Ilomastat contributes to the survival of mouse after irradiation via promoting the recovery of hematopoietic system.

Ilomastat, a broad-spectrum inhibitor of matrix metalloproteinases (MMPs), has drawn attentions for its function in alleviating radiation damage. However, the detailed mechanisms of Ilomastat's protection from animal model remain not fully clear. In this study, the C57BL/6 mice were pre-administrated with Ilomastat or vihicle for 2 h, and then total body of mice were exposed to 6 Gy of γ-rays. The protective effect of Ilomastat on the hematopoietic system in the irradiated mice were investigated. We found that pretreatment with Ilomastat significantly reduced the level of TGF-ß1 and TNF-α, and elevated the number of bone marrow (BM) mononuclear cells in the irradiated mice. Ilomastat pretreatment also increased the fraction of BM hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) at day 30 after irradiation, and protected the spleen of mouse from irradiation. These results suggest that Ilomastat promotes the recovery of hematopoietic injury in the irradiated mice, and thus contributes to the survival of mouse after irradiation.

Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients.

Chronic obstructive pulmonary disease (COPD) is due to structural changes and narrowing of small airways and parenchymal destruction (loss of the alveolar attachment as a result of pulmonary emphysema), which all lead to airflow limitation. Extracorporeal shock waves (ESW) increase cell proliferation and differentiation of connective tissue fibroblasts. To date no studies are available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects. We obtained primary bronchial fibroblasts from bronchial biopsies of 3 patients with mild/moderate COPD and 3 control smokers with normal lung function. 16HBE cells were also studied. Cells were treated with a piezoelectric shock wave generator at low energy (0.3 mJ/mm2, 500 pulses). After treatment, viability was evaluated and cells were recultured and followed up for 4, 24, 48, and 72 h. Cell growth (WST-1 test) was assessed, and proliferation markers were analyzed by qRT-PCR in cell lysates and by ELISA tests in cell supernatants and cell lysates. After ESW treatment, we observed a significant increase of cell proliferation in all cell types. C-Kit (CD117) mRNA was significantly increased in 16HBE cells at 4 h. Protein levels were significantly increased for c-Kit (CD117) at 4 h in 16HBE (p < 0.0001) and at 24 h in COPD-fibroblasts (p = 0.037); for PCNA at 4 h in 16HBE (p = 0.046); for Thy1 (CD90) at 24 and 72 h in CS-fibroblasts (p = 0.031 and p = 0.041); for TGFß1 at 72 h in CS-fibroblasts (p = 0.038); for procollagen-1 at 4 h in COPD-fibroblasts (p = 0.020); and for NF-κB-p65 at 4 and 24 h in 16HBE (p = 0.015 and p = 0.0002). In the peripheral lung tissue of a representative COPD patient, alveolar type II epithelial cells (TTF-1+) coexpressing c-Kit (CD117) and PCNA were occasionally observed. These data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in 16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects.

Radiation-Induced Amplification of TGFB1-Induced Mesenchymal Stem Cell-Mediated Sodium Iodide Symporter (NIS) Gene 131I Therapy.

PURPOSE: The innate tumor homing potential of mesenchymal stem cells (MSCs) has been used for a targeted delivery of the theranostic sodium iodide symporter (NIS) transgene into solid tumors. We have previously shown that external beam radiotherapy (EBRT) results in the enhanced recruitment of NIS-expressing MSCs into human hepatocellular carcinoma (HuH7). In parallel, the tumor-associated cytokine TGFB1 becomes strongly upregulated in HuH7 tumors in response to EBRT. EXPERIMENTAL DESIGN: We therefore evaluated the effects of combining focused EBRT (5 Gy) with MSC-mediated systemic delivery of the theranostic NIS transgene under control of a synthetic TGFB1-inducible SMAD-responsive promoter (SMAD-NIS-MSCs) using 123I-scintigraphy followed by 131I therapy in CD1 nu/nu mice harboring subcutaneous human hepatocellular carcinoma (HuH7). RESULTS: Following tumor irradiation and SMAD-NIS-MSC application, tumoral iodide uptake monitored in vivo by 123I-scintigraphy was enhanced as compared with nonirradiated tumors. Combination of EBRT and SMAD-NIS-MSC-mediated 131I therapy resulted in a significantly improved delay in tumor growth and prolonged survival in therapy mice as compared with the combined therapy using CMV-NIS-MSCs or to control groups receiving EBRT or saline only, or EBRT together with SMAD-NIS-MSCs and saline applications. CONCLUSIONS: MSC-based NIS-mediated 131I therapy after EBRT treatment dramatically enhanced therapeutic efficacy when a TGFB1-inducible SMAD-responsive promoter was used to drive NIS expression in adoptively applied MSCs. The remarkable therapeutic effect seen is thought to be linked in large part to the enhanced TGFB1 produced in this context, which leads to a highly selective and focused amplification of MSC-based NIS expression within the tumor milieu.

Effect of bronchial thermoplasty on structural changes and inflammatory mediators in the airways of subjects with severe asthma.

BACKGROUND: Bronchial thermoplasty (BT) is a novel technique used in the treatment of subjects with severe refractory asthma. Radiofrequency is provided to airway walls during bronchoscopy in order to reduce airway remodeling. Several clinical studies have reported an improvement in subjects' symptoms following BT. However, how BT affects the airway architectures and inflammatory mediators in the airways has not been yet fully elucidated. METHODS: Fourteen subjects with severe asthma were recruited in this study according to the criteria of ATS severe asthma definition. The study subjects undertook bronchial biopsy during the bronchoscopy procedure at baseline and 6 weeks after the initial BT treatment. The obtained samples were stained with antibodies for α-smooth muscle actin (α-SMA); protein gene product (PGP) 9.5, a specific nerve marker; von Willebrand factor (vWF), a marker for blood vessels; interleukin-17A (IL-17A) and transforming growth factor-ß1 (TGF-ß1). RESULTS: The expression of α-SMA and PGP9.5 were significantly reduced post-BT. There was no significant difference in the number of blood vessels between baseline and post-BT. In addition, BT did not affect the production of IL-17A and TGF-ß1 in the airways. The changes in the expression of α-SMA and PGP9.5 had no significant correlation with the improvement of pulmonary function. CONCLUSION: and Clinical Relevance: This study suggests that BT reduces airway smooth muscle mass and the airway innervation without affecting vasculature and the production of inflammatory mediators in the airways of subjects with severe asthma.

Differing energy densities with laser 670nm InGaP controls inflammation and collagen reorganization in burns.

PURPOSE: This study compared different energy densities of laser on second degrees burns in rats aiming to determine the most effective dosimetry in stimulation of the healing process. METHODS: Burns were induced in the dorsal skin of 54 animals divided into three groups (n: 18): 1-without treatment; 2-irradiated lesions by the Indium Gallium Phosphide (InGaP) 670nm (4.93J/cm2) laser; 3-irradiated lesions by the InGaP-670nm (9.86J/cm2) laser. Samples were collected on the 2, 10 and 18 days after injury for structural, morphometry, biochemical analysis and Western blotting. RESULTS: The energy densities examined were effective in significantly increasing the total number of fibroblasts and blood vessels and reduce the number of inflammatory cells particularly in irradiated lesions with 9.86J/cm2. This same energy density significantly increased the amount of GAGs (Glycosaminoglycans), decreased the TGF-ß1 (Transforming Growth Factor ß1) and increased the VEGF (Vascular and Endothelial Growth Factor) during the experimental period. This energy density also significantly increased the Collagen type I and decreased Collagen type III and the active isoform of metalloproteinase 9 (MMP-9). CONCLUSIONS: The energy density of 9.86J/cm2 was more effective in promoting cellular responses related to neoangiogenesis, decreasing inflammation and collagen fibers reorganization.

Vigna angularis water extracts protect against ultraviolet b-exposed skin aging in vitro and in vivo.

Exposure to ultraviolet (UV) radiation induces various pathological changes, such as thickened skin and wrinkle formation. In particular, UVB irradiation increases matrix metalloproteinase (MMP)-1 production and collagen degradation, leading to premature aging, termed photoaging. The azuki bean (Vigna angularis; VA) has been widely used as a food product as well as a traditional medicine. However, its activity needs additional study to confirm its functional application in foods and cosmetics for protecting skin. In this study, hot-water extract from VA (VAE) and its active component, rutin, were investigated to determine their antiphotoaging effects. VAE was found to have antioxidant activity. In UVB-exposed normal human dermal fibroblasts cells with VAE and rutin treatments, MMP-1 production was significantly suppressed (90% and 47%, respectively). The effects of both topical and oral administration of VAE were tested in UVB-irradiated hairless mice. VAE suppressed wrinkle formation and skin thickness by promoting elastin, procollagen type I, and TGF-ß1 expression (118%, 156%, and 136%, respectively) and by diminishing MMP-1 production. These results suggest that VAE may be effective for preventing skin photoaging accelerated by UVB radiation.

Photoactivation of endogenous latent transforming growth factor-ß1 directs dental stem cell differentiation for regeneration.

Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor-ß1 (TGF-ß1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-ß1 (LTGF-ß1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-ß1 was capable of differentiating human dental stem cells in vitro. Further, an in vivo pulp capping model in rat teeth demonstrated significant increase in dentin regeneration after LPL treatment. These in vivo effects were abrogated in TGF-ß receptor II (TGF-ßRII) conditional knockout (DSPP(Cre)TGF-ßRII(fl/fl)) mice or when wild-type mice were given a TGF-ßRI inhibitor. These findings indicate a pivotal role for TGF-ß in mediating LPL-induced dental tissue regeneration. More broadly, this work outlines a mechanistic basis for harnessing resident stem cells with a light-activated endogenous cue for clinical regenerative applications.

Albumin acts like transforming growth factor ß1 in microbubble-based drug delivery.

Unlike lipid-shelled microbubbles (MBs), albumin-shelled microbubbles (MBs) have not been reported to be actively targeted to cells without the assistance of antibodies. Recent studies indicate that the albumin molecule is similar to transforming growth factor ß (TGF-ß) both structurally and functionally. The TGF-ß superfamily is important during early tumor outgrowth, with an elevated TGF-ß being tumor suppressive; at later stages, this switches to malignant conversion and progression, including breast cancer. TGF-ß receptors I and II play crucial roles in both the binding and endocytosis of albumin. However, until now, no specific albumin receptor has been found. On the basis of the above-mentioned information, we hypothesized that non-antibody-conjugated albumin-shelled MBs can be used to deliver drugs to breast cancer cells. We also studied the possible roles of TGF-ß1 and radiation force in the behavior of cells and albumin-shelled MBs. The results indicate that albumin-shelled MBs loaded with paclitaxel (PTX) induce breast cancer cell apoptosis without the specific targeting produced by an antibody. Applying either an acoustic radiation force or cavitation alone to cells with PTX-loaded albumin MBs increased the apoptosis rate to 23.2% and 26.3% (p < 0.05), respectively. We also found that albumin-shelled MBs can enter MDA-MB-231 breast cancer cells and remain there for at least 24 h, even in the presence of PTX loading. Confocal micrographs revealed that 70.5% of the breast cancer cells took up albumin-shelled MBs spontaneously after 1 d of incubation. Applying an acoustic radiation force further increased the percentage to 91.9% in our experiments. However, this process could be blocked by TGF-ß1, even with subsequent exposure to the radiation force. From these results, we conclude that TGF-ß1 receptors are involved in the endocytotic process by which albumin-shelled MBs enter breast cancer cells. The acoustic radiation force increases the contact rate between albumin-shelled MBs and tumor cells. Combining a radiation force and cavitation yields an apoptosis rate of 31.3%. This in vitro study found that non-antibody-conjugated albumin-shelled MBs provide a useful method of drug delivery. Further in vivo studies of the roles of albumin MBs and TGF-ß in different stages of cancer are necessary.

The effect of ultraviolet radiation on the transforming growth factor beta 1/Smads pathway and p53 in actinic keratosis and normal skin.

Ultraviolet (UV) radiation is considered to be essential for the progression of actinic keratosis (AK) to squamous cell carcinoma (SCC); however, the mechanisms have not been fully elucidated. To understand this process, the effects of UV radiation on the transforming growth factor beta 1 (TGFß1)/Smads pathway and p53 in normal skin and AK were studied. Normal human skin and AK tissues were cultured and divided into the following four groups according to the UV radiation dose: 0 (control group), 5, 10, and 20 J/cm2. The tissues were radiated for four consecutive days; 24 h after radiation, the tissues were collected for investigation. Compared with the control group, greater proliferative inhibition and apoptosis were induced by UV radiation in normal skin than AK. The expression of TGFß1, Smad7, and p53 was increased in AK and normal skin, while the level of TßRII was decreased. Smad2 was reduced in AK only. The expressions of TßRI, Smad3, and Smad4 were not significantly changed. The results demonstrated that although p53 was induced, suppression of the TGFß1/Smads pathway by UV radiation might contribute to the progression of AK to SCC.

Tissue-engineered mucosa is a suitable model to quantify the acute biological effects of ionizing radiation.

The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.

Targeting the TGF-beta1 pathway to prevent normal tissue injury after cancer therapy.

With >10,000,000 cancer survivors in the U.S. alone, the late effects of cancer treatment are a significant public health issue. Over the past 15 years, much work has been done that has led to an improvement in our understanding of the molecular mechanisms underlying the development of normal tissue injury after cancer therapy. In many cases, these injuries are characterized at the histologic level by loss of parenchymal cells, excessive fibrosis, and tissue atrophy. Among the many cytokines involved in this process, transforming growth factor (TGF)-beta1 is thought to play a pivotal role. TGF-beta1 has a multitude of functions, including both promoting the formation and inhibiting the breakdown of connective tissue. It also inhibits epithelial cell proliferation. TGF-beta1 is overexpressed at sites of injury after radiation and chemotherapy. Thus, TGF-beta1 represents a logical target for molecular therapies designed to prevent or reduce normal tissue injury after cancer therapy. Herein, the evidence supporting the critical role of TGF-beta1 in the development of normal tissue injury after cancer therapy is reviewed and the results of recent research aimed at preventing normal tissue injury by targeting the TGF-beta1 pathway are presented.

Preoperative radiotherapy and extracellular matrix remodeling in rectal mucosa and tumour matrix metalloproteinases and plasminogen components.

BACKGROUND. Preoperative radiotherapy reduces recurrence but increases postoperative morbidity. The aim of this study was to explore the effect of radiotherapy in rectal mucosa and rectal tumour extracellular matrix (ECM) by studying enzymes and growth factors involved in ECM remodeling. MATERIALS AND METHODS. Twenty patients with short-term preoperative radiotherapy and 12 control patients without radiotherapy were studied. Biopsies from rectal mucosa and tumour were collected prior to radiotherapy and at surgery. Tissue MMP-1, -2, -9, TIMP-1, uPA, PAI-1, TGF-beta1 and calprotectin were determined by ELISA. Biopsies from irradiated and non-irradiated peritoneal areas were also analysed. RESULTS. Radiotherapy increased the tissue levels of MMP-2 and PAI-1 in both the rectal mucosa and tumours while calprotectin and uPA showed an increase only in the mucosa after irradiation. The increase of calprotectin was due to an influx of inflammatory cells as revealed by immunohistochemistry. Prior to irradiation, the tumour tissues had increased levels of MMP-1, -2, -9, total TGF-beta1, uPA, PAI-1 and calprotectin compared to mucosa, while TIMP-1 and the active TGF-beta1 fraction showed no statistical difference. CONCLUSIONS. This study indicates a radiation-induced effect on selected ECM remodeling proteases. This reaction may be responsible for early and late morbidity. Interference of this response might reduce these consequences.

[Effect of intense pulsed light on transforming growth factor-beta1 mRNA expression in rat skin].

OBJECTIVE: To observe the effect of intense pulsed light (IPL) on transforming growth factor-beta1 mRNA (TGF-beta1 mRNA) expression in rat skin and explore the molecular mechanisms of photorejuvenation. METHODS: Fifteen SD rats were exposed to IPL in 3 dermal regions with triple pulses (duration of 4, 5, and 6 ms) at the energy density of 34 J/cm2 and pulse delay of 20 or 25 ms. On days 1, 3, 5, 7, 15, and 30 after the treatment, skin specimens from the treated and non-treated areas were obtained to detect TGF-beta1 mRNA expression with in situ hybridization. RESULTS: In the UPL-exposed skin areas, TGF-beta1 mRNA expression was detected in the epidermal keratinocytes and dermal cells 1 day after the exposure, reaching the highest expression level on day 7 followed by gradual decrement since day 15, and till day 30, only weak expression was found in the dermal cells. In the non-exposed regions, the cells remained negative for TGF-beta1 mRNA. CONCLUSION: IPL can enhance TGF-beta1 mRNA expression in the skin, suggesting that TGF-beta1 plays an important role in dermal remodeling in photorejuvenation.

Flashlamp pulsed-dye laser suppressed TGF-beta1 expression and proliferation in cultured keloid fibroblasts is mediated by MAPK pathway.

BACKGROUND AND OBJECTIVES: Our previous clinical study indicated that transforming growth factor-beta1 (TGF-beta1) and mitogen-activated protein kinases (MAPK) are both involved in keloid regression following flashlamp pulsed-dye laser (PDL). To further characterize of this involvement, this work examined whether PDL suppression of TGF-beta1 expression was mediated through MAPK pathway in cultured keloid fibroblasts (KF). STUDY DESIGN/MATERIALS AND METHODS: Primary culture of KF harvested from keloid patients received various dosages of PDL treatment in 585-nm wavelength. TGF-beta1 expressions in KF following various dosages of PDL were assessed. Additionally, MAPK pathway activities were studied using the PD98059 (an ERK inhibitor), SB203580 (a p38 kinase inhibitor), and SP600125 (a JNK inhibitor), to determine the role in keloid following PDL treatment. Activator protein-1 (AP-1), a transcription factor of TGF-beta, was analyzed by electrophoretic mobility shift assay (EMSA). Phosphorylated c-Jun, one of the components of AP-1, was also detected. RESULTS: The observation results demonstrated that optimal dosages of PDL significantly suppressed KF proliferation and TGF-beta1 expression. EMSA study identified PDL downregulation of super-shift of AP-1. Three subtypes of MAPK cascades were augmented between 30 minutes and 4 hours following PDL treatment, particularly phosphorylation of ERK1/2 and p38. Pre-treatment with PD98059, SB203580, but not SP600125, markedly inhibited the downregulating effects of TGF-beta1 and phosphorylated c-Jun expression following PDL treatment. CONCLUSION: PDL induced keloid regression is mediated by triggering MAPK cascades and blockade of AP-1 transcription and TGF-beta expression. Modulation of TGF-beta and MAPK interaction in keloids may provide specific targets for therapeutic intervention.

Influence of quercetin and x-ray on collagen synthesis of cultured human keloid-derived fibroblasts.

OBJECTIVE: To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism. METHODS: Collagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen I and III expression. mRNA expression of collagen I and III, and transforming growth factor (TGF)-beta 1 were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS: Quercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent manner. Immunocytochemical staining indicated that collagen I and III were down-regulated by quercetin and X-ray (P < 0.05), particularly collagen I (P < 0.05). mRNA expression of both collagen I and III in quercetin groups significantly decreased compared with that in control group (P < 0.05), especially in the group treated with both quercetin and X-ray (P < 0.01). mRNA level of TGF-beta 1 gene was down-regulated by quercertin (P < 0.05). CONCLUSIONS: Quercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.