Sonic hedgehog signaling promotes angiogenesis of endothelial progenitor cells to improve pressure ulcers healing by PI3K/AKT/eNOS signaling.

BACKGROUND: Pressure ulcer is a severe disease in the paralyzed and aging populations. Endothelial progenitor cells (EPCs) are able to regulate ulcer healing by modulating angiogenesis, but the molecular mechanism is still obscure. Sonic hedgehog (SHH) signaling contributes to angiogenesis in various diseases and has been identified to modulate EPCs function. Here, we aimed to explore the significance of SHH signaling in EPCs function during pressure ulcers. METHODS: The EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1. Cell proliferation was detected by cell counting kit 8 (CCK-8). The DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 were analyzed by immunofluorescent analysis. The angiogenesis of EPCs was analyzed by tube formation assay. The pressure ulcers rat model was constructed, the wound injury was analyzed by H&E staining and angiogenesis was analyzed by the accumulation of CD31 based on immunofluorescent analysis. RESULTS: The expression of patched-1 and Gli-1 was enhanced by SHH activator SAG but reduced by SHH inhibitor cyclopamine in the EPCsThe PI3K, Akt, eNOS expression and the Akt phosphorylation were induced by SAG, while the treatment of cyclopamine presented a reversed result. The proliferation and migration of EPCs were enhanced by SAG but repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse the effect of SAG. CONCLUSIONS: Thus, we found that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. SHH signaling may serve as the potential target for attenuating pressure ulcers.

PSTPIP2 is associated with disease severity in patients with pressure ulcer sepsis and has anti-inflammatory effects

Background: One of the common adverse reactions in patients with pressure ulcers (PU) is sepsis, which is mainly related to microbial infections caused by pathogenic organisms. The activation of nuclear factor kappa-B (NF-κB) frequently occurs in conjunction with pathogenic microbial infections. Proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) is closely related to inflammatory disorders. The role and mechanism of PSTPIP2 in sepsis because of pressure ulcers is unclear. In this study, we discovered that PSTPIP2 was lowly expressed in peripheral blood of patients with sepsis induced by pressure ulcers. Methods: Peripheral blood was collected from 20 patients with sepsis due to pressure ulcers and 10 healthy controls, and the expression of PSTPIP2 in peripheral blood was discovered by polymerase chain reaction and Western blot analysis. Information on the clinical characteristics of patients was summarized, and the expression data of PSTPIP2 were correlated with the patients’ acute physiology and chronic health evaluation (APACHE) II score, sequential organ failure assessment (SOFA) score, and C-reactive protein (CRP) and procalcitonin (PCT) scores by Spearman’s correlation analysis. One of the main mediators of Gram-negative sepsis is lipopolysaccharide (LPS). In order to establish an in vitro sepsis model, THP-1 cells were treated with LPS, and the cells were transfected with PSTPIP2. Contents of interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor-α (TNF-α) in each group of cells were detected by enzyme-linked--immunosorbent serologic assay, and NF-κB-related proteins were detected by Western blot analysis (AU)

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.

Methacrylated gelatin shape-memorable cryogel subcutaneously delivers EPCs and aFGF for improved pressure ulcer repair in diabetic rat model.

Pressure ulcer (PU) in patients with diabetes mellitus (DM) is still a clinical intractable issue due to the complicated physiological characteristics by the prolonged high glucose level and impaired angiogenesis. The PU treatment includes surgical debridement, stem cell therapy and growth factors, leading to high cost and repeated professional involvement. Developing effective wound dressing combining the therapeutic cells and growth factors has become highly demanded. Herein, we reported the direct subcutaneous administration of endothelial progenitor cells (EPCs) and acid fibroblast growth factor (aFGF) with a shape-memorable methacrylated gelatin cryogel (EPCs/aFGF@GelMA) for the therapy of PU in rats with DM. This EPCs/aFGF@GelMA cryogel system presented microporous structure, elastic mechanical strength and enhanced cell migration property with controlled release of aFGF. Moreover, compared with EPCs/aFGF and GelMA alone, in vivo results showed that this EPCs/aFGF@GelMA system exhibited accelerated wound closure rate, enhanced granulation formation, collagen deposition as well as re-epithelization. Importantly, we found that the excellent positive performance of EPCs/aFGF@GelMA is due to its up-regulation of HIF-ɑ upon the wound site, modulating the microenvironment of wound site to initiate the impaired local angiogenesis. Collectively, this hybrid gelatin cryogels show great promise for biomedical applications, especially in tissue engineering and regenerative medicine.

Protective effects of conditioned media of immortalized stem cells from human exfoliated deciduous teeth on pressure ulcer formation.

Pressure ulcers (PUs) are increasing with aging worldwide, but there is no effective causal therapy. Although mesenchymal stem cells (MSCs) promote cutaneous wound healing, the effects of the conditioned medium (CM) of MSCs on cutaneous PU formation induced by ischemia-reperfusion injury have been poorly investigated. To address this issue, herein, we first established an immortalized stem cell line from human exfoliated deciduous teeth (SHED). This cell line was revealed to have superior characteristics in that it grows infinitely and vigorously, and stably and consistently secretes a variety of cytokines. Using the CM obtained from the immortalized SHED cell line, we investigated the therapeutic potential on a cutaneous ischemia-reperfusion mouse model for PU formation using two magnetic plates. This is the first study to show that CM from immortalized SHEDs exerts therapeutic effects on PU formation by promoting angiogenesis and oxidative stress resistance through vascular endothelial growth factor and hepatocyte growth factor. Thus, the CM of MSCs has potent therapeutic effects, whereas these therapies have not been implemented in human medicine. To try to meet the regulatory requirements for manufacturing and quality control as much as possible, it is necessary to produce CM that is consistently safe and effective. The immortalization of stem cells could be one of the breakthroughs to meet the regulatory requirements and consequently open up a novel avenue to create a novel type of cell-free regenerative medicine, although further investigation into the quality control is warranted.

Role of microRNAs in Pressure Ulcer Immune Response, Pathogenesis, and Treatment.

Pressure ulcers are preventable, yet highly prevalent, chronic wounds that have significant patient morbidity and high healthcare costs. Like other chronic wounds, they are characterized by impaired wound healing due to dysregulated immune processes. This review will highlight key biochemical pathways in the pathogenesis of pressure injury and how this signaling leads to impaired wound healing. This review is the first to comprehensively describe the current literature on microRNA (miRNA, miR) regulation of pressure ulcer pathophysiology.

Quantitative iTRAQ LC-MS/MS reveals muscular proteome profiles of deep pressure ulcers.

Pressure ulcers (PUs) are a common clinical issue lacking effective treatment and validated pharmacological therapy in hospital settings. Ischemia-reperfusion injury of deep tissue, especially muscle, plays a vital role in the formation and development of the overwhelming majority of PUs. However, muscular protein expression study in PUs has not been reported. Herein, we aimed to investigate the muscular proteins profiles in PUs and to explore the pathological mechanism of PUs. The iTRAQ LC-MS/MS was conducted to detect the protein profiles in clinical muscle samples of PUs. The GO and KEGG pathways analyses were performed for annotation of differentially expressed proteins. Protein-protein interaction (PPI) network was constructed by STRING online database, and hub proteins were validated by the immunoblotting. Based on proteomics results, we found a number of proteins that were differentially expressed in PU muscle samples compared with the normal and identified unique proteins expression patterns between these two groups, suggesting that they might involve in pathological process of the disease. Importantly, cathepsin B and D, as well as other autophagy-lysosome and apoptosis associated proteins were identified. Further experiments characterize the expression of these proteins and their regulation in the process of apoptosis and autophagy. These findings may provide novel insights into the mechanisms of lysosome-associated pathways involved in the initiation of PUs. This is the first study linking proteomics to PUs muscle tissues, which indicated cathepsin B and D might be key drug target for PUs.

Comprehensive Proteomic Profiling of Pressure Ulcers in Patients with Spinal Cord Injury Identifies a Specific Protein Pattern of Pathology.

Objective: Severe pressure ulcers (PUs) do not respond to conservative wound therapy and need surgical repair. To better understand the pathogenesis and to advance on new therapeutic options, we focused on the proteomic analysis of PU, which offers substantial opportunities to identify significant changes in protein abundance during the course of PU formation in an unbiased manner. Approach: To better define the protein pattern of this pathology, we performed a proteomic approach in which we compare severe PU tissue from spinal cord injury (SCI) patients with control tissue from the same patients. Results: We found 76 proteins with difference in abundance. Of these, 10 proteins were verified as proteins that define the pathology: antithrombin-III, alpha-1-antitrypsin, kininogen-1, alpha-2-macroglobulin, fibronectin, apolipoprotein A-I, collagen alpha-1 (XII) chain, haptoglobin, apolipoprotein B-100, and complement factor B. Innovation: This is the first study to analyze differential abundance protein of PU tissue from SCI patients using high-throughput protein identification and quantification by tandem mass tags followed by liquid chromatography tandem mass spectrometry. Conclusion: Differential abundance proteins are mainly involved in tissue regeneration. These proteins might be considered as future therapeutic options to enhance the physiological response and permit cellular repair of damaged tissue.

Vaccarin Regulates Diabetic Chronic Wound Healing through FOXP2/AGGF1 Pathways.

BACKGROUND: Diabetes mellitus is a growing global health issue nearly across the world. Diabetic patients who are prone to develop diabetes-related complications often exhibit progressive neuropathy (painless and sensory loss). It is usual for small wounds to progress to ulceration, which especially worsens with peripheral arterial disease and in the presence of anaerobic bacteria, culminating into gangrene. In our study, vaccarin (VAC), the main active monomer extracted from Chinese herb vaccariae semen, is proven to have a role in promoting diabetic chronic wound healing through a cytoprotective role under high glucose conditions. MATERIALS AND METHODS: We constructed a pressure ulcer on both VAC-treated and control mice based on a type 1 diabetes (T1DM) model. The wound healing index was evaluated by an experimental wound assessment tool (EWAT). We also determined the effect of VAC on the proliferation and cell migration of human microvascular endothelial cells (HMEC-1) by a cell counting kit (CCK-8), a scratch and transwell assay. RESULTS: The results demonstrated that VAC could promote the proliferation and migration of high glucose-stimulated HMEC-1 cells, which depend on the activation of FOXP2/AGGF1. Activation of the angiogenic factor with G patch and FHA domains 1 (AGGF1) caused enhanced phosphorylation of serine/threonine kinase (Akt) and extracellular regulated protein kinases (Erk1/2). By silencing the expression of forkhead box p2 (FOXP2) protein by siRNA, both mRNA and protein expression of AGGF1 were downregulated, leading to a decreased proliferation and migration of HMEC-1 cells. In addition, a diabetic chronic wound model in vivo unveiled that VAC had a positive effect on chronic wound healing, which involved the activation of the above-mentioned pathways. CONCLUSIONS: In summary, our study found that VAC promoted chronic wound healing in T1DM mice by activating the FOXP2/AGGF1 pathway, indicating that VAC may be a promising candidate for the treatment of the chronic wounds of diabetic patients.

Using Subepidermal Moisture Level as an Indicator of Early Pressure Damage to Local Skin and Tissue.

OBJECTIVE: Inflammation triggered by tissue injury increases blood flow to the injured area, resulting in fluid accumulation below the epidermis known as subepidermal moisture (SEM). This accumulation can be assessed using an SEM scanner. The primary aim of this study was to determine whether levels of SEM from repeated measures at a localized area confirm the absence of a pressure injury at that site in healthy participants. METHODS: Fifty patients attending a specialized pain clinic who were at very low risk of or unaffected by pressure injuries were enrolled. Subepidermal moisture readings were gathered at the center and near-proximate to the bony prominences of the heel and sacrum. MAIN RESULTS: Among the participants, repeated localized measurements did not differ significantly at or around a single anatomic site. The heel had a slightly higher variation than the sacrum, although the variance was less than 0.6 SEM units, indicating that there is likely no inflammation at these sites in patients who are not at risk of pressure injuries and who show no visual signs of localized tissue inflammation. Demographic and other potential confounder data were gathered, but no statistically significant findings emerged from these data. CONCLUSIONS: The relatively flat pattern of SEM values around the bony prominences of the heels and sacrum in healthy participants supports the hypothesis that healthy tissue is not inflamed.

The relationship between skin ultrasound images and muscle damage using skin blotting in wheelchair basketball athletes.

STUDY DESIGN: Secondary analysis of a cross-sectional observation study. OBJECTIVES: To determine the relationship between skin ultrasound images and muscle damage in wheelchair basketball athletes, using skin blotting examinations of the ischial regions. SETTING: Community, Japan. METHODS: Fourteen elite wheelchair basketball athletes were recruited. We obtained data regarding participants' characteristics. We undertook ultrasonographic images and quantitative skin blotting of the ischial region before and after training, and after rest. RESULTS: We identified Category II and III pressure injuries in 2 of the 12 participants. Structural features were classified into four categories based on ultrasonographic features, namely, normal skin structure, unclear superficial and deep fascia, cloudy fat layer, and fat infiltration and low-echoic lesion/anechoic lesions. The muscle-type creatinine kinase (CK-M) level (median [interquartile range: IQR], 2.98 [2.80-3.47]) in the fat infiltration and low-echoic lesion/anechoic lesion group was significantly higher (1.43 [1.41-1.49]) than in a nonfat infiltration and low-echoic lesion/anechoic lesion group after training (p = 0.03). The interleukin-6 (IL-6) level (median [IQR], 23.5 [16.15-58.97]) in the fat infiltration and low-echoic lesion/anechoic lesion group was significantly higher (1.94 [1.74-4.44]) than in the nonfat infiltration and low-echoic lesion/anechoic lesion group after rest (mean difference = -25.4, 95% CI -61.1 to 10.7, p = 0.03). CONCLUSIONS: The combination of ultrasonographic images and skin blotting using CK-M and IL-6, could detect early deep tissue damage in wheelchair athletes. These techniques could be potentially useful in the treatment and prevention of pressure injuries. SPONSORSHIP: This study was supported in part by YAMAHA Motor Foundation for Sports.

Anti-antioxidant impacts of circZNF609 silence in HaCaT cells through regulating miR-145.

Background: CircZNF609 (cZNF609) is previously revealed as an essential mediator in oxidative stress. This paper determined the role of cZNF609 in skin oxidative damage to evaluate its importance in pressure ulcer.Methods: HaCaT cells treated by H2O2 were considered as a cell model of pressure ulcer. The role of cZNF609 in the model was checked by conducting CCK-8 assay, FITC-PI double-staining, ROS detection and Western blot. The downstream gene and signalling of cZNF609 were studied by utilizing qRT-PCR and Western blot.Results: HaCaT cells were remarkably damaged by H2O2, as evidenced by the viability loss, apoptosis and ROS generation. It was coupled with the elevated expression of p53, p16, Bax and the activated forms of caspase-3 and PARP. Meanwhile, cZNF609 was high-expressed in response to H2O2. The oxidative stress driven by H2O2 was alleviated by transfection with cZNF609 specific siRNA. Further, the anti-antioxidant impacts of cZNF609 silence were impeded by miR-145 silence. The inhibition of JNK and p38MAPK pathways induced by cZNF609 silence was impeded by miR-145 silence.Conclusion: The protective function of cZNF609 silence in H2O2-injured HaCaT cells was revealed in vitro. Silence of cZNF609 exhibited its impact possibly through regulating miR-145, and JNK and p38MAPK pathways.

Chronological changes in rat heel skin following depressurization of pressure ulcer-like dermal lesions.

In our previous study, we proposed an animal model in which pressure ulcer-like dermal lesions can be produced by denervation of the sciatic nerve and application of a pressure load to rat heel skin. In the present study, we divided these animals into non-treated and pressure loading groups, and initiated hindlimb unloading (depressurization) by tail suspension at 1, 3, 5, 7, and 14 days after inflicting lesions (1-14d pressurization groups). Chronological changes in heel lesions were examined morphologically in all treatment groups after 1, 3, 7, 14, 28, and 40 days. Open dermal lesions were formed by 14 days in the loading group and numerous macrophages were present. In the 14d pressurization group, numerous macrophages were still distributed in and around lesions and Vascular endothelial cell growth factor (VEGF) expression was strongly detected by 3 days, but a thin germinal layer began to appear and CD68-positive macrophages and VEGF immunoreactions decreased gradually by 7 days later. By 14 days after depressurization, the germinal layer was repaired, and macrophages and immunoreactions of VEGF were similar to those of non-treated skin. These chronological changes were similar to those in human pressure ulcers, but from 5d after depressurization, different chronological changes were observed. Specifically, epidermis was thickened and macrophages were hardly detected at 5 days in the loading group, but the epidermis disappeared by 1 day in the 5d pressurization group. Subsequently, numerous macrophages aggregated and VEGF expression was increased by 3 days, and the remaining healing process was similar to that in the 14d pressurization group. Even when unloading was performed during the early stages (5d pressurization group), the epidermis disappeared and macrophages were then distributed before repair of the lesion was observed. These results suggest that earlier migration of macrophages to skin lesions might be associated with rapid wound healing.

Non-invasive detection of local tissue responses to predict pressure ulcer development in mouse models.

The development of pressure ulcers is associated with four different pathways: ischemia, ischemia-reperfusion injury, impaired interstitial fluid flow and lymphatic drainage, and cell deformation. For prediction of pressure ulcer development, it is important to detect the tissue response involved in the pathways at the molecular level. However, non-invasive techniques for detecting this tissue response are not available. This study aimed to demonstrate that the secretion of the candidate marker proteins in pressure-loaded mouse skin can be detected by skin blotting, and to propose a novel direct skin assessment method for predicting pressure ulcer development. We created three different tissue damage models: early stage pressure ulcers, blanchable erythema and intact skin. We confirmed the pathways involved in the pressure ulcer development by histological analyses in the pressure ulcer model. Interleukin-1α (IL-1α), vascular endothelial growth factor C (VEGF-C) and heat shock protein 90α (HSP90α) were expressed in the pressure ulcer model at a significantly different level compared to the blanchable erythema or intact skin during the time course. Detecting the secretion of these novel biomarkers by skin blotting can be a useful method for non-invasive prediction of pressure ulcer development.

The expression of anaerobic metabolites in sweat and sebum from human skin subjected to intermittent and continuous mechanical loading.

Pressure ulcers (PUs) represent a substantial burden to both patients and healthcare providers. Accordingly, effective prevention strategies should follow early detection of PUs. Anaerobic metabolites, such as lactate and pyruvate, are promising noninvasive biomarkers indicative of tissue ischaemia, one of the major mechanisms leading to PU development. The aim of this study was to investigate if the temporal release profile of these metabolites in sweat and sebum is sensitive to detect local tissue changes resulting from prolonged mechanical loads. The sacrum of healthy volunteers was subjected to two different loading protocols. After a baseline measurement, the left and right side of the sacrum were subjected to continuous and intermittent loading regimen, respectively, at a pressure of 100 mmHg. Biomarker samples were collected every 20 min, with a total experimental time of 140 min. Sweat was collected at 37 ∘C and 80% relative humidity, and sebum at ambient conditions, from 11 to 13 volunteers, respectively. Both samples were analysed for lactate and pyruvate concentrations using ultra-high performance supercritical fluid chromatography mass spectrometry. Prior to analysis metabolite concentrations were normalized to individual baseline levels and, in the case of sweat, additional normalization was performed to an unloaded control site to account for fatigue of sweat glands. Although substantial variability was present, the temporal release profiles of both sweat and sebum metabolites reflected the applied loading regimen with increased levels upon load application, and recovery to baseline levels following load removal. Highest relative increases were 20% and 30% for sweat lactate and pyruvate, respectively, and 41% for sebum lactate. Sebum pyruvate was not present in quantifiable amounts. There was a linear correlation between the individual responses to intermittent and continuous loading. The present study revealed that metabolite biomarkers in both sweat and sebum were sensitive to the application of mechanical loads, indicative of local ischaemia within skin and soft tissues. Similar trends in metabolic biomarkers were observed in response to intermittent and continuous loading regimens in both sweat and sebum. Metabolites represent a potential means to monitor the health of loaded skin and soft tissues informing timely interventions of PU prevention.

[Protein expressions of matrix metalloproteinase-9 and its inhibitor and their ratio changes in wound healing of patients with stages â ¢ and â £ pressure ulcers].

Objective: To explore the expression levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein and the change of MMP-9/TIMP-1 ratio in wound exudates of patients with stages Ⅲ and Ⅳ pressure ulcers during wound healing. Methods: From July 2017 to July 2018, 30 patients with stage Ⅲ pressure ulcers [30 wounds, 16 males and 14 females, aged (65±10) years] and 34 patients with stage Ⅳ pressure ulcers [50 wounds, 17 males and 17 females, aged (65±9) years] admitted to Hebei General Hospital who met the inclusion criteria were enrolled in this prospective cohort study. According to the principle of wound treatment and the characteristics and needs of wound in different periods, individualized intervention measures were formulated for patients and appropriate dressings were selected. At the time of admission and on 7, 14, 21, 28 days of treatment, the healing of pressure ulcer wounds was evaluated by Pressure Ulcer Healing Scale. Afterwards, the wound exudate was collected at each time point to detect the expression levels of MMP-9 and TIMP-1 protein by enzyme-linked immunosorbent assay, and the MMP-9/TIMP-1 ratio was calculated. Data were processed with analysis of variance for repeated measurements of single group and linear trend test. Results: (1) There were significantly statistical differences in wound healing scores of patients with stages Ⅲ and Ⅳ pressure ulcers among the time of admission and on 7, 14, 21, 28 days of treatment within each stage (F=145.382, 153.234, P<0.01), and they all showed a gradually decreasing trend (F=170.466, 284.585, P<0.01). (2) At the time of admission and on 7, 14, 21, 28 days of treatment, the expression levels of MMP-9 protein in wound exudates of patients with stages Ⅲ and Ⅳ pressure ulcers were (171±104), (138±88), (110±70), (85±55), (62±41) ng/L and (193±107), (173±104), (139±83), (114±70), (89±56) ng/L, respectively. There were significantly statistical differences within each stage (F=58.007, 111.680, P<0.01), and they all showed a gradually decreasing trend (F=62.901, 134.628, P<0.01). At the time of admission and on 7, 14, 21, 28 days of treatment, the expression levels of TIMP-1 protein in wound exudates of patients with stages Ⅲ and Ⅳ pressure ulcers were (6.2±3.9), (5.6±3.4), (5.1±3.1), (4.4±2.5), (3.8±2.3) ng/L and (4.8±2.5), (4.7±2.6), (4.4±2.6), (4.6±2.7), (4.1±2.4) ng/L, respectively. There were significantly statistical differences within each stage (F=25.479, 7.778, P<0.01), and there was a gradually decreasing trend in stage Ⅲ (F=62.901, P<0.01) and a decreasing trend in stage Ⅳ (F=134.628, P<0.01). At the time of admission, the expression levels of MMP-9 and TIMP-1 in wound exudates of patients with stage Ⅲ pressure ulcers were similar to those of patients with stage Ⅳ pressure ulcers (t=-1.03, 1.47, P>0.05). (3) At the time of admission and on 7, 14, 21, 28 days of treatment, the MMP-9/TIMP-1 ratios in the wound exudates of patients with pressure ulcers of stages Ⅲ and Ⅳ were 30±13, 25±9, 22±9, 20±8, 17±6 and 43±19, 37±13, 32±10, 26±9, 22±9, respectively. There were significantly statistical differences within each stage (F=37.173, 97.191, P<0.01), and they all showed a gradually decreasing trend (F=54.183, 130.088, P<0.01). At the time of admission, the MMP-9/TIMP-1 ratio in wound exudates of patients with stage Ⅳ pressure ulcers was significantly higher than that of patients with stage Ⅲ pressure ulcers (t=-3.42, P<0.01). Conclusions: During the wound healing process of patients with stages Ⅲ and Ⅳ pressure ulcers, the expression levels of MMP-9 and TIMP-1 protein and the MMP-9/TIMP-1 ratio in wound exudates show a decreasing trend. The stage of wound healing can be predicted according to the expression level of MMP-9 protein and the MMP-9/TIMP-1 ratio.

Assessing subclinical inflammation by peroxidase detection in patients with pressure ulcers.

OBJECTIVE: This study aimed to examine the superiority of peroxidase detection of macroscopic observations using rat wounds, and to test the external validity of the peroxidase analysis in pressure ulcers (PU) in humans. METHOD: In the animal study, rat wounds were analysed. A cross-sectional study analysed, by wound blotting, exudate samples from full-thickness PUs. Peroxidase activity was divided into two groups (ring and non-ring signals). Scores in the 'inflammation/infection' and 'necrotic tissue' components of DESIGN, a classification tool of PUs, were compared between the groups. RESULTS: In the animal study, 20 rat wounds were assessed and in the clinical study, 62 samples were collected from 26 full-thickness PUs of 21 patients aged ≥ 65 years. In the animal study, five of six wounds with clinical inflammation signs showed ring signal (defined as a signal on the wound edge and no signal on the wound bed). While the tissue sections of three wounds with a ring signal showed inflammatory features, they showed no clinical signs of 'inflammation/infection'. In the clinical study, which analysed 630 ring and 32 non-ring signals, 13 samples in the ring signal group and five in the non-ring signal group had 'inflammation/infection; scores of ≥1 (p=0.016). Despite having no clinical signs, 17 samples showed the ring signal. CONCLUSION: This study revealed the external validity of the wound blotting analysis of peroxidase and demonstrated its use to detect subclinical inflammation.

[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.

Prediction of healing in Category I pressure ulcers by skin blotting with plasminogen activator inhibitor 1, interleukin-1α, vascular endothelial growth factor C, and heat shock protein 90α: A pilot study.

The prevention of progression of Category I pressure ulcers (PUs) to Category II or higher is important, as Category II or higher PUs are open wounds and have a higher infection risk. Prognosis prediction of Category I PUs is necessary to provide successful intensive care for PUs with impaired healing. We focused on skin blotting using plasminogen activator inhibitor 1 (PAI1), interleukin-1α (IL-1α), vascular endothelial growth factor C (VEGF-C), and heat shock protein 90α (HSP90α). This pilot study was conducted at long-term-care and general hospitals to examine the applicability of DESIGN-R and thermography; the feasibility of skin blotting technique; the biomarker candidates, PAI1, IL-1α, VEGF-C, and HSP90α; and sample size for prognosis prediction for Category I PUs. Patients aged >65 years underwent skin blotting, scoring for DESIGN-R, and took thermography images of their Category I PU site. Albumin signals were not detected in one out of three participants. PAI1, IL-1α, VEGF-C, and HSP90α were detected in 19 participants, among whom 11 participants could be followed up after one week. There was no difference in DESIGN-R score and skin surface temperature between normal and impaired healing groups, and the sample size was calculated as 16. In conclusion, the feasibility of skin blotting was confirmed. PAI1, IL-1α, VEGF-C, and HSP90α could be biomarker candidates for prognosis prediction for Category I PU and the combination of VEGF-C and HSP90α could be associated with the prognosis of Category I PU. We need to investigate 842 patients in a future study.

[Analysis of differential gene expressions of inflammatory and repair-related factors in chronic refractory wounds in clinic].

Objective: To compare the tissue morphology and gene expressions of inflammatory and repair-related factors in chronic refractory wound tissue including pressure ulcers and diabetic feet. Methods: During August 2016 to September 2017, 10 samples of prepuce were collected after circumcision of 10 urological patients [all male, aged (38±4) years old] admitted in the First Affiliated Hospital of Nanchang University and included in normal skin group, samples of tissue around the edge of wounds with blood supply were collected from 9 heat or electric burn patients [6 male patients, 3 female patients, aged (51±8) years old], 13 pressure ulcer patients [9 male patients, 4 female patients, aged (51±14) years old] and 10 diabetic foot patients [8 male patients, 2 female patients, aged (61±10) years old] during the operations. The samples were divided into burn wound group (9 samples), pressure ulcer group (13 samples), and diabetic foot group (10 samples). Ten slices were taken from pressure ulcer group and diabetic foot group respectively, and 5 slices in each group were used to observe the tissue morphology and expressions of Ki67 and CD31 of wounds respectively with immunofluorescence method. Ten samples from normal skin group, 9 samples from burn wound group, 13 samples from pressure ulcer group, and 10 samples from diabetic foot group were collected for analysis of mRNA expressions of vascular endothelial growth factor 192 (VEGF192), transforming growth factor ß (TGF-ß), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) , interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α) by real time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with Mann-Whitney U test and Kruskal-Wallis rank-sum test. Results: (1) The expression level of Ki67 in diabetic foot group (390±100) was higher than that of pressure ulcer group (182±14, Z=-2.611, P<0.01). (2) Although there were a large number of vascular endothelial cells (CD31 positive cells) in wounds of diabetic foot group, their distribution was disordered and failed to form intact lumen. There were less vascular endothelial cells in wounds of pressure ulcer group than those of diabetic foot group, but the complete lumen was formed. (3) The mRNA expression levels of VEGF192 in wounds of burn wound group, pressure ulcer group, and diabetic foot group were significantly lower than the level in normal skin group (H=13.72, 30.50, 15.20, P<0.05 or P<0.01), and the level was the lowest in pressure ulcer group. The mRNA expression level of VEGF192 in wounds of pressure ulcer group was significantly lower than that of diabetic foot group (H=15.30, P<0.01). Compared with that of normal skin group, the mRNA expression level of TGF-ß in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), while the mRNA expression levels of TGF-ß in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P<0.01). The mRNA expression level of TGF-ß in wounds of pressure ulcer group was similar to that of diabetic foot group (H=3.54, P>0.05). (4) Compared with those of normal skin group, the mRNA expression levels of VCAM-1 in wounds of burn wound group and pressure ulcer group were significantly increased (H=-22.50, -11.50, P<0.05 or P<0.01), and there was no significant difference in the mRNA expression level of VCAM-1 in wounds of diabetic foot group (H=10.00, P>0.05); the mRNA expression level of ICAM-1 in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), and the levels of ICAM-1 in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=16.50, 16.50, P<0.01). The mRNA expression level of VCAM-1 in wounds of pressure ulcer group was significantly higher than that of diabetic foot group (H=-21.50, P<0.01), the mRNA expression level of ICAM-1 in wounds of pressure ulcer group was similar to that of diabetic foot group (H=0, P>0.05). (5) Compared with those of normal skin group, except for the mRNA expression level of IL-1ß in wounds of diabetic foot group showed no significant difference (H=-10.00, P>0.05), the mRNA expression levels of IL-1ß in wounds of burn wound group and pressure ulcer group were significantly increased (H=-32.50, -21.50, P<0.01); the mRNA expression levels of IL-6 were significantly increased in wounds of burn wound group, pressure ulcer group, and diabetic foot group (H=-17.50, -30.50, -11.80, P<0.05 or P<0.01); except for the mRNA expression level of TNF-α in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), the mRNA expression levels of TNF-α in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P<0.01). The mRNA expression levels of IL-1ß and TNF-α in wounds of pressure ulcer group were significantly lower than those of burn wound group (H=11.00, 27.54, P<0.05 or P<0.01), while the mRNA expression level of IL-6 was significantly higher (H=-13.00, P<0.05). The mRNA expression levels of IL-1ß and TNF-α in wounds of diabetic foot group were significantly lower than those of burn wound group (H=22.50, 24.00, P<0.01), while the mRNA expression level of IL-6 showed no significant difference (H=5.70, P>0.05). Conclusions: The phenotypes of diabetic foot and pressure ulcer vary from the expressions levels of proliferating cell nuclear antigen and blood vessels forming ability to the expression levels of growth factors, cell adhesion factors, and inflammatory cytokines.