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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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Antimicrobial peptides gene expression in cell cultures from DFU biopsies.Primary cells cultures from diabetic foot ulcers biopsies were performed and stimulated with L-isoleucine (50 µg/ml) and 1,25(OH)2D3 (10−9 M) for 18 h and DEFB4 (A) and CAMP (B) gene expression was measured by qPCR. Data are expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15. *p<0.05; ** p<0.01.
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pone-0111355-g002: Antimicrobial peptides gene expression in cell cultures from DFU biopsies.Primary cells cultures from diabetic foot ulcers biopsies were performed and stimulated with L-isoleucine (50 µg/ml) and 1,25(OH)2D3 (10−9 M) for 18 h and DEFB4 (A) and CAMP (B) gene expression was measured by qPCR. Data are expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15. *p<0.05; ** p<0.01.

Mentions: Real time qPCR gene expression analysis showed that L-isoleucine was able to induce DEFB4 gene expression varying from 1.223 to 56.10-fold versus no treated cells. Nevertheless, 73.3% of same primary cultures responded to 1,25(OH)2D3 stimulation, increasing gene expression of this peptide within ranges from 1.2 to 51.8-fold with p value of <0.05 (Figure 2A). In regards with CRAMP, when primary cell cultures were stimulated with L-isoleucine, only six cultures responded to the treatment increasing modestly the gene expression whereas 80% of these cultures responded to 1,25(OH)2D3 increasing the gene expression form 1.20 to 118.80-fold when compared with untreated cell cultures (Figure 2B). These results demonstrate that the gene expression of each AMPs is restore under the treatment with 1,25(OH)2D3 in primary cultures of skin biopsies from DFU.


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)

Antimicrobial peptides gene expression in cell cultures from DFU biopsies.Primary cells cultures from diabetic foot ulcers biopsies were performed and stimulated with L-isoleucine (50 µg/ml) and 1,25(OH)2D3 (10−9 M) for 18 h and DEFB4 (A) and CAMP (B) gene expression was measured by qPCR. Data are expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15. *p<0.05; ** p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111355-g002: Antimicrobial peptides gene expression in cell cultures from DFU biopsies.Primary cells cultures from diabetic foot ulcers biopsies were performed and stimulated with L-isoleucine (50 µg/ml) and 1,25(OH)2D3 (10−9 M) for 18 h and DEFB4 (A) and CAMP (B) gene expression was measured by qPCR. Data are expressed as median ± interquartile range. Statistics were calculated by Kruskal-Wallis test. In each experimental group, n = 15. *p<0.05; ** p<0.01.
Mentions: Real time qPCR gene expression analysis showed that L-isoleucine was able to induce DEFB4 gene expression varying from 1.223 to 56.10-fold versus no treated cells. Nevertheless, 73.3% of same primary cultures responded to 1,25(OH)2D3 stimulation, increasing gene expression of this peptide within ranges from 1.2 to 51.8-fold with p value of <0.05 (Figure 2A). In regards with CRAMP, when primary cell cultures were stimulated with L-isoleucine, only six cultures responded to the treatment increasing modestly the gene expression whereas 80% of these cultures responded to 1,25(OH)2D3 increasing the gene expression form 1.20 to 118.80-fold when compared with untreated cell cultures (Figure 2B). These results demonstrate that the gene expression of each AMPs is restore under the treatment with 1,25(OH)2D3 in primary cultures of skin biopsies from DFU.

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