Horacio Ramirez

Comparative Genomic, MicroRNA, and Tissue Analyses Reveal Subtle Differences between Non-Diabetic and Diabetic Foot Skin

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.

Integrative analysis of miRNA and mRNA paired expression profiling of primary fibroblast derived from diabetic foot ulcers reveals multiple impaired cellular functions.

Diabetic foot ulcers (DFUs) are one of the major complications of diabetes. Its molecular pathology remains poorly understood, impeding the development of effective treatments. Although it has been established that multiple cell types, including fibroblasts, keratinocytes, macrophages, and endothelial cells, all contribute to inhibition of healing, less is known regarding contributions of individual cell type. Thus, we generated primary fibroblasts from nonhealing DFUs and evaluated their cellular and molecular properties in comparison to nondiabetic foot fibroblasts (NFFs). Specifically, we analyzed both micro‐RNA and mRNA expression profiles of primary DFU fibroblasts. Paired genomic analyses identified a total of 331 reciprocal miRNA–mRNA pairs including 21 miRNAs (FC > 2.0) along with 239 predicted target genes (FC > 1.5) that are significantly and differentially expressed. Of these, we focused on three miRNAs (miR‐21‐5p, miR‐34a‐5p, miR‐145‐5p) that were induced in DFU fibroblasts as most differentially regulated. The involvement of these microRNAs in wound healing was investigated by testing the expression of their downstream targets as well as by quantifying cellular behaviors in prospectively collected and generated cell lines from 15 patients (seven DFUF and eight NFF samples). We found large number of downstream targets of miR‐21‐5p, miR‐34a‐5p, miR‐145‐5p to be coordinately regulated in mRNA profiles, which was confirmed by quantitative real‐time PCR. Pathway analysis on paired miRNA–mRNA profiles predicted inhibition of cell movement and cell proliferation, as well as activation of cell differentiation and senescence in DFU fibroblasts, which was confirmed by cellular assays. We concluded that induction of miR‐21‐5p, miR‐34a‐5p, miR‐145‐5p in DFU dermal fibroblasts plays an important role in impairing multiple cellular functions, thus contributing to overall inhibition of healing in DFUs.

A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers

A bioengineered bilayered living cellular construct promotes ulcer healing by modulating inflammation, stimulating wound edge keratinocytes, and attenuating Wnt/β-catenin signaling to activate an acute wound response. Activating healing in chronic wounds Effective therapies for chronic venous leg ulcers (VLUs) remain elusive, in part, because of the incomplete understanding of the pathophysiology of nonhealing wounds. Stone et al. conducted a postmarket clinical trial using transcriptomics to understand the mechanisms of action of an FDA-approved bilayered living cellular construct (BLCC) in nonhealing VLUs. After 1 week of BLCC treatment in addition to standard of care compression therapy, nonhealing VLUs showed changes in inflammation and gene expression characteristic of acutely healing wounds. This study provides mechanistic insight into how the acute healing process can be activated by a cell therapy in chronic nonhealing wounds. Chronic nonhealing venous leg ulcers (VLUs) are widespread and debilitating, with high morbidity and associated costs; about

Epithelialization in Wound Healing: A Comprehensive Review

15 billion is spent annually on the care of VLUs in the United States. Despite this, there is a paucity of treatments for VLUs because of the lack of pathophysiologic insight into ulcer development as well as the lack of knowledge regarding biologic actions of existing VLU-targeted therapies. The bioengineered bilayered living cellular construct (BLCC) skin substitute is a U.S. Food and Drug Administration–approved biologic treatment for healing VLUs. To elucidate the mechanisms through which the BLCC promotes healing of chronic VLUs, we conducted a clinical trial (NCT01327937) in which patients with nonhealing VLUs were treated with either standard of care (compression therapy) or the BLCC together with standard of care. Tissue was collected from the VLU edge before and 1 week after treatment, and the samples underwent comprehensive microarray mRNA and protein analyses. Ulcers treated with the BLCC skin substitute displayed three distinct transcriptomic patterns, suggesting that BLCC induced a shift from a nonhealing to a healing tissue response, involving modulation of inflammatory and growth factor signaling, keratinocyte activation, and attenuation of Wnt/β-catenin signaling. In these ways, BLCC application orchestrated a shift from the chronic nonhealing ulcer microenvironment to a distinctive healing milieu resembling that of an acute, healing wound. Our findings provide in vivo evidence in VLU patients of pathways that can be targeted in the design of new therapies to promote healing of chronic VLUs.