Limbal tissue was used to obtain different conditioned media (CM). For the in vitro assay, corneal epithelial cells were treated with CM and the epithelial migration was analyzed. Growth factors in the CM were identified with ELISA and multiplex. For the in vivo assay in rats, total limbal stem cell deficiency (LSCD) was induced with an abrasive injury to the ocular surface, and the animals were treated with different CM. Clinical and histological analyses were performed. In the in vitro assay, treatment with limbal fibroblast (LF CM) was more effective compared to the other CM, and analysis revealed high concentrations of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF). In the in vivo assay, animals treated with LF CM showed epithelial defect improvement, maintenance of thickness, and decreased opacity and neovascularization. This treatment also allowed better ocular surface tissue organization when compared to the other treatments. The in vitro and in vivo experiments showed better outcomes in the corneal wound healing for the LF CM treatment. The high concentrations of KGF and HGF, linked to epithelial cell migration and proliferation, may correlate to the best results found in this treatment.

Growth Factor Concentration in Keratocyte Supernatant after Incubation with Human Serum In Vitro

Application of serum eye drops is an alternative treatment option for therapy-resistant corneal epithelial defects. In case of persisting epithelial defects, cytokines, which are secreted from stromal keratocytes, may play a role in epithelial wound healing. Our aim was to analyze fibroblast growth factor basic (FGFb), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and transforming growth factor β1 (TGF-β1) concentration in the supernatant of keratocytes, after incubation with human serum (HS). Serum eye drops of 10 patients were prepared using the standards of the LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz, and were stored at - 80 °C. Primary human keratocytes were isolated from human corneoscleral rings using collagenase A (1 mg/ml) (n = 1) and were cultured in DMEM/Ham's culture medium with 10% fetal bovine serum (FBS). Thereafter, keratocyte cultures were incubated in 15 or 30% HS (in DMEM/F14 without FBS) and FGFb, HGF, KGF and TGF-β1 concentration was determined with an enzyme-linked immunoabsorbent assay (ELISA) from the supernatant of the culture after 24 hours. We used 15 or 30% HS without keratocytes (under the same storage conditions) as controls. HGF concentration was, for both HS concentrations, significantly higher in the supernatant of keratocytes, than in HS controls (without keratocytes) following 24 hours (p < 0.01). FGFb concentration was significantly increased in 30% HS with keratocytes compared to 30% HS without keratocytes after 24 hours (p < 0.01). TGF-β1 and KGF concentrations remained unchanged through keratocytes. HGF and FGFb concentrations increase in the supernatant of keratocytes, 24 hours after incubation with human serum. These concentration changes may play a role in wound healing of epithelial defects.

Synergetic Effect of α-Lipoic Acid with Keratinocyte Growth Factor on Protecting Alveolar Epithelial Type II Cells of Rat Fetus from Hyperoxia -Induced Injury

Aim: To explore the potential mechanism of the synergetic effect of α-lipoic acid with keratinocyte growth factor (KGF) on protecting alveolar epithelial type II cells (ATIICs) from hyperoxia-induced injury. Methods: Primary culture of ATIICs from the Sprague-Dawley rat fetuses was examined under room air and 95% of O₂. Various KGF concentrations (0 to 100 ng/mL) and 0.5 mM of α-lipoic were added into the cell culture. Levels of intracellular reactive oxygen species, necrosis, and proliferation of ATIICs were measured using flow cytometry, ELISA, and MTT assays, respectively. RT-PCR was performed to detect KGFR mRNA expression. Western blot was employed to detect the expression of KGFR, phospho-p53, HDAC1, and acetylated H3 and H4. Results: KGF promoted the proliferation and inhibited the apoptosis of ATIICs in room air or under temporary exposure to hyperoxia. However, the resistance of ATIICs to KGF was observed after prolonged exposure. Further investigation demonstrated that down-regulation of KGF receptor via activation of p53 and recruitment of HDAC1 induced by oxidative stress contributed to KGF resistance. This resistance could be attenuated by α-lipoic acid, a powerful antioxidant. Conclusion: Application of KGF combined with α-lipoic acid could inhibit KGF resistance to provide maximum protection to ATIICs from hyperoxic injury.

Expression of Keratinocyte Growth Factor in Goat Ovaries and Its Effects on Preantral Follicles Within Cultured Ovarian Cortex

The aims of this study were to evaluate the expression of keratinocyte growth factor (KGF) in goat ovaries and to study its effects on preantral follicle survival and development. The ovaries were used for immunohistochemistry or for in vitro culture for 1 or 7 days with KGF (0, 1, 10, 50, 100, 150, or 200 ng/mL). Noncultured (fresh control) and cultured ovarian slices were processed for histological analysis and transmission electron microscopy (TEM). The results showed that after 7 days of in vitro culture, all treatments had a significant reduction in the percentage of normal follicles compared with the fresh control. After 7 days of culture, the highest KGF concentrations (150 and 200 ng/mL) induced a significant reduction in the percentage of normal follicles compared with the tissues cultured in the absence (α-MEM(+) alone) or presence of 1, 10, and 50 ng/mL KGF. Transmission electron microscopy confirmed follicular integrity after 7 days of culture in 1 ng/mL KGF. In addition, compared with the fresh control, the percentage of growing follicles was significantly increased in all treatments after 1 or 7 days of culture. Immunohistochemical analyses showed the expression of KGF in oocytes and granulosa cells in all follicle developmental stages as well as in thecal and stromal cells. In conclusion, this study demonstrated that, at the lowest concentration (1 ng/mL), KGF maintained the ultrastructure of goat preantral follicles cultured in vitro for up to 7 days. Furthermore, the KGF protein was widely distributed in goat ovaries, especially in ovarian follicles.

Determining the protein drug release characteristics and cell adhesion to a PLLA or PLGA biodegradable polymer membrane

Biodegradable polymers are of interest for developing controlled protein drug delivery platforms. In this study, two poly (alpha-hydroxy) esters were formulated with Aerosol-OT, a surfactant stabilizer, to encapsulate the protein keratinocyte growth factor (KGF) for controlled release KGF is involved in a number of crucial biologic processes, most notably epithelial growth and repair. The concentration of KGF that caused a biological response in vitro was determined (optimally 10 ng/mL) and compared with the release of KGF from the two biodegradable polymer membrane formulations. Each polymer formulation released biologically relevant levels, 10 ng/mL of active KGF, although with different times release kinetics. The membrane composed of PLGA/AOT/KGF exhibited a faster release rate of KGF into solution after 120 h of degradation time than the release rate of the PLLA/AOT/KGF matrices. Cell seeding assays showed that both polymer matrices, when formulated with AOT, sustained cell growth. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was used to characterize the distribution of AOT and KGF through the polymer membrane. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Aqueous humor keratinocyte growth factor levels in patients with acute anterior uveitis.

Abstract Purpose Keratinocyte growth factor (KGF) is produced by cells of mesenchymal origin and has an important role in repairing tissues and possesses anti‐inflammatory properties. The aim of this study was to evaluate the levels of KGF in the aqueous humor in eyes with acute anterior uveitis (AAU). Methods A prospective, comparative control study. Aqueous humor was collected from 16 eyes of 16 patients with AAU. The level of KGF was determined with a commercially available enzyme‐linked immunoabsorbent assay kit. The control group comprised 16 aqueous samples from 16 patients about to undergo cataract surgery and without any other ocular or systemic diseases. Results The concentration of KGF in aqueous humor was markedly higher in patients with UUA than in control subjects (Mann‐Withney U test, P&lt; 0.001). The level of KGF was 38.06 ± 37.46 (mean ± SD) pg/ml in eyes with UUA and 3.50 ± 1.3 pg/ml in the eyes of the control group. Conclusion The aqueous humor KGF level is increased in eyes with AAU. These results imply that KGF is associated with the regulation of inflammation in patients with AAU.

Inhibition of proliferation of Pseudomonas aeruginosa by KGF in an experimental burn model using human cultured keratinocytes

Experimental models showed the ability of Pseudomonas aeruginosa to interact with epidermal keratinocytes [Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979;76(11):5665–8], stimulating these cells to produce specific peptides that start an immunological chain reaction in the epidermis [O’Connor NE, Mulliken JB, Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;(1):75–8]. The immune reaction causes the release of cytokines and growth factors. The objective of this study was to test whether the presence of keratinocyte growth factor (KGF) alters P. aeruginosa proliferation in an experimental burn model. Methods Human keratinocytes derived from neonatal foreskins were isolated and cultured following standard methods [Gallico III, GG, O’Connor NE, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311(7):448–51]. Some of these cells were genetically modified to produce KGF, and the other cells were supplemented with different KGF concentrations in the culture media. Both groups of keratinocytes were seeded in collagen matrices and cultured to form stratified epithelia. A hot plate was used to produce burn defects. Each matrix was inoculated with luminescent P. aeruginosa strain. Experiments were made using keratinocytes without KGF, keratinocytes supplemented with different concentrations of KGF, and keratinocytes genetically modified to produce KGF. Statistical analyses were made using Wilcoxon paired test. Results When KGF was added to P. aeruginosa in the presence of keratinocytes, bacterial growth was inhibited, and the same was observed when genetically modified keratinocytes were used. Conclusion Many studies have been done on KGF, where its known properties were defined as a mitogen for keratinocytes [Munster AM. Cultured skin for massive burns: a prospective, controlled trial. Ann Surg 1996;224(3):372–7]. This method allows for a qualitative and quantitative evaluation in real time of the bacterial growth in wound sites after bacterial inoculation. KGF was involved in the reduction of bacterial viability. However, as KGF alone did not produce any effect on P. aeruginosa, it seems to modulate the skin innate immune response.

Interaction of exogenous liposomal insulin‐like growth factor‐I cDNA gene transfer with growth factors on collagen expression in acute wounds

Growth factors have been shown to modulate the complex cascade of wound healing, however, interaction between different growth factors during dermal and epidermal regeneration is still not entirely defined. We have recently shown that exogenous liposomal gene transfer of cDNA results in physiologic expression and response in an acute wound. In the present study we determined the interaction between insulin-like growth factor-I (IGF-I), a mesenchymal growth factor, administered as liposomal cDNA, with other dermal and epidermal growth factors on collagen synthesis in an acute wound. Sprague-Dawley rats were given a scald burn to inflict an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus a beta-galactosidase containing plasmid (Lac Z [0.2 microg, vehicle]), or liposomes plus the IGF-I cDNA containing plasmid (2.2 microg) and Lac Z (0.2 microg). Immunological assays, histological and immunohistochemical techniques were used to determine growth factor concentration and different types of collagen (I, III, and IV) after IGF-I cDNA gene transfer. IGF-I cDNA transfer accelerated reepithelization and was associated with increased levels of IGF-I, fibroblast growth factor, keratinocyte growth factor, vascular endothelial cell growth factor, and platelet-derived growth factor protein expression. IGF-I cDNA had no effect on transforming growth factor-beta. IGF-I cDNA significantly increased type IV collagen while it had no effect on types I and III collagen. Exogenously administered IGF-I cDNA increased protein concentrations of keratinocyte growth factor, fibroblast growth factor, platelet-derived growth factor, and type IV collagen. We conclude that liposomal IGF-I gene transfer can accelerate wound healing without causing an increase in types I and III collagen expression.

275 Epithelial Repair in The Preterm Lung is Decreased in The Early Phase of Bronchopulmonary Dysplasia

Top of pageAbstract Background: In the early phases bronchopulmonary dysplasia (BPD) is characterized by epithelial and endothelial damage and inflammation. A recent study showed an absent or decreased concentration of keratinocyte growth factor, a factor contributing to epithelial repair, in early BPD (Danan C et al, Am J Respir Crit Care Med. 2002;165:1384–7). Aim: To investigate whether bronchoalveolar lavage fluid (BALF) from preterm infants who develop BPD has a decreased capacity to stimulate epithelial repair compared to BALF from infants who recover from respiratory distress syndrome (RDS). Methods: A standardized blind bronchoalveolar lavage was performed with 2x1 ml/kg saline in ventilated preterm infants with a gestational age <30 weeks on day 2 to 4 of life. Infants who recovered from RDS were all extubated within the first week of life. BPD was defined as oxygen requirement at 28 days of life. A bronchial epithelial cell line (BEAS) was grown to confluence and a scratch of known surface area was made in the epithelium (Geiser et al, Am J Physiol Lung Cell Mol Physiol 2000;279:L1184–90). BALF (or control medium without serum) was added to the cell cultures for 18 hours and the closure of the epithelial wound was assessed (expressed as % closure above control medium). Results: BALF from infants who developed BPD (n=13) had a significantly lower capacity to stimulate epithelial wound closure than BALF from infants who recovered from RDS (n=10): 25±5% versus 48±4.5% closure above control medium (p=0.02 by t-test) (mean±SEM). Conclusion: BALF on day 2 to 4 from preterm infants who develop BPD has a decreased capacity to stimulate epithelial repair in an in vitro assay compared to BALF from infants who recover from RDS. This suggests that besides epithelial injury also decreased epithelial repair plays a role in the early phase of BPD development. Further investigations are necessary to elucidate which growth factors or other factors are important for the decreased epithelial repair in the early phase of BPD development.

Secretion of keratinocyte growth factor by cultured human endometrial stromal cells is induced through a cyclic adenosine monophosphate-dependent pathway

Objective: To evaluate the effects of known modulators of endometrial function on the production of keratinocyte growth factor by endometrial stromal cells. Design: The effects of dibutyryl-cyclic adenosine monophosphate (db-cAMP), 12- O-tetradecanoylphorbol 13-acetate (TPA), ethinyl estradiol-17α (EE), and medroxyprogesterone acetate (MPA) on the secretion of keratinocyte growth factor by endometrial stromal cells were investigated. Setting: Research laboratory at a university medical school. Patient(s): Eleven endometrial specimens in the late proliferative phase. Intervention(s): Endometrial stromal cells were incubated for 24 hours with db-cAMP, TPA, EE, or MPA. Main Outcome Measure(s): The concentration of keratinocyte growth factor in the culture media was measured using an ELISA. Result(s): Small amounts of keratinocyte growth factor were detected in the culture media of unstimulated endometrial stromal cells. The production of keratinocyte growth factor by endometrial stromal cells was stimulated with db-cAMP in a dose-dependent manner. The stimulatory effect of db-cAMP was inhibited by Rp-adenosine 3′,5′-cyclic monophosphothioate. None of TPA, EE, nor MPA affected the keratinocyte growth factor production by these cells. Conclusion(s): These results suggest that a cAMP-dependent pathway may play an important role in the regulation of keratinocyte growth factor production by endometrial stromal cells. Keratinocyte growth factor secreted by endometrial stromal cells may be involved in the regeneration of the endometrium during the normal menstrual cycle and early pregnancy.

Evidence for the presence of keratinocyte growth factor (KGF) in human ovarian follicles.

The presence of keratinocyte growth factor (KGF) in human follicular fluid (FF) was investigated in a total of 145 FFs obtained during oocyte retrieval for in vitro fertilization (IVF) from 29 patients with no apparent endocrine disorders. The concentrations of KGF, estradiol, progesterone, testosterone and human chorionic gonadotropin (hCG) in FF were measured by enzyme-linked immunosorbent assay. FF samples contained relatively higher amounts of KGF (2194+/-87 pg/ml), whereas its concentrations in serum were below assay limit (<31.2 pg/ml). Concentrations of KGF in FF were positively correlated with both progesterone (r=0.311, p<0.0005) and testosterone (r=0.230, p<0.01) concentrations in FF. However, KGF concentrations were not significantly correlated with estradiol and hCG concentrations. KGF in FF was detected as a broad band (26-29 kD) by immunoblotting, the size being reduced by 7kD after N-glycosidase treatment. In an in vitro experiment, KGF suppressed the basal and hCG-stimulated progesterone production by cultured human luteinized granulosa cells. summary, we demonstrated the presence of KGF in human ovarian follicles, suggesting its possible role as a local factor in regulating human ovarian functions.

Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients.

To determine bronchoalveolar lavage (BAL) fluid concentrations of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), two potent growth factors for alveolar type II epithelial cells, in patients with acute respiratory distress syndrome (ARDS). Prospective study. An adult trauma/surgical intensive care unit in an urban teaching hospital. A total of 32 ventilated patients with pulmonary infiltrates prospectively identified with ARDS (n = 17) or without ARDS (n = 15), including eight patients with hydrostatic edema (HE), and ten nonventilated patients serving as controls. None. BAL was performed 2.88 days +/- 2.4, 3.5 days +/- 2.4, and 2.3 days +/- 2.2 after the lung insult in ARDS, HE, and other non-ARDS patients respectively (p = .32). KGF was detected in BAL fluid in 13 of the 17 ARDS patients (median, 31.6 pg/mL), in one patient with HE, and in none of other non-ARDS patients. In ARDS patients, detection of KGF in BAL was associated in BAL fluid with the detection of type III procollagen peptide (PIIIP), a biological marker of fibroproliferation. In ARDS patients, detection of KGF in BAL was associated with death (p = .02). HGF was detected in 15 ARDS patients (median, 855 pg/mL), in seven patients with HE (median, 294 pg/mL; p = .05 for the comparison with ARDS group), in six of other non-ARDS patients (median, 849 pg/mL; p = .32 with ARDS group). HGF concentrations were higher in nonsurvivors than in survivors (p = .01). None of the ten BAL of controls contained either KGF or HGF. KGF was detected almost exclusively in BAL fluid from ARDS patients and correlated with a poor prognosis in this group. In contrast, HGF was detected in the BAL fluid from a majority of patients with or without ARDS. Elevated HGF concentrations were associated with a poor prognosis in the overall group.

Hepatocyte growth factor and keratinocyte growth factor in the pulmonary edema fluid of patients with acute lung injury. Biologic and clinical significance.

Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are among the most potent mitogens identified for alveolar type II epithelial cells and may have other important functions in repair of the alveolar epithelium in acute lung injury (ALI). However, neither growth factor has been identified in the distal air spaces or plasma of patients with ALI. The goals of this study were to determine: (1) whether HGF and KGF are present in pulmonary edema fluid from patients with ALI and control patients with hydrostatic pulmonary edema; (2) whether HGF and KGF are biologically active in pulmonary edema; and (3) whether HGF or KGF levels are associated with clinical outcome. Pulmonary edema and plasma samples were obtained within 48 h of onset of acute pulmonary edema requiring mechanical ventilation in 26 patients with ALI and 11 control patients with hydrostatic edema. HGF and KGF concentrations were measured with enzyme-linked immunosorbent assays (ELISAs). The median (25th to 75th percentiles) concentration of HGF in pulmonary edema fluid was 21.4 (8.3 to 41.3) ng/ml in ALI and 6.6 (4.8 to 11.4) ng/ml in hydrostatic edema fluid (p < 0.01). The HGF concentration was 7-fold higher in the edema fluid than in the plasma of patients with ALI. In contrast, KGF was detected in low concentrations in edema fluid of patients with ALI and hydrostatic pulmonary edema; the concentration of KGF did not differ in ALI edema (0.6 [0.3 to 2.1] ng/ml) and hydrostatic edema fluid (0.2 [0.0 to 2.6] ng/ml) (p = NS). HGF and KGF were partly purified from four edema-fluid samples by heparin-Sepharose chromatography. Partly purified edema fluids were potent stimuli of DNA synthesis in cultured rat type II alveolar cells; addition of neutralizing antibodies to HGF and KGF attenuated this increase in DNA synthesis by 66% and 53%, respectively. Interestingly, higher edema-fluid levels of HGF were associated with higher mortality in patients with ALI. These novel results show that HGF and KGF are active in the alveolar space early in ALI, probably mediating early events in lung repair, and that increased levels of HGF in edema fluid may have prognostic value early in ALI.