ResearchPad - wound-healing https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Functional and structural consequences of epithelial cell invasion by <i>Bordetella pertussis</i> adenylate cyclase toxin]]> https://www.researchpad.co/article/elastic_article_7693 Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.

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<![CDATA[Molecular insights in the pathogenesis of classical Ehlers-Danlos syndrome from transcriptome-wide expression profiling of patients’ skin fibroblasts]]> https://www.researchpad.co/article/5c61e8b3d5eed0c48496efc5

Classical Ehlers-Danlos syndrome (cEDS) is a dominant inherited connective tissue disorder mainly caused by mutations in the COL5A1 and COL5A2 genes encoding type V collagen (COLLV), which is a fibrillar COLL widely distributed in a variety of connective tissues. cEDS patients suffer from skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. Most of the causative variants result in a non-functional COL5A1 allele and COLLV haploinsufficiency, whilst COL5A2 mutations affect its structural integrity. To shed light into disease mechanisms involved in cEDS, we performed gene expression profiling in skin fibroblasts from four patients harboring haploinsufficient and structural mutations in both disease genes. Transcriptome profiling revealed significant changes in the expression levels of different extracellular matrix (ECM)-related genes, such as SPP1, POSTN, EDIL3, IGFBP2, and C3, which encode both matricellular and soluble proteins that are mainly involved in cell proliferation and migration, and cutaneous wound healing. These gene expression changes are consistent with our previous protein findings on in vitro fibroblasts from other cEDS patients, which exhibited reduced migration and poor wound repair owing to COLLV disorganization, altered deposition of fibronectin into ECM, and an abnormal integrin pattern. Microarray analysis also indicated the decreased expression of DNAJB7, VIPAS39, CCPG1, ATG10, SVIP, which encode molecular chaperones facilitating protein folding, enzymes regulating post-Golgi COLLs processing, and proteins acting as cargo receptors required for endoplasmic reticulum (ER) proteostasis and implicated in the autophagy process. Patients’ cells also showed altered mRNA levels of many cell cycle regulating genes including CCNE2, KIF4A, MKI67, DTL, and DDIAS. Protein studies showed that aberrant COLLV expression causes the disassembly of itself and many structural ECM constituents including COLLI, COLLIII, fibronectin, and fibrillins. Our findings provide the first molecular evidence of significant gene expression changes in cEDS skin fibroblasts highlighting that defective ECM remodeling, ER homeostasis and autophagy might play a role in the pathogenesis of this connective tissue disorder.

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<![CDATA[Sleeping through anything: The effects of unpredictable disruptions on mouse sleep, healing, and affect]]> https://www.researchpad.co/article/5c5ca2b9d5eed0c48441e977

Many aspects of the laboratory environment are not tailored to the needs of rodents, which may cause stress. Unpredictable stressors can cause ulcers, prolonged pituitary-adrenal activation, and anhedonia. Similarly, pain has been demonstrated to slow wound healing, and mice experiencing pain exhibit altered behavior. However it is unknown how husbandry, which occurs when the mice are inactive, and lack of analgesia, specifically in a punch biopsy procedure, effects animal physiology, behavior, and welfare, particularly as it relates to sleep fragmentation. We hypothesized that sleep fragmentation, induced by unpredictable husbandry and lack of pain management will slow wound healing. Two main treatments were tested in a factorial design in C57BL/6 mice of both sexes (64 mice total); 1) analgesia (carprofen and saline) and 2) sleep disruptions (random and predictable). Mice were singly housed in a non-invasive sleep monitoring apparatus on arrival (Day -4). Disruption treatments were applied from Day -3 to 2. All mice received a punch biopsy surgery (Day 0) with topical lidocaine gel and their analgesic treatment prior to recovery, and on Days 1 and 2. Nesting behavior was assessed daily and a sugar cereal consumption test, as a measure of anhedonia, was conducted on Days -1 to 2. On Day 3, mice were euthanized and wound tissue and adrenal glands were collected. We found that the disruption predictability had no effect on mouse sleep, wound healing, or adrenal cortex:medulla ratio. It’s possible that the disruption period was not long enough to induce chronic stress. However, male mice who received analgesia slept more than their female counterparts; this may be related to sex differences in pain perception. Overall, it does not appear that the predictability of disturbance effects sleep fragmentation or stress responses, indicating that husbandry activities do not need to occur at set predictable times to improve welfare.

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<![CDATA[Dual role of iodine, silver, chlorhexidine and octenidine as antimicrobial and antiprotease agents]]> https://www.researchpad.co/article/5c5ca2e7d5eed0c48441ece4

Objectives

The majority of human chronic wounds contain bacterial biofilms, which produce proteases and retard the resolution of inflammation. This in turn leads to elevated patient protease activity. Chronic wounds progressing towards closure show a reduction in proteolytic degradation. Therefore, the modulation of protease activity may lead to the faster healing of chronic wounds. Antimicrobials are used to control biofilm-based infection; however, some of them also exhibit the inhibition of matrix metalloproteinases and bacterial proteases. We investigated the antimicrobial agents used in wound healing for their potential to inhibit bacterial and host proteases relevant to chronic wounds.

Methods

Using in vitro zymography, we tested the ability of povidone-iodine, silver lactate, chlorhexidine digluconate, and octenidine hydrochloride to inhibit selected human proteases and proteases from Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, and Serratia liquefaciens. We investigated penetration and skin protease inhibition by means of in situ zymography.

Results

All the tested antimicrobials inhibited both eukaryotic and prokaryotic proteases in a dose-dependent manner in vitro. The tested compounds were also able to penetrate into skin ex vivo and inhibit the resident proteases. Silver lactate and chlorhexidine digluconate showed an inhibitory effect ex vivo even in partial contact with skin in Franz diffusion cells.

Conclusions

Our in vitro and ex vivo results suggest that wound healing devices which contain iodine, silver, chlorhexidine, and octenidine may add value to the antibacterial effect and also aid in chronic wound healing. Antiprotease effects should be considered in the design of future antimicrobial wound healing devices.

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<![CDATA[Topical essential fatty acid oil on wounds: Local and systemic effects]]> https://www.researchpad.co/article/5c390bafd5eed0c48491dd3a

Background

The use of medicinal plants and their derivatives is increasing, and approximately one-third of all traditional herbal medicines are intended for wound treatment. Natural products used in these treatments include vegetable oils, which are rich in essential fatty acids. Once in contact with an ulcerative surface, the oil reaches the blood and lymphatic vessels, thus eliciting systemic effects.

Objective

This study evaluated the local and possible systemic effects of essential fatty acids (sunflower oil) applied topically to rat wounds.

Methods

Cutaneous punch wounds (6 mm) were produced on the dorsa of 30 rats. Saline (SS), mineral oil (MO) or essential fatty acid (EFA) solutions were applied topically. Healing was evaluated after 2, 4 and 10 days (n = 5 per group) by visual and histological/morphometric examination, second harmonic generation (SHG) microscopy, and cytokine and growth factor quantification in the scar tissue (real-time PCR) and in serum (ELISA).

Results

MO/EFA-treated animals had higher IGF-1, leptin, IL-6 and IFN-γ mRNA expression and lower serum IL-6 levels than the control (SS/MO) animals. SHG analysis showed no difference in collagen density between the animals treated with MO and EFA.

Conclusion

EFA treatment induces topical (observed by local IGF-1, leptin, IL-6 and IFN-γ production) and systemic effects, lowering IL-6 levels in the serum. As the oil is widely used to shorten ulcer healing time, studies are needed to evaluate the treatment safety and possible undesired effects.

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<![CDATA[Microbiome Composition and Function Drives Wound-Healing Impairment in the Female Genital Tract]]> https://www.researchpad.co/article/5989da37ab0ee8fa60b869de

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.

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<![CDATA[Accelerated Wound Closure - Differently Organized Nanofibers Affect Cell Migration and Hence the Closure of Artificial Wounds in a Cell Based In Vitro Model]]> https://www.researchpad.co/article/5989d9feab0ee8fa60b730a7

Nanofiber meshes holds great promise in wound healing applications by mimicking the topography of extracellular matrix, hence providing guidance for crucial cells involved in the regenerative processes. Here we explored the influence of nanofiber alignment on fibroblast behavior in a novel in vitro wound model. The model included electrospun poly-ε-caprolactone scaffolds with different nanofiber orientation. Fibroblasts were cultured to confluency for 24h before custom-made inserts were removed, creating cell-free zones serving as artificial wounds. Cell migration into these wounds was evaluated at 0-, 48- and 96h. Cell morphological analysis was performed using nuclei- and cytoskeleton stainings. Cell viability was assessed using a biochemical assay. This study demonstrates a novel in vitro wound assay, for exploring of the impact of nanofibers on wound healing. Additionally we show that it’s possible to affect the process of wound closure in a spatial manner using nanotopographies, resulting in faster closure on aligned fiber substrates.

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<![CDATA[Comparison of corneal epitheliotrophic capacities among human platelet lysates and other blood derivatives]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc507

Purpose

To evaluate the corneal epitheliotropic abilities of two commercialized human platelet lysates (HPLs) and to compare the results with other blood derivatives, including human peripheral serum (HPS) and bovine fetal serum (FBS).

Methods

In vitro, human corneal epithelial cells were incubated in various concentrations (0%, 3%, 5% and 10%) of blood derivatives. Two commercialized HPLs, including UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were tested and compared with HPS and FBS. Scratch-induced directional wounding assay was performed to evaluate cellular migration. MTS assay was used to evaluate cellular proliferation. Cellular differentiation was examined by scanning electron microscopy, inverted microscopy and transepithelial electrical resistance. Sprague-Dawley rats were used to evaluate the effects of the blood derivatives on corneal epithelial wound healing in vivo. Different blood derivatives were applied topically every 2 hours for 2 days after corneal epithelial debridement. The concentrations of epidermal growth factor (EGF), transforming growth factor -β1 (TGF-β1), fibronectin, platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, and hyaluronic acid in different blood derivatives were evaluated by enzyme-linked immunosorbent assay (ELISA).

Results

In vitro experiments demonstrated statistically comparable epitheliotropic characteristics in cellular proliferation, migration, and differentiation for the two commercialized HPLs compared to FBS and HPS. Cells cultured without any serum were used as control group. The epitheliotropic capacities were statistically higher in the two commercialized HPLs compared to the control group (p<0.05). Among the different concentrations of blood derivatives, the preparations with 3% yielded better outcomes compared to 5% and 10%. In rats, HPLs also caused improved but not statistically significant wound healing compared to HPS. All the blood derivatives had better wound healing ratios than the control group (p<0.05). In the quantification of epitheliotropic factors, UltraGRO and PLTMax had significantly higher levels of EGF, TGF- β1, fibronectin than human peripheral serum (p<0.05).

Conclusions

Both commercialized HPLs showed comparable corneal epitheliotropic abilities and wound healing rates compared to HPS and FBS in the in vivo and in vitro studies. Our results suggest that HPLs may have the potential to replace HPS in the treatment of corneal epithelial problems.

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<![CDATA[Physiological and Pathological Impact of Blood Sampling by Retro-Bulbar Sinus Puncture and Facial Vein Phlebotomy in Laboratory Mice]]> https://www.researchpad.co/article/5989db42ab0ee8fa60bd7070

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals' physiological condition, which should be considered whenever blood samples are obtained.

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<![CDATA[Photodynamic Therapy Using Systemic Administration of 5-Aminolevulinic Acid and a 410-nm Wavelength Light-Emitting Diode for Methicillin-Resistant Staphylococcus aureus-Infected Ulcers in Mice]]> https://www.researchpad.co/article/5989da3dab0ee8fa60b88ba0

Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds.

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<![CDATA[Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbcd4

During wound healing, skin function is restored by the action of several cell types that undergo differentiation, migration, proliferation and/or apoptosis. These dynamics are tightly regulated by the evolution of the extra cellular matrix (ECM) contents along the process. Pharmacologically active flavonoids have shown to exhibit useful physiological properties interesting in pathological states. Among them, oleanolic acid (OA), a pentacyclic triterpene, shows promising properties over wound healing, as increased cell migration in vitro and improved wound resolution in vivo. In this paper, we pursued to disclose the molecular mechanisms underlying those effects, by using an in vitro scratch assay in two epithelial cell lines of different linage: non-malignant mink lung epithelial cells, Mv1Lu; and human breast cancer cells, MDA-MB-231. In every case, we observed that OA clearly enhanced cell migration for in vitro scratch closure. This correlated with the stimulation of molecular pathways related to mitogen-activated protein (MAP) kinases, as ERK1,2 and Jun N-terminal kinase (JNK) 1,2 activation and c-Jun phosphorylation. Moreover, MDA-MB-231 cells treated with OA displayed an altered gene expression profile affecting transcription factor genes (c-JUN) as well as proteins involved in migration and ECM dynamics (PAI1), in line with the development of an epithelial to mesenchymal transition (EMT) status. Strikingly, upon OA treatment, we observed changes in the epidermal growth factor receptor (EGFR) subcellular localization, while interfering with its signalling completely prevented migration effects. This data provides a physiological framework supporting the notion that lipophilic plant extracts used in traditional medicine, might modulate wound healing processes in vivo through its OA contents. The molecular implications of these observations are discussed.

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<![CDATA[Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells]]> https://www.researchpad.co/article/5989db4bab0ee8fa60bda620

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.

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<![CDATA[The Heat Shock Transcription Factor HSF1 Induces Ovarian Cancer Epithelial-Mesenchymal Transition in a 3D Spheroid Growth Model]]> https://www.researchpad.co/article/5989db0cab0ee8fa60bca8ff

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. The proteotoxic stress-responsive transcription factor HSF1 is frequently overexpressed in a variety of cancers and is vital to cellular proliferation and invasion in some cancers. Upon analysis of various patient data sets, we find that HSF1 is frequently overexpressed in ovarian tumor samples. In order to determine the role of HSF1 in ovarian cancer, inducible HSF1 knockdown cell lines were created. Knockdown of HSF1 in SKOV3 and HEY ovarian cancer cell lines attenuates the epithelial-to-mesenchymal transition (EMT) in cells treated with TGFβ, as determined by western blot and quantitative RT-PCR analysis of multiple EMT markers. To further explore the role of HSF1 in ovarian cancer EMT, we cultured multicellular spheroids in a non-adherent environment to simulate early avascular tumors. In the spheroid model, cells more readily undergo EMT; however, EMT inhibition by HSF1 becomes more pronounced in the spheroid model. These findings suggest that HSF1 is important in the ovarian cancer TGFβ response and in EMT.

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<![CDATA[Plasminogen activator activity in tears of pregnant women]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf5ba

Purpose

Plasminogen activator activity (PAA) in tears of pregnant women was investigated at various gestation times to assess the availability of plasminogen activator for aiding potential corneal wound healing processes during pregnancy.

Methods

PAA was measured by a spectrophotometric method. The analysis used 91 tear samples from pregnant and non-pregnant women, supplemented with 10 additional tear PAA measurements from non-pregnant women obtained in a previous study.

Results

Tear levels of PAA in pregnant women formed a bimodal distribution. Either the tear PAA level was zero or non-zero during pregnancy. When non-zero, the tear PAA level was dissociated from gestation time and not different than non-pregnant and post-pregnant levels. The frequency of occurrence of zero level tear PAA increased with gestation: 16%, 17% and 46% had zero tear PAA in samples taken from women in the first, second and third trimester, respectively.

Conclusions

Overall, of the tear samples taken from women during pregnancy, a total of 26% were at zero tear PAA. The remaining tear samples had non-zero tear PAA values throughout gestation equivalent to non-pregnant tear PAA values, suggesting local control of the source of PAA in tears. Given the importance of the plasminogen activator system in tears to wound healing in the cornea, and the high occurrence of zero tear PAA in our sample of pregnant women, elective corneal surgery would be contraindicated. If corneal surgery is nevertheless necessary, the tear PAA level would be worth checking and patients with low level should be closely observed during the postoperative period.

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<![CDATA[Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdcfe7

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.

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<![CDATA[Impact of diabetes on gingival wound healing via oxidative stress]]> https://www.researchpad.co/article/5bf279b2d5eed0c484fc2fdc

The aim of this study is to investigate the mechanisms linking high glucose to gingival wound healing. Bilateral wounds were created in the palatal gingiva adjacent to maxillary molars of control rats and rats with streptozotocin-induced diabetes. After evaluating postsurgical wound closure by digital imaging, the maxillae including wounds were resected for histological examinations. mRNA expressions of angiogenesis, inflammation, and oxidative stress markers in the surgical sites were quantified by real-time polymerase chain reaction. Primary fibroblast culture from the gingiva of both rats was performed in high glucose and normal medium. In vitro wound healing and cell proliferation assays were performed. Oxidative stress marker mRNA expressions and reactive oxygen species production were measured. Insulin resistance was evaluated via PI3K/Akt and MAPK/Erk signaling following insulin stimulation using Western blotting. To clarify oxidative stress involvement in high glucose culture and cells of diabetic rats, cells underwent N-acetyl-L-cysteine treatment; subsequent Akt activity was measured. Wound healing in diabetic rats was significantly delayed compared with that in control rats. Nox1, Nox2, Nox4, p-47, and tumor necrosis factor-α mRNA levels were significantly higher at baseline in diabetic rats than in control rats. In vitro study showed that cell proliferation and migration significantly decreased in diabetic and high glucose culture groups compared with control groups. Nox1, Nox2, Nox4, and p47 expressions and reactive oxygen species production were significantly higher in diabetic and high glucose culture groups than in control groups. Akt phosphorylation decreased in the high glucose groups compared with the control groups. Erk1/2 phosphorylation increased in the high glucose groups, with or without insulin treatment, compared with the control groups. Impaired Akt phosphorylation partially normalized after antioxidant N-acetyl-L-cysteine treatment. Thus, delayed gingival wound healing in diabetic rats occurred because of impaired fibroblast proliferation and migration. Fibroblast dysfunction may occur owing to high glucose-induced insulin resistance via oxidative stress.

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<![CDATA[RhoA promotes epidermal stem cell proliferation via PKN1-cyclin D1 signaling]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbb76

Objective

Epidermal stem cells (ESCs) play a critical role in wound healing, but the mechanism underlying ESC proliferation is not well defined. Here, we explore the effects of RhoA on ESC proliferation and the possible underlying mechanism.

Methods

Human ESCs were enriched by rapid adhesion to collagen IV. RhoA(+/+)(G14V), RhoA(-/-)(T19N) and pGFP control plasmids were transfected into human ESCs. The effect of RhoA on cell proliferation was detected by cell proliferation and DNA synthesis assays. Induction of PKN1 activity by RhoA was determined by immunoblot analysis, and the effects of PKN1 on RhoA in terms of inducing cell proliferation and cyclin D1 expression were detected using specific siRNA targeting PKN1. The effects of U-46619 (a RhoA agonist) and C3 transferase (a RhoA antagonist) on ESC proliferation were observed in vivo.

Results

RhoA had a positive effect on ESC proliferation, and PKN1 activity was up-regulated by the active RhoA mutant (G14V) and suppressed by RhoA T19N. Moreover, the ability of RhoA to promote ESC proliferation and DNA synthesis was interrupted by PKN1 siRNA. Additionally, cyclin D1 protein and mRNA expression levels were up-regulated by RhoA G14V, and these effects were inhibited by siRNA-mediated knock-down of PKN1. RhoA also promoted ESC proliferation via PKN in vivo.

Conclusion

This study shows that the effect of RhoA on ESC proliferation is mediated by activation of the PKN1-cyclin D1 pathway in vitro, suggesting that RhoA may serve as a new therapeutic target for wound healing.

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<![CDATA[Optimization of Non-Thermal Plasma Treatment in an In Vivo Model Organism]]> https://www.researchpad.co/article/5989db10ab0ee8fa60bcbdcc

Non-thermal plasma is increasingly being recognized for a wide range of medical and biological applications. However, the effect of non-thermal plasma on physiological functions is not well characterized in in vivo model systems. Here we use a genetically amenable, widely used model system, Drosophila melanogaster, to develop an in vivo system, and investigate the role of non-thermal plasma in blood cell differentiation. Although the blood system in Drosophila is primitive, it is an efficient system with three types of hemocytes, functioning during different developmental stages and environmental stimuli. Blood cell differentiation in Drosophila plays an essential role in tissue modeling during embryogenesis, morphogenesis and also in innate immunity. In this study, we optimized distance and frequency for a direct non-thermal plasma application, and standardized doses to treat larvae and adult flies so that there is no effect on the viability, fertility or locomotion of the organism. We discovered that at optimal distance, time and frequency, application of plasma induced blood cell differentiation in the Drosophila larval lymph gland. We articulate that the augmented differentiation could be due to an increase in the levels of reactive oxygen species (ROS) upon non-thermal plasma application. Our studies open avenues to use Drosophila as a model system in plasma medicine to study various genetic disorders and biological processes where non-thermal plasma has a possible therapeutic application.

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<![CDATA[Loss of TRPV4 Function Suppresses Inflammatory Fibrosis Induced by Alkali-Burning Mouse Corneas]]> https://www.researchpad.co/article/5989da27ab0ee8fa60b81461

In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor β, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice.

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<![CDATA[Insulin treatment prevents wounding associated changes in tissue and circulating neutrophil MMP-9 and NGAL in diabetic rats]]> https://www.researchpad.co/article/5b36e10c463d7e60298b5940

Neutrophils are important for wound repair, but their persistence can impair the healing process. Neutrophils express matrix metalloproteinases including MMP-9 and its regulator neutrophil gelatinase associated lipocalin (NGAL). Whether wounding affects neutrophil MMP-9 and NGAL in diabetic animals is not known. Skin wound tissue MMP-9 and NGAL was examined by qRT-PCR and immunohistochemistry in control, diabetic and insulin treated diabetic rats. The temporal expression of MMP-9 and NGAL mRNA, MMP-9 activity and the NGAL/MMP-9 complex was also investigated in an implant model and their circulating neutrophils. The cellular localisation of MMP-9 and NGAL was confirmed by immunofluorescence and the ability of glucose to regulate these factors was examined in isolated neutrophils. In skin wound tissue compared with control, diabetes increased neutrophil infiltration, NGAL mRNA and MMP-9 protein (P<0.05). Diabetes significantly increased implant neutrophil NGAL and MMP-9 protein as well as NGAL mRNA, wound fluid NGAL/MMP-9 complex and MMP-9 activity (all <0.05). Circulating neutrophil MMP-9 and NGAL was also increased in these diabetic animals (P<0.05). These changes were prevented by insulin treatment. Ex vivo, high glucose (25mM) increased neutrophil NGAL and MMP-9 (both by 2 fold, P<0.05). NGAL and MMP-9 are increased in wound and circulating neutrophils in diabetic rodents. These changes and the association between higher NGAL and increased wound fluid MMP-9 activity suggest that increased neutrophil NGAL may contribute to increased MMP-9 in poorly healing diabetic wounds. Whether targeting neutrophil NGAL or MMP-9 can improve diabetic wound healing remains to be investigated.

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