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.
Highlights
It is estimated that more than 356 million people worldwide have type 2 diabetes mellitus (NIDDM) and 15–25% of these patients will develop diabetic foot ulcerations (DFU) during their lifetime [1]
We recently showed that patients with NIDDM have lower levels of CAMP (LL-37) and DEFB4 (HBD-2) gene expression in peripheral blood cells [15] which probably is translated into susceptibility to infectious diseases
Results showed that keratinocyte-conditioned medium (KCM) from DFUs induced keratinocyte migration mainly at 48 hours post-stimulation (Figure 6A–B), worthwhile to mention that migrating cells had an evident invasive phenotype, which is seen during in vivo wound healing (Figure 6B, arrow heads)
Summary
It is estimated that more than 356 million people worldwide have type 2 diabetes mellitus (NIDDM) and 15–25% of these patients will develop diabetic foot ulcerations (DFU) during their lifetime [1]. Peripheral neuropathy, vascular insufficiency and diminished immune response are major factors in the development of skin ulceration and infection in these patients [2]. Is estimated that 50% of these chronic ulcerations will become infected by several microorganisms, which together with vascular insufficiency and poor wound healing lead to high hospitalization rates and increased the probability of lower extremity amputation [3]. The use of new molecules that have antimicrobial, angiogenic and wound healing activity are highly desirable for the DFU treatment
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