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1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Gonzalez-Curiel I, Trujillo V, Montoya-Rosales A, Rincon K, Rivas-Calderon B, deHaro-Acosta J, Marin-Luevano P, Lozano-Lopez D, Enciso-Moreno JA, Rivas-Santiago B - PLoS ONE (2014)

Bottom Line: Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant.These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration.In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico; Department of Immunology, Faculty of Medicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico.

ABSTRACT
Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

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Keratinocyte-conditioned medium mediate proliferation and keratinocyte migration.Vitamin D- treated KCM from DFU were tested for cell migration induction. HaCaT cells were grown to confluence on 24-well tissue culture plates. Then each well was denuded by half from cells and re-coated with fibronectin, then we measured wound closure at 24, 48 and 72 hours post-incubation using as controls Epithelial Growth Factor (EGF), LL-37 or HBD-2, and the KCM with blocking antibodies either for HBD-2 (7A) or for LL-37 (7B). BrdU incorporation was used as an index of cellular proliferation (7C). Data are expressed as a percentage of the BrdU-positive cells (BrdU incorporation). Each value represents the median ± interquartile range. Values are the mean of 3 independent experiments. Asterisks show the treated groups with statistical difference when compared with KCM for each time point. * p<0.05, ** p<0.01.
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pone-0111355-g007: Keratinocyte-conditioned medium mediate proliferation and keratinocyte migration.Vitamin D- treated KCM from DFU were tested for cell migration induction. HaCaT cells were grown to confluence on 24-well tissue culture plates. Then each well was denuded by half from cells and re-coated with fibronectin, then we measured wound closure at 24, 48 and 72 hours post-incubation using as controls Epithelial Growth Factor (EGF), LL-37 or HBD-2, and the KCM with blocking antibodies either for HBD-2 (7A) or for LL-37 (7B). BrdU incorporation was used as an index of cellular proliferation (7C). Data are expressed as a percentage of the BrdU-positive cells (BrdU incorporation). Each value represents the median ± interquartile range. Values are the mean of 3 independent experiments. Asterisks show the treated groups with statistical difference when compared with KCM for each time point. * p<0.05, ** p<0.01.

Mentions: Subsequently, we measured the percentage of wound closure at 24, 48 and 72 hours post-incubation. Results showed that KCMs increase wound closure reaching 40%, whereas none-treated cultures reached 18–22%. Positive control (EGF) reached percentages above 55%. To evaluate whether this activity is AMPs-dependent, we blocked LL-37 and HBD-2 with specific antibodies. Results showed that the use of blocking antibodies decreases wound closure for both LL-37 and HBD-2, albeit is more evident for the case of LL-37 (Figure 7A and 7B).


1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Gonzalez-Curiel I, Trujillo V, Montoya-Rosales A, Rincon K, Rivas-Calderon B, deHaro-Acosta J, Marin-Luevano P, Lozano-Lopez D, Enciso-Moreno JA, Rivas-Santiago B - PLoS ONE (2014)

Keratinocyte-conditioned medium mediate proliferation and keratinocyte migration.Vitamin D- treated KCM from DFU were tested for cell migration induction. HaCaT cells were grown to confluence on 24-well tissue culture plates. Then each well was denuded by half from cells and re-coated with fibronectin, then we measured wound closure at 24, 48 and 72 hours post-incubation using as controls Epithelial Growth Factor (EGF), LL-37 or HBD-2, and the KCM with blocking antibodies either for HBD-2 (7A) or for LL-37 (7B). BrdU incorporation was used as an index of cellular proliferation (7C). Data are expressed as a percentage of the BrdU-positive cells (BrdU incorporation). Each value represents the median ± interquartile range. Values are the mean of 3 independent experiments. Asterisks show the treated groups with statistical difference when compared with KCM for each time point. * p<0.05, ** p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206472&req=5

pone-0111355-g007: Keratinocyte-conditioned medium mediate proliferation and keratinocyte migration.Vitamin D- treated KCM from DFU were tested for cell migration induction. HaCaT cells were grown to confluence on 24-well tissue culture plates. Then each well was denuded by half from cells and re-coated with fibronectin, then we measured wound closure at 24, 48 and 72 hours post-incubation using as controls Epithelial Growth Factor (EGF), LL-37 or HBD-2, and the KCM with blocking antibodies either for HBD-2 (7A) or for LL-37 (7B). BrdU incorporation was used as an index of cellular proliferation (7C). Data are expressed as a percentage of the BrdU-positive cells (BrdU incorporation). Each value represents the median ± interquartile range. Values are the mean of 3 independent experiments. Asterisks show the treated groups with statistical difference when compared with KCM for each time point. * p<0.05, ** p<0.01.
Mentions: Subsequently, we measured the percentage of wound closure at 24, 48 and 72 hours post-incubation. Results showed that KCMs increase wound closure reaching 40%, whereas none-treated cultures reached 18–22%. Positive control (EGF) reached percentages above 55%. To evaluate whether this activity is AMPs-dependent, we blocked LL-37 and HBD-2 with specific antibodies. Results showed that the use of blocking antibodies decreases wound closure for both LL-37 and HBD-2, albeit is more evident for the case of LL-37 (Figure 7A and 7B).

Bottom Line: Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant.These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration.In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico; Department of Immunology, Faculty of Medicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico.

ABSTRACT
Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Show MeSH
Related in: MedlinePlus