Diabetes Mellitus (DM) is a chronic, severe disease rapidly increasing in incidence and prevalence and is associated with numerous complications. Patients with DM are at high risk of developing diabetic foot ulcers (DFU) that often lead to lower limb amputations, long term disability, and a shortened lifespan. Despite this, the effects of DM on human foot skin biology are largely unknown. Thus, the focus of this study was to determine whether DM changes foot skin biology predisposing it for healing impairment and development of DFU. Foot skin samples were collected from 20 patients receiving corrective foot surgery and, using a combination of multiple molecular and cellular approaches we performed comparative analyses of non-ulcerated non-neuropathic diabetic foot skin (DFS) and healthy non-diabetic foot skin (NFS). MicroRNA (miR) profiling of laser captured epidermis and primary dermal fibroblasts from both DFS and NFS samples identified 5 miRs de-regulated in the epidermis of DFS though none reached statistical significance. MiR-31-5p and miR-31-3p were most profoundly induced. Although none were significantly regulated in diabetic fibroblasts, miR-29c-3p showed a trend of up-regulation, which was confirmed by qPCR in a prospective set of 20 skin samples. Gene expression profiling of full thickness biopsies identified 36 de-regulated genes in DFS (>2 fold-change, unadjusted p-value ≤ 0.05). Of this group, three out of seven tested genes were confirmed by qPCR: SERPINB3 was up-regulated whereas OR2A4 and LGR5 were down-regulated in DFS. However no morphological differences in histology, collagen deposition, and number of blood vessels or lymphocytes were found. No difference in proliferative capacity was observed by quantification of Ki67 positive cells in epidermis. These findings suggest DM causes only subtle changes to foot skin. Since morphology, mRNA and miR levels were not affected in a major way, additional factors, such as neuropathy, vascular complications, or duration of DM, may further compromise tissue’s healing ability leading to development of DFUs.
Overall design: Total RNA including the miRNA fraction was extracted from the samples using the QIAGEN miRNeasy mini kit and following the manufacturer’s instructions. The RNA quality was assessed using the AGILENT bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) to estimate the RNA integrity number (RIN). Samples with a RIN higher than 5 were used for mRNA profiling as described below. We previously described methods for tissue specimen preparation and hybridization. All processing and analysis of microarrays utilized standard protocols at the University of Miami Microarray Core Facility. Briefly, between 100 to 300 ng of total RNA was reverse transcribed, amplified, then the sense strand cDNA synthesized, labeled, and hybridized on arrays. The amplified, fragmented and biotin-labeled cDNAs were hybridized to the Affymetrix GeneChip Human Gene 2.0 ST microarray according to the manufacturer’s recommendations. Arrays were washed and stained using Affymetrix Fluidic stations 450 and scanned using Affymetrix GeneChip scanner 3000 7G. Image analysis was performed using the Affymetrix Command Console Software (AGCC). Resulting CEL files was imported into Expression Console™ Software (Affymetrix, Santa Clara, CA, USA) and underwent gene level normalization and signal summarization. The output files from this step were imported in Transcriptome Analysis Console (TAC) 2.0 Software (Affymetrix, Santa Clara, CA, USA) to identify differentially expressed genes and carry out clustering analysis. Only genes with a p-value lower than 0.05 and a fold-change greater than 2 were confirmed by qPCR in a prospective set of 10 NFS and 10 DFS.
Less...