Lumican Accelerates Wound Healing by Enhancing α2β1 Integrin-Mediated Fibroblast Contractility

Lumican is a dermatan sulfate proteoglycan highly expressed in connective tissue and has the ability to regulate collagen fibril assembly. Previous studies have shown that lumican is involved in wound healing, but the precise effects of lumican on reepithelialization and wound contraction, the two pivotal aspects of skin wound healing, have not been investigated. Here we explored the roles of lumican in fibroblast contractility, a main aspect of skin wound healing, by adopting mice skin wound healing model and the corresponding in vitro cellular experiments. Our results showed that lumican can promote skin wound healing by facilitating wound fibroblast activation and contraction but not by promoting keratinocyte proliferation and migration. Silencing of integrin α2 completely abolished the pro-contractility of lumican, indicating lumican enhances fibroblast contractility via integrin α2. Our study for the first time demonstrated that lumican can affect fibroblast’s mechanical property, which is pivotal for many important pathological processes, such as wound healing, fibrosis, and tumor development, suggesting that lumican might have a potential to be used to modulate these processes.     

Adipose tissue extract shows potential for wound healing: in vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma

Growth factors are the key elements in wound healing signaling for cell migration, differentiation and proliferation. Platelet-rich plasma (PRP), one of the most studied sources of growth factors, has demonstrated to promote wound healing in vitro and in vivo. Adipose tissue is an alternative source of growth factors. Through a simple lipoaspirate method, adipose derived growth factor-rich preparation (adipose tissue extract; ATE) can be obtained. The authors set out to compare the effects of these two growth factor sources in cell proliferation and migration (scratch) assays of keratinocyte, fibroblast, endothelial and adipose derived stem cells. Growth factors involved in wound healing were measured: keratinocyte growth factor, epidermal growth factor, insulin-like growth factor, interleukin 6, platelet-derived growth factor beta, tumor necrosis factor alfa, transforming growth factor beta and vascular endothelial growth factor. PRP showed higher growth factor concentrations, except for keratinocyte growth factor, that was present in adipose tissue in greater quantities. This was reflected in vitro, where ATE significantly induced proliferation of keratinocytes at day 6 (p < 0.001), compared to plasma and control. Similarly, ATE-treated fibroblast and adipose stem cell cultures showed accelerated migration in scratch assays. Moreover, both sources showed accelerated keratinocyte migration. Adipose tissue preparation has an inductive effect in wound healing by proliferation and migration of cells involved in wound closure. Adipose tissue preparation appears to offer the distinct advantage of containing the adequate quantities of growth factors that induce cell activation, proliferation and migration, particularly in the early phase of wound healing.     

Endoglin Haploinsufficiency Promotes Fibroblast Accumulation during Wound Healing through Akt Activation

Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng^+/−) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng^+/− MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng^+/− mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng^+/− mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.     

Xanthine Oxidoreductase Function Contributes to Normal Wound Healing

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H₂O₂, 0.15%) or allopurinol (30 μg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H₂O₂ and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H₂O₂ restored wound healing in tungsten-fed mice. In vitro, nitrite and H₂O₂ both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.     

Chaperonin Containing T-Complex Polypeptide Subunit Eta (CCT-eta) Is a Specific Regulator of Fibroblast Motility and Contractility

Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult) wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta) is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF) stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA) reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (α-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less α-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased α-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of α-SMA with siRNA reduced both basal and growth factor-induced fibroblast motility. These results indicate that CCT-eta is a specific regulator of fibroblast motility and contractility and may be a key determinant of the scarless wound healing phenotype by means of its specific regulation of α-SMA expression.     

Fluctuation of ROS regulates proliferation and mediates inhibition of migration by reducing the interaction between DLC1 and CAV-1 in breast cancer cells

The aim of our present study was to elucidate the effects of up-regulation and down-regulation of intracellular reactive oxygen species (ROS) level on proliferation, migration, and related molecular mechanism. Breast cancer cells were treated by catalase or H₂O₂. MTT, colony formation assay, and Hoechst/PI staining were used to evaluate proliferation and apoptosis. The level of intracellular ROS was measured by dichlorodihydrofluorescein diacetate probes. The ability of migration was detected by wound healing. Western blotting and coimmunoprecipitation (co-IP) were used to determine the expression of DLC1 and CAV-1 and their interaction. Our data indicated that up-regulation of intracellular ROS induced by H₂O₂ significantly inhibited proliferation and induced apoptosis accompanying G1 cell cycle arrest and elevated expression of p53. For cell migration, either up-regulation or down-regulation of ROS induced migration inhibition with reduction of interaction between DLC1 and CAV-1. Our results suggested that up-regulation of intracellular ROS inhibited proliferation by promoting expression of p53 and induced G1 cycle arrest and apoptosis. Fluctuation of ROS inhibited migration through reducing the interaction between DLC1 and CAV-1.     

Proliferation and wound healing of vascular cells trigger the generation of extracellular reactive oxygen species and LDL oxidation

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (O₂^•− and H₂O₂) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.     

Spatial Analysis of 3′ Phosphoinositide Signaling in Living Fibroblasts: II. Parameter Estimates for Individual Cells from Experiments

Fibroblast migration is directed by gradients of platelet-derived growth factor (PDGF) during wound healing. As in other chemotactic systems, it has been shown recently that localized stimulation of intracellular phosphoinositide (PI) 3-kinase activity and production of 3′ PI lipids in the plasma membrane are important events in the signaling of spatially biased motility processes. In turn, 3′ PI localization depends on the effective diffusion coefficient, D, and turnover rate constant, k, of these lipids. Here we present a systematic and direct comparison of mathematical model calculations and experimental measurements to estimate the values of the effective 3′ PI diffusion coefficient, D, turnover rate constant, k, and other parameters in individual fibroblasts stimulated uniformly with PDGF. In the context of our uniform stimulation model, the values of D and k in each cell were typically estimated within 10–20% or less, and the mean values across all of the cells analyzed were D = 0.37 ± 0.25 μm²/s and k = 1.18 ± 0.54 min⁻¹. In addition, we report that 3′ PI turnover is not affected by PDGF receptor signaling in our cells, allowing us to focus our attention on the regulation of 3′ PI production as this system is studied further.     

PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22^phox, p40^phox, p47^phox and p67^phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.     

Protectin DX promotes epithelial injury repair and inhibits fibroproliferation partly via ALX/PI3K signalling pathway

Acute respiratory distress syndrome/acute lung injury (ARDS/ALI) is histologically characterized by extensive alveolar barrier disruption and excessive fibroproliferation responses. Protectin DX (PDX) displays anti‐inflammatory and potent inflammation pro‐resolving actions. We sought to investigate whether PDX attenuates LPS (lipopolysaccharide)‐induced lung injury via modulating epithelial cell injury repair, apoptosis and fibroblasts activation. In vivo, PDX was administered intraperitoneally (IP) with 200 ng/per mouse after intratracheal injection of LPS, which remarkedly stimulated proliferation of type II alveolar epithelial cells (AT II cells), reduced the apoptosis of AT II cells, which attenuated lung injury induced by LPS. Moreover, primary type II alveolar cells were isolated and cultured to assess the effects of PDX on wound repair, apoptosis, proliferation and transdifferentiation in vitro. We also investigated the effects of PDX on primary rat lung fibroblast proliferation and myofibroblast differentiation. Our result suggests PDX promotes primary AT II cells wound closure by inducing the proliferation of AT II cells and reducing the apoptosis of AT II cells induced by LPS, and promotes AT II cells transdifferentiation. Furthermore, PDX inhibits transforming growth factor‐β₁ (TGF‐β₁) induced fibroproliferation, fibroblast collagen production and myofibroblast transformation. Furthermore, the effects of PDX on epithelial wound healing and proliferation, fibroblast proliferation and activation partly via the ALX/ PI3K signalling pathway. These data present identify a new mechanism of PDX which targets the airway epithelial cell and fibroproliferation are potential for treatment of ARDS/ALI.     

Expression of TGF-β3 in isolated fibroblasts from foreskin

Background:

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s).

Methods:

We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

Conclusions:

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.Key Words: Fibroblasts, Gene expression, TGF-β3     

bFGF Regulates PI3-Kinase-Rac1-JNK Pathway and Promotes Fibroblast Migration in Wound Healing

Fibroblast proliferation and migration play important roles in wound healing. bFGF is known to promote both fibroblast proliferation and migration during the process of wound healing. However, the signal transduction of bFGF-induced fibroblast migration is still unclear, because bFGF can affect both proliferation and migration. Herein, we investigated the effect of bFGF on fibroblast migration regardless of its effect on fibroblast proliferation. We noticed involvement of the small GTPases of the Rho family, PI3-kinase, and JNK. bFGF activated RhoA, Rac1, PI3-kinase, and JNK in cultured fibroblasts. Inhibition of RhoA did not block bFGF-induced fibroblast migration, whereas inhibition of Rac1, PI3-kinase, or JNK blocked the fibroblast migration significantly. PI3-kinase-inhibited cells down-regulated the activities of Rac1 and JNK, and Rac1-inhibited cells down-regulated JNK activity, suggesting that PI3-kinase is upstream of Rac1 and that JNK is downstream of Rac1. Thus, we concluded that PI3-kinase, Rac1, and JNK were essential for bFGF-induced fibroblast migration, which is a novel pathway of bFGF-induced cell migration.     

Surface molecules on HaCaT keratinocytes after interaction with non‐thermal atmospheric pressure plasma

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α₂‐ and β₁‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H₂DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.     

Valproic Acid Induces Cutaneous Wound Healing In Vivo and Enhances Keratinocyte Motility

Background

Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK) signaling pathways. Valproic acid (VPA) is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown.

Methods and Findings

We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA), collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathways.

Conclusions

VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes.     

Epithelial Cells Are Active Participants in Vocal Fold Wound Healing: An In Vivo Animal Model of Injury

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.     

Hyaluronidase Modulates Inflammatory Response and Accelerates the Cutaneous Wound Healing

Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2^nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.     

Upregulation of miR-760 and miR-186 Is Associated with Replicative Senescence in Human Lung Fibroblast Cells

We have previously shown that microRNAs (miRNAs) miR-760, miR-186, miR-337-3p, and miR-216b stimulate premature senescence through protein kinase CK2 (CK2) down-regulation in human colon cancer cells. Here, we examined whether these four miRNAs are involved in the replicative senescence of human lung fibroblast IMR-90 cells. miR-760 and miR-186 were significantly upregulated in replicatively senescent IMR-90 cells, and their joint action with both miR-337-3p and miR-216b was necessary for efficient downregulation of the α subunit of CK2 (CK2α) in IMR-90 cells. A mutation in any of the four miRNA-binding sequences within the CK2α 3′-untranslated region (UTR) indicated that all four miRNAs should simultaneously bind to the target sites for CK2α downregulation. The four miRNAs increased senescence-associated β-galactosidase (SA-β-gal) staining, p53 and p21^Cip1/WAF1 expression, and reactive oxygen species (ROS) production in proliferating IMR-90 cells. CK2α over-expression almost abolished this event. Taken together, the present results suggest that the upregulation of miR-760 and miR-186 is associated with replicative senescence in human lung fibroblast cells, and their cooperative action with miR-337-3p and miR-216b may induce replicative senescence through CK2α downregulation-dependent ROS generation.