Molecular characterization of chronic cutaneous wounds reveals subregion- and wound type-specific differential gene expression.

A limited understanding of the pathology underlying chronic wounds has hindered the development of effective diagnostic markers and pharmaceutical interventions. This study aimed to elucidate the molecular composition of various common chronic ulcer types to facilitate drug discovery strategies. We conducted a comprehensive analysis of leg ulcers (LUs), encompassing venous and arterial ulcers, foot ulcers (FUs), pressure ulcers (PUs), and compared them with surgical wound healing complications (WHCs). To explore the pathophysiological mechanisms and identify similarities or differences within wounds, we dissected wounds into distinct subregions, including the wound bed, border, and peri-wound areas, and compared them against intact skin. By correlating histopathology, RNA sequencing (RNA-Seq), and immunohistochemistry (IHC), we identified unique genes, pathways, and cell type abundance patterns in each wound type and subregion. These correlations aim to aid clinicians in selecting targeted treatment options and informing the design of future preclinical and clinical studies in wound healing. Notably, specific genes, such as PITX1 and UPP1, exhibited exclusive upregulation in LUs and FUs, potentially offering significant benefits to specialists in limb preservation and clinical treatment decisions. In contrast, comparisons between different wound subregions, regardless of wound type, revealed distinct expression profiles. The pleiotropic chemokine-like ligand GPR15L (C10orf99) and transmembrane serine proteases TMPRSS11A/D were significantly upregulated in wound border subregions. Interestingly, WHCs exhibited a nearly identical transcriptome to PUs, indicating clinical relevance. Histological examination revealed blood vessel occlusions with impaired angiogenesis in chronic wounds, alongside elevated expression of genes and immunoreactive markers related to blood vessel and lymphatic epithelial cells in wound bed subregions. Additionally, inflammatory and epithelial markers indicated heightened inflammatory responses in wound bed and border subregions and reduced wound bed epithelialization. In summary, chronic wounds from diverse anatomical sites share common aspects of wound pathophysiology but also exhibit distinct molecular differences. These unique molecular characteristics present promising opportunities for drug discovery and treatment, particularly for patients suffering from chronic wounds. The identified diagnostic markers hold the potential to enhance preclinical and clinical trials in the field of wound healing.

Genomic Biomarkers Can Provide a Deeper Understanding of Recurrent Pressure Injuries.

OBJECTIVE: To identify genetic biomarkers predisposing individuals with spinal cord injury (SCI) to recurrent pressure injuries (PIs). METHODS: Repeated measures of the transcriptome profile of veterans with SCI at three Veterans Spinal Cord Injuries and Disorders Centers. Exclusion criteria included having significant active systemic disease at time of enrollment. Researchers obtained comprehensive profiles of clinical and health factors and demographic information relevant to PI history at enrollment and at each follow-up visit by reviewing patients' medical charts. Whole blood samples were collected at 6- to 12-month intervals for 2 to 4 years. In addition to DNA profiling with whole genome sequencing of the patients, RNA sequencing was performed to assess pathways associated with PI risk. RESULTS: Whole genome sequencing analysis identified 260 genes that showed increased prevalence of single-nucleotide variations in exonic regions with high (>20) combined annotation-dependent depletion scores between persons with high versus low intramuscular adipose tissue levels when cross-referenced with persons who had recurrent PIs. Gene set enrichment analysis using Hallmark and KEGG (Kyoto Encyclopedia of Genes and Genomes) gene sets of these candidate genes revealed enrichment in genes encoding proteins involved in fatty acid metabolism (P < .01). Further, RNA sequencing revealed upregulated activity in biological senescence pathways and downregulated activity in antimicrobial protection pathways. CONCLUSIONS: Genomic biomarkers may complement electronic health records to support management of complex interactive health issues such as risk of recurrent PIs in people with SCI. These findings may also be leveraged for homogeneous phenotypic grouping of higher-risk individuals.

Therapeutic Effects and Underlying Mechanism of SOCS-com Gene-Transfected ADMSCs in Pressure Ulcer Mouse Models.

Although the proportion of ulcer patients with medical problems among the elderly has increased with the extension of human life expectancy, treatment efficiency is drastically low, incurring substantial social costs. MSCs have independent regeneration potential, making them useful in clinical trials of difficult-to-treat diseases. In particular, ADMSCs are promising in the stem cell therapy industry as they can be obtained in vast amounts using non-invasive methods. Furthermore, studies are underway to enhance the regeneration potential of ADMSCs using cytokines, growth factors, and gene delivery to generate highly functional ADMSCs. In this study, key regulators of wound healing, SOCS-1, -3, and -5, were combined to maximize the regenerative potential of ADMSCs in pressure ulcer treatments. After transfecting SOCS-1, -3, -5, and SOCS-com into ADMSCs using a non-viral method, the expression of the inflammatory factors TNF-alpha, INF-gamma, and IL-10 was confirmed. ADMSCs transfected with SOCS-com showed decreased overall expression of inflammatory factors and increased expression of anti-inflammatory factors. Based on these results, we implanted ADMSCs transfected with SOCS-com into a pressure ulcer mouse model to observe their subsequent wound-healing effects. Notably, SOCS-com improved wound closure in ulcers, and reconstruction of the epidermis and dermis was observed. The healing mechanism of ADMSCs transfected with SOCS-com was examined by RNA sequencing. Gene analysis results confirmed that expression changes occurred in genes of key regulators of wound healing, such as chemokines, MMP-1, 9, CSF-2, and IL-33, and that such genetic changes enhanced wound healing in ulcers. Based on these results, we demonstrate the potential of ADMSCs transfected with SOCS-com as an ulcer treatment tool.

Changes in gene expression in pressure ulcers debrided by different approaches - a pilot study.

Pressure ulcers (PUs), also known as pressure injuries, are chronic wounds that represent potential lifelong complications. Pressure ulcers of a deep category (III and IV) are often indicated for surgical treatment - debridement and surgical reconstruction. Sharp surgical debridement is widely used in the debridement of PUs; however, the Versajet® hydrosurgery system is becoming an increasingly popular tool for tangential excision in surgery due to its numerous advantages. This work focused on the expression of selected genes, especially those associated with oxidative stress, in PUs debrided by two approaches - sharp surgical debridement and debridement using Versajet® hydrosurgery system. Expression of following genes was evaluated: NFE2L2, ACTA2, NFKB1, VEGFA, MKI67, HMOX1, HMOX2, HIF1A, and SOD2. ACTB and PSMB were used as housekeeping genes. So far, five patients have been enrolled in the study. Preliminary results suggest no significant difference in gene expression with different pressure ulcer treatment approaches except NFE2L2, despite the macroscopic differences. However, the results revealed correlations between the expression of some genes, namely HIF1A and SOD2, VEGFA and SOD2 and VEGFA and HIF1A. These results may indicate a connection between hypoxia, oxidative stress, pressure ulcer healing processes and angiogenesis.

Polymorphism analysis of candidate risk genes for pressure injuries in older Japanese patients: A cross-sectional study at a long-term care hospital.

Advances in patient care for pressure injuries (PIs) have reduced the prevalence of PIs in Japan, although not in recent years. Several single-nucleotide polymorphisms (SNPs) have been identified in genes potentially associated with PIs. However, individual variance among PI risks require targeted investigations that may lead to the identification of PI susceptibilities or preventive care options that directly influence PI development pathways. This cross-sectional study examined the association between PIs and SNPs in genes related to tissue tolerance in patients in a long-term care hospital in Japan. A total of 178 participants (130 control, 20 with superficial PI history, and 28 with deep PI history) were enrolled in this study of eight SNPs in hypoxia inducible factor 1 subunit alpha (HIF1A), vascular endothelial growth factor C (VEGFC), heat shock protein 90 alpha family class A member 1 (HSP90AA1), myostatin (MSTN), and vitamin D receptor (VDR). The primary outcome was a history of superficial and deep PIs in the last 6 months. SNPs were examined by real-time polymerase chain reaction, followed by multivariate logistic regression analyses of the associations between the SNPs and PI history. The results showed a significant association between VEGFC rs1485766 and the history of superficial PIs (odds ratio = 2.95; 95% confidence interval = 1.07-8.11; p = 0.04). Stratified analysis using the Braden Scale (≤14) indicated a significant association between HIF1A rs11549465 and deep PIs (p = 0.04). Our study demonstrated that VEGFC rs1485766 and HIF1A rs11549465 were associated with superficial and deep PI susceptibilities, respectively.

Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization.

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3-7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.

[Expression and significance of hypoxia-inducible factor-1α and vascular endothelial growth factor and receptor 2 in stage 3 pressure injury of rats].

OBJECTIVE: To analyze the expression and relationship of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR) in local skin tissues of pressure injury and investigate the possible mechanism of stage 3 pressure injury refractory wound. METHODS: Forty male SD rats were randomly divided into normal control group, compressed 3 d, 5 d, 7 d, and 9 d groups. Stage 3 pressure injury animal model were established by magnet compression. The morphology of skin was observed by HE staining. The expression of VEGF was detected by immunohistochemistry. The expression levels of HIF-1α, VEGF and KDR protein in skin tissue were detected by Western blot. One-way analysis of variance and LSD test were performed on the data. RESULTS: ①The HE results showed that compared with the normal control group, the epidermis of the compressed group was gradually thickened, the number of blood vessels was decreased, the collagen arrangement disordered and inflammatory cells infiltration were increased. ②Immunohistochemical results showed that the expression of VEGF protein in the 3 d group was significantly higher than that in the normal control group (P＜0.01). The expression of VEGF protein in the skin tissue of 5 d, 7 d and 9 d groups was lower than that in normal control group (P＜0.05). WB results were consistent with immunohistochemistry results. ③WB results showed that the expression of HIF-1α in the skin tissues of the rats in 3 d, 5 d and 7 d groups was higher than that in the normal control group (P＜0.01 or P＜0.05). The expression of KDR protein was lower than that of the normal control group (P＜0.05 or P＜0.01). CONCLUSION: HIF-1α mediated reduction of VEGF and KDR protein expression and decreased tissue angiogenesis may be one of the important causes of chronic dysfunction of stage 3 pressure injury.

Human embryonic stem cell-derived exosomes promote pressure ulcer healing in aged mice by rejuvenating senescent endothelial cells.

BACKGROUND: Angiogenesis, as an endogenous repair mechanism, plays crucial roles in wound healing and tissue regeneration. However, this process is impaired in the elderly due to aging-related vascular endothelial dysfunction. This study was aimed to explore the pro-angiogenic effects of exosomes from human embryonic stem cells (ESC-Exos) in aged mice of pressure-induced ulcer model and the underlying mechanism. METHODS: Pressure ulcer wounds were created on the back of D-galactose-induced aging mice. ESC-Exos were locally applied onto the wound beds, with PBS as control. The effects of ESC-Exos on wound healing were analyzed by measuring wound closure rates, histological and immunofluorescence analyses. Then, the anti-aging effect of ESC-Exos on vascular endothelial cells was tested in an in vitro D-galactose-induced HUVEC senescence model. RESULTS: ESC-Exos could accelerate wound closure and enhance angiogenesis, and the senescence of vascular endothelial cells was significantly ameliorated after ESC-Exos treatment. In vitro, ESC-Exos could rejuvenate the senescence of endothelial cells and recover compromised proliferation, migratory capacity, and tube formation. This recovery was Nrf2-activation-dependent, since cotreatment with Nrf2 inhibitor Brusatol could abolish the rejuvenative effects of ESC-Exos. Further study revealed that miR-200a was highly enriched in ESC-Exos and played a crucial role in ESC-Exos-mediated rejuvenation through downregulating Keap1, which negatively regulates Nrf2 expression. CONCLUSIONS: ESC-Exos ameliorate endothelial senescence by activating Nrf2 and recover aging-related angiogenic dysfunction, thereby accelerating wound healing in aged mice. ESC-Exos might be a natural nano-biomaterial for aging-related diseases therapy.

Hydrogen gas inhalation protects against cutaneous ischaemia/reperfusion injury in a mouse model of pressure ulcer.

Pressure ulcer formation depends on various factors among which repetitive ischaemia/reperfusion(I/R) injury plays a vital role. Molecular hydrogen (H2 ) was reported to have protective effects on I/R injuries of various internal organs. In this study, we investigated the effects of H2 inhalation on pressure ulcer and the underlying mechanisms. H2 inhalation significantly reduced wound area, 8-oxo-dG level (oxidative DNA damage) and cell apoptosis rates in skin lesions. H2 remarkably decreased ROS accumulation and enhanced antioxidant enzymes activities by up-regulating expression of Nrf2 and its downstream components in wound tissue and/or H2 O2 -treated endothelia. Meanwhile, H2 inhibited the overexpression of MCP-1, E-selectin, P-selectin and ICAM-1 in oxidant-induced endothelia and reduced inflammatory cells infiltration and proinflammatory cytokines (TNF-α, IL-1, IL-6 and IL-8) production in the wound. Furthermore, H2 promoted the expression of pro-healing factors (IL-22, TGF-ß, VEGF and IGF1) and inhibited the production of MMP9 in wound tissue in parallel with acceleration of cutaneous collagen synthesis. Taken together, these data indicated that H2 inhalation suppressed the formation of pressure ulcer in a mouse model. Molecular hydrogen has potentials as a novel and alternative therapy for severe pressure ulcer. The therapeutic effects of molecular hydrogen might be related to its antioxidant, anti-inflammatory, pro-healing actions.

A functional polymorphism at miR­491­5p binding site in the 3'UTR of MMP9 gene confers increased risk for pressure ulcers after hip fracture.

The roles of matrix metalloproteinase (MMP)9 in the control of pressure ulcers (PU) after hip fracture as well as how the rs1056629 in MMP9 3'UTR compromises the interaction between MMP9 and miR­491 were explored. Online miRNA database (http://www.bioguo.org) was utilized to explore gene polymorphism in MMP9 3'UTR that might break the interaction between MMP9 and miRNA. Luciferase assay was utilized to confirm the miRNA targeted MMP9. Real­time PCR, western blot analysis and immunohistochemistry were carried out to understand the roles of MMP9 in PU as well as how rs1056629 in MMP9 3'UTR compromises the interaction between MMP9 and miR­491. rs1056629 in MMP9 3'UTR that compromised the interaction between MMP9 and four miRNAs including miR­194­3p, miR­491, miR­1915­3p and miR­941, and only miR­491 among miR­194­3p, miR­491, miR­1915­3p and miR­941 decreased luciferase activity of wild­type MMP9 3'UTR, and luciferase activities of mutant­3 and mutant­4 MMP9 3'UTR in miR­491 overexpressing cells was comparable with scramble control. miR­194­3p, miR­491, miR­1915­3p and miR­941 levels in PU group was comparable with healthy control, and miR­194­3p, miR­491, miR­1915­3p and miR­941 in subjects carrying AA genotype was similar with those in AC and CC groups. MMP9 mRNA and protein, and histology score in subjects with PU were much higher, and were also much higher in AA group. Only miR­491 mimic among miR­194­3p, miR­491, miR­1915­3p and miR­941 mimics downregulated the MMP9 level, and only miR­491 inhibitor among miR­194­3p, miR­491, miR­1915­3p and miR­941 inhibitors upregulated the MMP9 level. Our study indicated that rs1056629 polymorphism could be a novel biomarker for predicting the occurrence of PU after a hip fracture.

Laser photobiomodulation in pressure ulcer healing of human diabetic patients: gene expression analysis of inflammatory biochemical markers.

Pressure ulcers (PU) are wounds located mainly on bone surfaces where the tissue under pressure suffers ischemia leading to cellular lesion and necrosis , its causes and the healing process depend on several factors. The aim of this study was evaluating the gene expression of inflammatory/reparative factors: IL6, TNF, VEGF, and TGF, which take part in the tissue healing process under effects of low-level laser therapy (LLLT). In order to perform lesion area analysis, PUs were photographed and computer analyzed. Biochemical analysis was performed sa.mpling ulcer border tissue obtained through biopsy before and after laser therapy and quantitative real-time PCR (qRT-PCR) analysis. The study comprised eight individuals, mean age sixty-two years old, and sacroiliac and calcaneous PU, classified as degree III and IV according to the National Pressure Ulcer Advisory Panel (NPUAP). PUs were irradiated with low-level laser (InGaAIP, 100 mW, 660 nm), energy density 2 J/cm2, once a day, with intervals of 24 h, totaling 12 applications. The lesion area analysis revealed averaged improvement of the granulation tissue size up to 50% from pre- to post-treatment. qRT-PCR analysis revealed that IL6 values were not significantly different before and after treatment, TNF gene expression was reduced, and VEFG and TGF-ß gene expression increased after treatment. After LLLT, wounds presented improvement in gross appearance, with increase in factors VEFG and TGF-ß, and reduction of TNF; despite our promising results, they have to be analyzed carefully as this study did not have a control group.

Rs739837 Polymorphism in MiR-885-3p Binding Site Within 3'-Untranslated Region of Vitamin D Receptor is Associated with a Decreased Risk of Pressure Ulcers.

BACKGROUND/AIMS: Accumulative evidence has shown that miR-885-3p plays a crucial role in human carcinogenesis. From miRNA database, we also found that rs739837 polymorphism in miR-885-3p binding site within 3'-untranslated region of Vitamin D receptor (VDR), compromising the suppressive effect of miR-885-3p on VDR. Moreover, Vitamin D is involved in controlling the cell immune response and may play a role in pressure ulcers development. However, whether this polymorphism is actually linked with pressure ulcers remains unclear. The aim of the present study was to investigate the potential association between the rs739837 polymorphism and pressure ulcers, and to explore molecular mechanism of VDR in pressure ulcers. METHODS: Luciferase assays were performed to validate the relationship between miR-885 and VDR, which was confirmed by western-blotting analysis. The relationship between rs739837 and the risk of pressure ulcers was explored using logistic regression analysis. RESULTS: We first conducted statistical analysis to explore the association between the rs739837 genotype and risk of pressure ulcers, and found that the polymorphism genotype was significantly associated with the risk of pressure ulcers (OR 0.68, 95% CI 0.43-0.95, P value = 0.02). We then searched the miRNA database online and identified VDR as a direct target. We established the negative regulatory relationship between miR-885-3p and VDR using luciferase assays. Meanwhile, we transfected cells with scramble control, miR-885-3p mimics, VDR siRNA and miR-885-3p inhibitors. The results further confirmed the negative regulatory relationship between miR-885-3p and VDR. CONCLUSION: VDR is a virtual target of miR-885-3p, and rs739837 might be a predictive biomarker for the risk of pressure ulcers.

Molecular finds of pressure ulcer: A bioinformatics approach in pressure ulcer.

BACKGROUND: Understanding the biological processes underlying Pressure Ulcer (PU) is an important strategy to identify new molecular targets. Bioinformatics has emerged as an important screening tool for a broad range of diseases. OBJECTIVE: This study aim of the current study is to investigate the protein-protein interaction in the PU context by bioinformatics. METHODS: We performed a search in gene databases, and bioinformatics algorithms were used to generate molecular targets for PU based in silico investigation. Interactions networks between protein-coding genes were built and compared to skin. RESULTS: TNFA, MMP9, and IL10 genes have higher disease-related connectivity than a connectivity general global. MAGOH, UBC, and PTCH1 as were leader genes related to skin. Ontological analysis demonstrated different mechanisms associated, such as response to oxidase stress. CONCLUSION: TNFA, MMP9, and IL10 are possible therapeutic targets for pressure ulcer. Additional investigation of cell post-transcriptional machinery should be investigated in PU.

Evaluation of physiological risk factors, oxidant-antioxidant imbalance, proteolytic and genetic variations of matrix metalloproteinase-9 in patients with pressure ulcer.

Pressure ulcer (PU) remains a common worldwide problem in all health care settings, it is synonymous with suffering. PU is a complex disease that is dependent on a number of interrelated factors. It involves multiple mechanisms such as physiological risk factors, chronic inflammation, oxidant-antioxidant imbalance and proteolytic attack on extracellular matrix by matrix metalloproteinases (MMP). Therefore, we propose that these wounds lead to molecular variations that can be detected by assessing biomarkers. In this study, we aimed to evaluate the major clinical elements and biological scars in Tunisian patients suffering from PU. Consistently, non-healing wound remains a challenging clinical problem. The complex challenges of the wound environment, involving nutrient deficiencies, bacterial infection, as well as the critical role played by inflammatory cells, should be considered because of their negative impact on wound healing. In addition, an imbalance between pro-oxidants and antioxidant systems seems to be more aggravated in patients with PU compared to healthy subjects. Of interest, this study provides further evidence to support a core role of the biological activity of MMP-9 in the pathogenesis of PU and indicates that the MMP9-1562 C/T (rs 3918242) functional polymorphism is associated with protection against this disease.

Hypoxia-Inducible Factor-1α Gene Expression and Apoptosis in Ischemia-Reperfusion Injury: A Rat Model of Early-Stage Pressure Ulcer.

BACKGROUND: Pressure ulcer (PU) is a significant and complicated health problem. Although ischemia-reperfusion (I/R) injury has been identified as the primary mechanism in the early stage of pressure ulceration, the cellular and molecular events contributing to I/R injury of PU development are unclear. OBJECTIVES: The aim of this study was to characterize hypoxia-inducible factor-1α (HIF-1α) expression profile and elaborate the apoptotic pathways based on an I/R injury model of PU in rats. METHODS: Two related experiments were conducted. The purpose of Experiment 1 was to establish an animal model of early-stage PU (Stage I PU). Forty-eight rats were randomly divided into six groups of eight. Each group received three cycles of I/R, but with different magnitudes of pressure-which ranged from 100 to 270 mm Hg-except for Control Group A. On the basis of this established animal model, Experiment 2 focused on the I/R mechanism of early-stage PU. The 36 rats used in Experiment 2 were randomly divided into one control and two experimental groups; the experimental groups received the same magnitude of pressure (170 mm Hg), but either 3 or 5 I/R cycles. Tissue underneath the compressed region, including the skin and muscle tissue, was incised for immunohistochemistry, enzyme-linked immunosorbent assay, TdT-mediated dUTP nick-end labeling, and real-time polymerase chain reaction gene expression analysis. RESULTS: HIF-1α gene expression in the compressed muscle tissue was slightly reduced in the 3 I/R group, but significantly elevated in the 5 I/R group (p < .05). The compressed tissue showed inflammation and evidence of oxidative stress reaction. ATPase activity was lower when cycles of I/R were prolonged. TdT-mediated dUTP nick-end labeling analysis showed a significant increase in the compressed muscle tissue relative to the control. Bax and Bcl-2 proteins expressed reversely. DISCUSSION: These results suggest that mitochondria-mediated apoptotic pathways may be involved in the development of early-stage PU, and HIF-1α might play a potential role for promoting apoptosis. Inflammation, oxidative stress reaction, and energy depletion may cooperate in the process of I/R injury of early-stage PU.

Analysis of Gene Expression in Experimental Pressure Ulcers in the Rat with Special Reference to Inflammatory Cytokines.

Pressure ulcers have been investigated in a few animal models, but the molecular mechanisms of pressure ulcers are not well understood. We hypothesized that pressure results in up-regulation of inflammatory cytokines and those cytokines contribute to the formation of pressure ulcers. We measured genome-wide changes in transcript levels after compression, and focused especially on inflammatory cytokines. The abdominal wall of rats was compressed at 100 mmHg for 4 hours by two magnets. Specimens were obtained 12 hours, 1, or 3 days after compression, and analyzed by light microscopy, microarray, Real-Time PCR, and ELISA. The skin and subcutaneous tissue in the compressed area were markedly thickened. The microarray showed that numerous genes were up-regulated after the compression. Up-regulated genes were involved in apoptosis, inflammation, oxidative stress, proteolysis, hypoxia, and so on. Real-Time PCR showed the up-regulation of granulocyte-macrophage colony stimulating factor (GM-CSF), interferon γ (IFN-γ), interleukin 1ß (IL-1ß), interleukin 1 receptor antagonist gene (IL1Ra), interleukin 6 (IL-6), interleukin 10 (IL-10), matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinase 1 (TIMP-1), and tumor necrosis factor α (TNF-α) at 12 hours, IFN-γ, IL-6, IL-10, MMP-3, and TIMP-1 at 1 day, and IFN-γ, IL-6, and MMP-3 at 3 days. Some genes from subcutaneous tissue were up-regulated temporarily, and others were kept at high levels of expression. ELISA data showed that the concentrations of IL-1ß and IL-6 proteins were most notably increased following compression. Prolonged up-regulation of IL-1ß, and IL-6 might enhance local inflammation, and continuous local inflammation may contribute to the pressure ulcer formation. In addition, GM-CSF, IFN-γ, MMP-3, and TIMP-1 were not reported previously in the wound healing process, and those genes may have a role in development of the pressure ulcers. Expression data from Real-Time PCR were generally in good agreement with those of the microarray. Our microarray data were useful for identifying genes involved in pressure ulcer formation. However, the expression levels of the genes didn't necessarily correspond with protein production. As such, the functions of these cytokines need to be further investigated.

Muscle apoptosis is induced in pressure-induced deep tissue injury.

Pressure ulcer is a complex and significant health problem. Although the factors including pressure, shear, and ischemia have been identified in the etiology of pressure ulcer, the cellular and molecular mechanisms that contribute to the development of pressure ulcer are unclear. This study tested the hypothesis that the early-onset molecular regulation of pressure ulcer involves apoptosis in muscle tissue. Adult Sprague-Dawley rats were subjected to an in vivo protocol to mimic pressure-induced deep tissue injury. Static pressure was applied to the tibialis region of the right limb of the rats for 6 h each day on two consecutive days. The compression force was continuously monitored by a three-axial force transducer equipped in the compression indentor. The contralateral uncompressed limb served as intra-animal control. Tissues underneath the compressed region were collected for histological analysis, terminal dUTP nick-end labeling (TUNEL), cell death ELISA, immunocytochemical staining, and real-time RT-PCR gene expression analysis. The compressed muscle tissue generally demonstrated degenerative characteristics. TUNEL/dystrophin labeling showed a significant increase in the apoptotic muscle-related nuclei, and cell death ELISA demonstrated a threefold elevation of apoptotic DNA fragmentation in the compressed muscle tissue relative to control. Positive immunoreactivities of cleaved caspase-3, Bax, and Bcl-2 were evident in compressed muscle. The mRNA contents of Bax, caspase-3, caspase-8, and caspase-9 were found to be higher in the compressed muscle tissue than control. These results demonstrated that apoptosis is activated in muscle tissue following prolonged moderate compression. The data are consistent with the hypothesis that muscle apoptosis is involved in the underlying mechanism of pressure-induced deep tissue injury.

Reduced up-regulation of cytoprotective genes in rat cutaneous tissue during the second cycle of ischemia-reperfusion.

Chronic wounds are major health problems that affect millions of people in the United States every year. Management of these wounds costs billions of dollars annually in the United States. Despite their clinical importance, the molecular mechanisms underlying these clinical conditions remain elusive. Repetitive ischemia-reperfusion (I-R) may play a pivotal role in chronic wound formation. The development of therapies for these wounds is hindered by the lack of animal models that allow identification of the molecular mechanisms underlying chronic wound formation. In the first study of its kind, we adapted our rat pressure sore model by imposing two cycles of ischemia (2 hours) and two cycles of reperfusion (24 hours), and we examined gene expression to better understand the molecular events that occur at the very early stages of cutaneous I-R injury with a goal of devising preventing strategies. We successfully tested our hypothesis and demonstrated that while cytoprotective genes, such as heat shock protein 70, heat shock protein 90, hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and heme oxygenase-1, were initially up-regulated during the first cycle of I-R, their up-regulation was subsequently reduced or completely abolished during the second cycle of I-R. These findings raise the possibility that reduced up-regulation of these cytoprotective genes may be causally linked to cutaneous I-R injury.