Expression Of Procollagen Type Research Articles

Actinidia chinensis Planch Ameliorates Photoaging in UVB-Irradiated NIH-3T3 Cells and SKH-1 Hairless Mice by Controlling the Reactive Oxygen Species/AKT Pathway.

In this study, we evaluated the antiphotoaging properties of Actinidia chinensis Planch (ACP) and the molecular mechanisms underlying its ability to prevent UVB-mediated photoaging. Administration of the ethanolic extract of ACP (EEACP) to the dorsal area of hairless mice effectively ameliorated UVB-mediated wrinkle formation, epidermal thickening, and loss of lipid droplets in the epidermis. Additionally, the UVB-induced loss of collagen content in the epidermis was significantly attenuated in mouse skin treated with EEACP. The expression of procollagen type 1 and metalloproteinase-1a, which are related to collagen content in the epidermis, was restored by EEACP treatment in UVB-irradiated mice and NIH-3T3 mouse skin fibroblast cells. Interestingly, EEACP effectively ameliorated UVB-induced reactive oxygen species overproduction. Furthermore, the activation/phosphorylation of AKT, rather than mitogen-activated protein kinases, has been identified as a major target of EEACP in preventing UVB-mediated photoaging. Additionally, N-(1 deoxy-1-fructosyl) valine and phenethylamine glucuronide were identified as analytical indicators of EEACP using high-performance liquid chromatography/mass spectrometry. These results suggest that EEACP can be developed as a functional natural agent capable of preventing photoaging by attenuating UVB-induced activation of the reactive oxygen species/AKT pathway.

Impact of Immunosuppressive Drugs on Fibroblasts: An In Vitro Study.

Background: The aim of this study was to compare the direct impact of different agents for immunosuppressive therapy on mouse fibroblasts as a possible cause of drug-induced gingival overgrowth (DIGO). Methods: 3T3 mouse fibroblasts were cultivated in cell-specific media (2 × 104 cells/mL) and treated for 6, 24, 48 and 72 h with one of three immunosuppressive drugs (IsDs): cyclosporin a (CsA), tacrolimus (TaC) and sirolimus (SiR). Different concentrations (10–750 ng/mL) were used to mimic serum levels under active immunosuppressive therapy conditions. Cell population characteristics (cell number, viability and morphology) were assessed using computer-assisted cell analysis. Expression of pro-collagen type I carboxy-terminal propeptide (PICP) was identified using an ELISA assay. Results: The influence of IsDs on the biological status of 3T3 fibroblasts was time- and dose-dependent. Comparing CsA and TaC, the total cell amount was enhanced using concentrations in the range of 10–150 ng/mL (p > 0.05). In contrast, treatment with SiR resulted in a decrease in the average cell number (p < 0.01). PICP and cell diameter of fibroblasts were not susceptible to IsD treatment (p > 0.05). Conclusions: Our results revealed time-dependent effects of IsDs, with distinct influences on cell number. The cell morphology and the PICP balance of the investigated fibroblast cell line remained unaffected. Hence, the potential role of IsDs is not a unilateral mechanism of action but rather a multifactorial process.

Pro-fibrotic phenotype of bone marrow stromal cells in Modic type 1 changes.

Modic type 1 changes (MC1) are painful vertebral bone marrow lesions frequently found in patients suffering from chronic low-back pain. Marrow fibrosis is a hallmark of MC1. Bone marrow stromal cells (BMSCs) are key players in other fibrotic bone marrow pathologies, yet their role in MC1 is unknown. The present study aimed to characterise MC1 BMSCs and hypothesised a pro-fibrotic role of BMSCs in MC1. BMSCs were isolated from patients undergoing lumbar spinal fusion from MC1 and adjacent control vertebrae. Frequency of colony-forming unit fibroblast (CFU-F), expression of stem cell surface markers, differentiation capacity, transcriptome, matrix adhesion, cell contractility as well as expression of pro-collagen type I alpha 1, α-smooth muscle actin, integrins and focal adhesion kinase (FAK) were compared. More CFU-F and increased expression of C-X-C-motif-chemokine 12 were found in MC1 BMSCs, possibly indicating overrepresentation of a perisinusoidal BMSC population. RNA sequencing analysis showed enrichment in extracellular matrix proteins and fibrosis-related signalling genes. Increases in pro-collagen type I alpha 1 expression, cell adhesion, cell contractility and phosphorylation of FAK provided further evidence for their pro-fibrotic phenotype. Moreover, a leptin receptor high expressing (LEPRhigh) BMSC population was identified that differentiated under transforming growth factor beta 1 stimulation into myofibroblasts in MC1 but not in control BMSCs. In conclusion, pro-fibrotic changes in MC1 BMSCs and a LEPRhigh MC1 BMSC subpopulation susceptible to myofibroblast differentiation were found. Fibrosis is a hallmark of MC1 and a potential therapeutic target. A causal link between the pro-fibrotic phenotype and clinical characteristics needs to be demonstrated.

Hedgehog signaling regulates the basement membrane remodeling during folliculogenesis in the neonatal mouse ovary.

In the mouse ovary, interactions between oocytes and somatic cells are essential for folliculogenesis and subsequent follicle development. The polyovular follicle (PF), which contains more than two oocytes in a follicle, can be induced in the neonatal mouse ovary when interactions between oocytes and somatic cells are disrupted by agents such as the potent synthetic estrogen diethylstilbestrol (DES) acting through estrogen receptor (ER) β. Hedgehog signaling is known to regulate granulosa cell proliferation, thecal cell differentiation, and follicle growth. To investigate the role of hedgehog signaling in the early folliculogenesis and in PF induction by DES, neonatal mouse ovaries were cultured with or without 10 μM cyclopamine (CPA), an inhibitor of hedgehog signaling, and grafted under the kidney capsule of adult ovariectomized host mice. The number and the incidence of PFs were significantly increased in organ-cultured ovaries post-grafting. Expression of procollagen type IV, alpha 1 (Col4a1) in organ-cultured ovaries was significantly reduced by CPA, but not by DES. The expression of two hedgehog ligands, Desert hedgehog (Dhh) and Indian hedgehog (Ihh), and a target gene, Hedgehog interacting protein (Hhip), was significantly increased by DES both in WT and ERβ KO mice. Therefore, we infer that DES can affect expression of those genes through ERα but not via suppression of hedgehog signaling. Thus, PFs are induced by DES or CPA, but the induction mechanism is different. Our results revealed an important role of hedgehog signaling in basement membrane remodeling during folliculogenesis even before thecal cell differentiation.

Adiponectin-Based Peptide (ADP355) Inhibits Transforming Growth Factor-β1-Induced Fibrosis in Keloids.

Keloids, benign cutaneous overgrowths of dermal fibroblasts, are caused by pathologic scarring of wounds during healing. Current surgical and therapeutic modalities are unsatisfactory. Although adiponectin has shown an antifibrotic effect, its large size and insolubility limit its potential use in keloid treatment. We investigated the effect of a smaller and more stable adiponectin-based peptide (ADP355) on transforming growth factor β1 (TGF-β1)-induced fibrosis in a primary culture of keloid fibroblasts prepared from clinically obtained keloid samples. Xenograft of keloid tissues on athymic nude mice was used to investigate the effect of intralesional injection of ADP355. ADP355 significantly attenuated the TGF-β1-induced expression of procollagen type 1 in keloid fibroblasts (p < 0.05). Moreover, it inhibited the TGF-β1-induced phosphorylation of SMAD3 and ERK, while amplifying the phosphorylation of AMP-activated protein kinase (p < 0.05). Knockdown of adiponectin receptor 1 reversed the attenuation of procollagen expression in ADP355-treated TGF-β1-induced fibrosis (p < 0.05). ADP355 also significantly reduced the gross weight and procollagen expression of keloid tissues in xenograft mice compared to control animals. These results demonstrate the therapeutic potential of the adiponectin peptide ADP355 for keloids.

Abstract 654: Loss of SPRR3 In ApoE -/- Mice Leads to Increased Atheroma Vulnerability and Evidence of Plaque Rupture and Cardiac Infarcts

Atheroma rupture is the leading cause of myocardial infarction. While studies have examined inflammatory cell-mediated effects on plaque vulnerability, less is known about the role of vascular smooth muscle cells (VSMCs) or specific molecular players in the maintenance of atheroma stability. We reported that loss of small proline-rich repeat protein 3 (SPRR3), enriched in atheroma VSMCs, leads to increased VSMC death and significantly accelerates atherosclerosis progression in ApoE -/- mice. Here, we show that loss of SPRR3 promotes features in plaques of brachiocephalic arteries common to unstable lesions, such as increased necrotic core size, reduced cap collagen content, and reduced VSMC content. Moreover, ApoE -/- mice lacking SPRR3 develop coronary artery lesions with advanced features, including intraplaque hemorrhage. In addition, Sprr3 -/- ApoE -/- mice fed a high-fat diet for 6 months develop spontaneous myocardial infarction. In vitro , SPRR3 deficient VSMCs show reduced expression of procollagen type I, an event associated with Akt activation. SPRR3-deficient VSMCs also show increased expression of MMP2 transcripts, and aortic root lesions of Sprr3 -/- ApoE -/- mice have increased gelatinase activity consistent with MMP2 activation. Our data demonstrate that SPRR3 loss in ApoE -/- mice decreases VSMC survival and collagen I synthesis while increasing MMP2 synthesis and activity, resulting in atheroma instability with evidence of downstream myocardial infarction. Taken together the results present the Sprr3 -/- ApoE -/- mouse as an experimental model of plaque rupture. This model will be used for additional experimental studies including in vivo genetic modulation of the Akt pathway as well as in vitro studies to determine phenotypic outcome, i.e. coronary arterial lesions, myocardial infarction, VSMC survival and collagen synthesis. We hope to establish a mechanistic link between altered Akt signaling and matrix integrity in the context of atheroma rupture, as well as potentially use SPRR3 as a molecular marker which could lead to detection of plaque instability as well as therapeutic intervention methodologies.

Caffeine Inhibits the Activation of Hepatic Stellate Cells Induced by Acetaldehyde via Adenosine A2A Receptor Mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK Signal Pathway

Hepatic stellate cell (HSC) activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR). Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine’s inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway. Conclusions: Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III.

Macroscopic effect of blue light cure on wound healing in NMRI mice NMRI.

Background:Wound healing is a complex process and has been an ongoing challenge all over the world. Some studies have suggested light cure as a modality to accelerate wound repair. It can induce fibroblast proliferation, increase collagen synthesis and activate cellular processes involved in expression of procollagen type I and III mRNA. This study was designed to assess the macroscopic effect of halogen dental curing blue light on full-thickness open wound healing in NMRI mice.Materials and Methods:Forty male NMRI mice were divided into control and treatment groups. A full-thickness wound of 6 mm in diameter was induced on the lower back of all mice under general anesthesia and sterile conditions. The mice of the treatment group received a 5-min exposure of halogen light Coltolux II (QHL), 420-500 nm, daily for 7 days. The diameter of the wound was measured in both the treatment and the control groups every second day up to Day 14. Data were analyzed by SPSS version 12 software using Student's t-test. A significance level of P ≤ 0.05 was considered for each comparison.Results:There was a significant difference in wound diameter between the control and the treatment groups at all measurements after Day 3 (P ≤ 0/05).Conclusion:The results of this study suggest improvement of full-thickness wound healing by daily irradiation of halogen dental curing blue light of 420-500 nm for 7 days.

Association of Cardiotrophin-1 With Myocardial Fibrosis in Hypertensive Patients With Heart Failure

Cardiotrophin-1 has been shown to be profibrogenic in experimental models. The aim of this study was to analyze whether cardiotrophin-1 is associated with left ventricular end-diastolic stress and myocardial fibrosis in hypertensive patients with heart failure. Endomyocardial biopsies from patients (n=31) and necropsies from 7 control subjects were studied. Myocardial cardiotrophin-1 protein and mRNA and the fraction of myocardial volume occupied by collagen were increased in patients compared with controls (P<0.001). Cardiotrophin-1 overexpression in patients was localized in cardiomyocytes. Cardiotrophin-1 protein was correlated with collagen type I and III mRNAs (r=0.653, P<0.001; r=0.541, P<0.01) and proteins (r=0.588, P<0.001; r=0.556, P<0.005) in all subjects and with left ventricular end-diastolic wall stress (r=0.450; P<0.05) in patients. Plasma cardiotrophin-1 and N-terminal pro-brain natriuretic peptide and serum biomarkers of myocardial fibrosis (carboxy-terminal propeptide of procollagen type I and amino-terminal propeptide of procollagen type III) were increased (P<0.001) in patients compared with controls. Plasma cardiotrophin-1 was correlated with N-terminal pro-brain natriuretic peptide (r=0.386; P<0.005), carboxy-terminal propeptide of procollagen type I (r=0.550; P<0.001), and amino-terminal propeptide of procollagen type III (r=0.267; P<0.05) in all subjects. In vitro, cardiotrophin-1 stimulated the differentiation of human cardiac fibroblast to myofibroblasts (P<0.05) and the expression of procollagen type I (P<0.05) and III (P<0.01) mRNAs. These findings show that an excess of cardiotrophin-1 is associated with increased collagen in the myocardium of hypertensive patients with heart failure. It is proposed that exaggerated cardiomyocyte production of cardiotrophin-1 in response to increased left ventricular end-diastolic stress may contribute to fibrosis through stimulation of fibroblasts in heart failure of hypertensive origin.

Abstract 4052: Targeting procollagen secretory pathway for the treatment of primary brain tumor through ER stress.

Abstract We have previously shown that primary brain tumor from patients and cell lines possess type I alpha 1 procollagen. Procollagen type I, III, and IV have also been reported to be expressed in certain glioma subtypes. Although the function of these procollagens is not known, they are most likely involved in invasion and signaling. We have further investigated whether manipulation of the secretion of these procollagens can be used to kill glioma cells. We have studied the expression of procollagen type I alpha 1 and2 (Pro-col1A1 &amp; A2), procollagen type III alpha 1 (Pro-col3A1) and procollagen type IV alpha 1&amp;2 (Proc-col4A1 &amp; A2) in 5 cell lines (Glioma-1, U-373, U-118, U-87, and A-172) using real time RT-PCR (qPCR) and western blot when antibody was available. Our data showed that A-172 expressed all 5 types of procollagen, while U-87, U-118 and Glioma-1 expressed primary Pro-colA1 and A2, and U-373 expressed only pro-col1A2 and pro-col4A1 and A2. Hsp47, which is a major chaperone in the folding of procollagen type I, is not expressed in U-373. This cell line also possessed very high levels of GRP-78 which is indicative of increased ER stress. We also investigated the presence of collagen receptor Endo 180 and DDR1 (discodin domain receptor 1) in these cell lines. Endo 180 was present in all 5 cell lines, but to a much lesser extent in U-373. The DDR1 expression by qPCR was the highest in A-172 followed by U-118 (&amp;gt;5 arbitrary units) while U-373, U-87 and Glioma-1 have less than 3 arbitrary units. It is known that procollagen needs to be folded at ER and then secreted out of the cells, where it will be processed to form collagen. Collagen can then bind DDR1, a receptor tyrosine kinase which plays a role in attachment, migration, proliferation and survival. In this regard, we have detected pro-col1A1 and A2 in the media which verified that pro-col1A1 and A2 are secreted outside the cells. We hypothesize that inability to secrete procollagen will result in both ER stress and inability for signaling. To accomplish this, we have studied the cytotoxic effect of brefeldin (BFA) which is known to block the transport of secretory protein from ER/Golgi network to the cell surface in these five cell lines. All five cell lines are very sensitive to brefeldin with IC50 of 3.0 ng/ml for U-118, A-172 and U-87. In U-373 and Glioma-1 the IC50 was 5.0 ng/ml. Interestingly, cancer stem cells isolated from U-87 did not possess pro-co1A1, but DDR1 mRNA was present and was 3 times higher than in the non stem cells. They also have different metabolic profiling than their parental cells, such as they do not express argininosuccinate synthetase, a key enzyme to synthesize arginine which made them sensitive to arginine deprivation treatment. Whether these differences exist in vivo is not known. Overall, our data indicate that the expression of procollagen and its secretory pathway can be used as a new paradigm to treat primary brain tumor through ER stress-mediated cell death. Citation Format: Min You, Shu-Mei Chen, Chunjing Wu, Ying-Ying Li, Lynn Feun, Medhi Wangpaithitr, Vy Dinh, Niramol Savaraj. Targeting procollagen secretory pathway for the treatment of primary brain tumor through ER stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4052. doi:10.1158/1538-7445.AM2013-4052

Caspase-3 activation and increased procollagen type I in irradiated hearts

The caspase-3-cleaved presence was evaluated in this study in the heart of irradiated rats, during the decline of ventricular function. Female Wistar rats were irradiated with a single dose of radiation (15 Gy) delivered directly to the heart and the molecular, histological and physiological evaluations were performed at thirteen months post-irradiation. The expressions of procollagen type I, TGF-ß1 and caspase-3-cleaved were analyzed using Western blotting. Cardiac structural and functional alterations were investigated by echocardiography and electron microscopy. In the irradiated group, the levels of procollagen type I, TGF-ß1 and caspase-3-cleaved are increased. Significant histological changes (degeneration of heart tissue and collagen deposition) and functional (reduced ejection fraction) were observed. Data suggest that the cardiac function decline after exposure to ionizing radiation is related, in part, to increased collagen and increased caspase-3-cleaved.

A comparison of Q-switched 1064 nm Nd:YAG laser and intense pulsed light in the nonablative rejuvenation on rat model

Background and objective: A variety of laser and IPL treatments with continued progress have been applied for nonablative skin rejuvenation; however, the complete understanding of working mechanisms and clinical appliance strategy is not clear. Materials and methods: The rats were divided into three groups and irradiated with Q-switched 1064 nm Nd:YAG laser and IPL. Image analysis, chemical colorimetry method, and real-time PCR (RT-PCR) were used to detect the dermal thickness, hydroxyproline, and the expression of III procollagen mRNA, respectively, at sequential time points following irradiation. In addition, the ultra-structure changes of rat skin were observed by TEM at 3 weeks after irradiation. Results: Two-light treatment contributed to increase in the dermal thickness, the hydroxyproline contents and the expression of III procollagen mRNA, and the dense arrangement of collagen. The effect of collagen synthesis and remodeling could last for at least 3 months after treatment, and the YAG group is more efficient than the IPL group. The expressions of procollagen type III mRNA reached peak level at 2 weeks. Conclusion: The effect of different lights depends on the wavelength and the penetrated depth; the best referential treatment interval of two kinds of lights for nonablative skin rejuvenation on rat skin is 2 weeks.

TGF-β1 and IGF-1 influence the re-differentiation capacity of human chondrocytes in 3D pellet cultures in relation to different oxygen concentrations

To prevent de-differentiation of chondrocytes in vitro, the 3D environment, growth factors and different oxygen concentrations were considered. In this in vitro study, we quantified the influence of insulin-like growth factor (IGF)-1 and/or transforming growth factor (TGF)-β1 under differing oxygen (5/21% O(2)) levels on the proliferation and synthesis rates of hyaline extracellular matrix (ECM) components in chondrogenic pellet cultures. Human chondrocytes isolated from articular cartilage were transferred into conical tubes to form pellets. Pellets were stimulated with TGF-β1 and/or IGF-1. After 2 and 5 weeks of cultivation the DNA concentration and expression of pro-collagen type 1, type 2 and aggrecan were analysed. Under hypoxia the DNA content remained stable. In contrast, under normoxia, cells showed an increase of DNA concentration after stimulation with TGF-β1/IGF-1 and TGF-β1. Nevertheless, DNA contents under normoxia did not reach the values of hypoxic-cultivated cells. Under both culture conditions a reduced synthesis of pro-collagen type 1 could be determined. Although the expression of pro-collagen type 2 was significantly higher under normoxia, a decrease in the case of TGF-β1/IGF-1- and IGF-1-stimulated cells was observed. Under hypoxia pro-collagen type 2 contents remained stable or increased for TGF-β1/IGF-1-stimulated cells. Furthermore, incubation with growth factors resulted in aggrecan accumulation under hypoxia, while a reduced expression under normoxia could be determined for TGF-β1/IGF-1- and IGF-1-stimulated cells. Our results demonstrate that the treatment with growth factors causes differences in the expression of ECM compounds within pellet cultures. While under normoxia TGF-β1 alone leads to a positive effect of the expression of hyaline cartilage-specific ECM components, an additive effect of both growth factors was only determined under hypoxia.

Influence of lipopolysaccharide on collagen metabolism of normal skin fibroblasts of human

Objective To observe the influence of lipopolysaccharide (LPS) on collagen metabolism of normal human skin fibroblasts and its biological role in the formation of hypertrophic scar.Methods Fibroblasts were isolated and cultured in vitro,and then exposed to different doses of LPS (0.005,0.01,0.05,0.1,0.5,1.0 μg/ml) from E.coli.055:B5 respectively.The expression of proccllagen type Ⅰ,Ⅲand collagenase mRNAs was tested by RT -PCR.Fibroblasts from hypertrophic scar tissue obtained from the same patients in the same culture passage were used as control.Results Compared with control group,the expression of procollagen type Ⅰ,Ⅲ mRNAs in normal skin fibroblasts increased (0.323 ± 0.041,0.303 ± 0.063,0.391 ± 0.071,0.344 ± 0.086,0.488 ± 0.059,0.401 ± 0.087,0.616 ± 0.107,0.434 ±0.084,0.823 ±0.092,0.542 ± 0.082),while the expression of collagenase mRNAs of normal skin fibroblasts depressed(0.598 ± 0.068,0.556 ± 0.049,0.441 ± 0.043,0.372 ± 0.083,0.260 ± 0.027 ).When LPS was set to the concentration of 0.005 μg/ml,it showed a concentration dependent manner.However,when the concentration of LPS was set to 0.5 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts began to decrease (0.451 ± 0.063,0.374 ± 0.072,0.360 ± 0.062).When the concentration of LPS was set to 1.0 μg/ml,the expression of procollagen type Ⅰ,Ⅲ mRNAs (0.162 ± 0.025,0.171 ± 0.061 )were inhibited and the expression of collagenase mRNAs began to increase (0.444 ±0.114).When the concentration of LPS was set to 0.1 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts(0.823 ±0.092,0.542 ±0.082,0.260 ±0.027)was similar to that of hypertrophic scar tissue fibroblasts(0.829 ±0.049,0.569 ±0.038,0.277 ±0.059).Conclusions This result supported that LPS may be an important factor in collagen metabolism of normal skin fibroblasts and it plays an important role in hypertrophic scar formation. Key words: Lipopolysaccharides/PD ; Collagen/ME ; Skin/CY/DE; Fibroblasts/ME/DE

Hepatitis C virus core protein induces fibrogenic actions of hepatic stellate cells via toll-like receptor 2

Hepatic stellate cells (HSCs) represent the main fibrogenic cell type accumulating extracellular matrix in the liver. Recent data suggest that hepatitis C virus (HCV) core protein may directly activate HSCs. Therefore, we examined the influence of recombinant HCV core protein on human HSCs. Primary human HSCs and the human HSC line LX-2 were stimulated with recombinant HCV proteins core and envelope 2 protein. Expression of procollagen type I α-1, α-smooth muscle actin, cysteine- and glycine-rich protein 2, glial fibrillary acidic protein, tissue growth factor β1, matrix metalloproteinases 2 (MMP2) and 13, tissue inhibitor of metalloproteinases 1 and 2 was investigated by real-time PCR. Intracellular signaling pathways of ERK1/2, p38 and, jun-amino-terminal kinase (JNK) were analyzed by western blot analysis. Recombinant HCV core protein induced upregulation of procollagen type I α-1, α-smooth muscle actin, MMP 2 and 13, tissue inhibitor of metalloproteinases 1 and 2, tissue growth factor β1, cysteine- and glycine-rich protein 2, and glial fibrillary acidic protein mRNA expression, whereas HCV envelope 2 protein did not exert any significant effect. Blocking of toll-like receptor 2 (TLR2) with a neutralizing antibody prevented mRNA upregulation by HCV core protein confirming that the TLR2 pathway was involved. Furthermore, western blot analysis revealed HCV-induced phosphorylation of the TLR2-dependent signaling molecules ERK1/2, p38 and JNK mitogen-activated kinases. Our in vitro results demonstrate a direct effect of HCV core protein on activation of HSCs toward a profibrogenic state, which is mediated via the TLR2 pathway. Manipulating the TLR2 pathway may thus provide a new approach for antifibrotic therapies in HCV infection.

Abstract 3324: Epithelial mesenchymal transition is associated with chemoresistance of poorly differentiated hepatoma cells

Abstract BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. It is the most rapidly increasing type of cancers in the US. Chemotherapy has been largely ineffective for HCC treatment, especially in cases with poorly-differentiated HCC. Cancer stem cells (CSCs) are a critical subpopulation that may be responsible for the initiation, progression, metastasis and recurrence of malignancies. CD133 and aldehyde dehydrogenase (ALDH) are useful markers to identify CSCs in solid tumors, such as lung and pancreatic cancers. AIMS: To separate CD133+/ALDHhigh cells in well-defined hepatoma cell lines, and to evaluate chemosentivity based on their CSC marker profile. METHODS: Hepatoma cell lines: HepG2, Hep3B, Huh-7, HLE, HLF and SKHep1 were separated into CD133+/ALDHhigh and CD133−/ALDHlow subpopulations by FACS. The resulting subpopulations were seeded on non-attachable plates for spheroid formation or used for chemosentivity assays. Epithelial mesenchymal transition (EMT) was determined by immunocytochemical staining of E-cadherin and vimentin. Gene expression of procollagen type I (α1) (Procoll-I) and transforming growth factor β1 (TGF-β1) was determined by quantitative RT-PCR. RESULTS: In well-differentiated hepatoma cell lines, HepG2, Hep3B and Huh-7, the positivity for CD133+/ALDHhigh was 1.1, 13.4 and 15.6%; whereas, poorly-differentiated hepatoma cells were almost all negative for these two markers. The positivity for CD133/EpCAM was 2.3, 10.3 and 18.3% in HepG2, Hep3B and Huh-7 cells, whereas it was almost negative in HLE, HLF and SKHep1 cells. FACS-enriched CD133+/ALDHhigh HepG2, Huh-7 and Hep3B cells were able to form spheroids in non-attachable culture plates; whereas their negative counterparts failed. In contrast, CD133−/ALDHlow SKHep1, HLF and HLE cells formed larger spheroids with negative α-feto protein staining. More CD133−/ALDHlow HLE cells survived in the exposure to cisplatin or doxorubicin than their positive counterparts (p&amp;lt;0.05-0.01). Moreover, CD133+/ALDHhigh HepG2, Hep3B and Huh-7 cells were positive for E-cadherin, but negative for vimentin; whereas, their CD133−/ALDHlow cells displayed an opposite expression pattern of E-cadherin and vimentin. CD133−/ALDHlow HLE, HLF and SKHep1 cells were E-cadherin-negative and vimentin-positive. In accordance, mRNA levels of Procoll-I and TGF-β1 in CD133−/ALDHlow HLE cells were one-fold higher than their positive counterparts. CONCLUSIONS: In well-differentiated hepatoma cells, CD133+/ALDHhigh or CD133+/EpCAM+ cells appear to be a CSC/progenitor subpopulation; whereas, in poorly-differentiated hepatoma cell lines which are negative for these CSC markers, EMT may be responsible for their chemoresistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3324.

The role of interleukin-1β and platelet-derived growth factor-AB in antifibrosis mediated by native human interferon α

Commercial preparations of native human interferon alpha (nHuIFN-alpha) contain several subtypes of interferon-alpha (IFN-alpha) and traces of other cytokines. Recently, we described its antifibrotic potential and showed nHuIFN-alpha to have a greater effect than that of recombinant human IFN-alpha (rHuIFN-alpha). We hypothesized that cooperation between different cytokines in the nHuIFN-alpha preparation is essential for this effect. Considerable concentrations of interleukin-1beta (IL-1beta) and platelet-derived growth factor AB (PDGF-AB) are present in the nHuIFN-alpha preparations. We tested the viability and the expression of procollagen type I messenger RNA (mRNA) in MRC5 fibroblasts treated with interleukin-1 beta (IL-1beta) and/or PDGF-AB, or the corresponding antibodies in combination with rHuIFN-alpha or nHuIFN-alpha. We showed that neither IL-1beta nor PDGF-AB significantly affect the viability of MRC5 cells. Furthermore, cell viability was not affected when IL-1beta or PDGF-AB were applied along with rHuIFN-alpha, relative to the viability of cells treated with rHuIFN-alpha only. In contrast, both cytokines suppressed the synthesis of procollagen type I mRNA. When coadministered with rHuIFN-alpha, IL-1beta enhanced the suppression induced by rHuIFN-alpha. Conversely, PDGF-AB acted as an antagonist of rHuIFN-alpha and restored partially the synthesis of procollagen type I mRNA. Interestingly, the addition of IL-1beta to the PDGF-AB/rHuIFN-alpha mix not only abolished the antagonistic activity of PDGF-AB but also decreased the synthesis of procollagen type I mRNA beyond the level achieved by IL-1beta/rHuIFN-alpha. Therefore, IL-1beta was able to reverse the activity of PDGF-AB. Our study suggests that IL-1beta is an important component of nHuIFN-alpha preparations, acting directly and indirectly to modulate the action of other components. This study provides insight into these complex cytokine networks, which is necessary for better and safer antifibrotic therapy.

141 Potential Use of Nitric Oxide Gas as an Antimicrobial Agent in Cutaneous Wound Infection

Despite recent advances in wound care, chronic infected ulcers still remain a huge challenge to the modern society. Nitric oxide has been identified as a mediator for important biological processes such as vasodilation, antimicrobial, and wound repair. The aim of the present study was to explore the antimicrobial properties of exogenous nitric oxide gas (gNO) as well as its effect on skin cells in vitro. To test this, a specialized gNO chamber was designed to expose S. aureus and P. aeruginosa in saline continuously to 200 ppm gNO or air. A colony count was obtained following gNO treatment. Furthermore, fibroblast-populated collagen gel, keratinocytes, and endothelial cells were cultured in DMEM, KSFM, and M199, respectively. Endothelial migration was tested by a 3D Matrigel tubule formation assay. Fibroblast matrix production and keratinocyte differentiation were monitored by mRNA expression of procollagen type I, interstitial collagenase, and involucrin. All cells were exposed to 200 ppm gNO for 8 hours a day for 3 consecutive days. A MTT assay was used to observe cell proliferation. The results revealed 100% bacterial kill following 4 hour exposure to 200 ppm gNO. Interestingly this dose did not exhibit any significant toxic effect on fibroblast, keratinocyte, and endothelial proliferation. Dermal fibroblasts migration out of the collagen gel was not affected by gNO treatment when compared to the control. The keratinocytes in both groups were able to express involucrin, an indicator of cell’s ability to differentiate. The migration and tube formation by endothelial cells within the matrigel was not compromised following gNO treatment. In conclusion, the present study provided evidence for potential application of gNO for reducing bacterial burden in chronic infected wounds without compromising re-epithilialization, proliferation, and angiogenesis in the wound healing process. Acknowledgment: This study was funded by PulmoNOx Medical Inc.

Expression of heat shock protein 47 in the fibrous tissue adjacent to mouse tumour subjected to photodynamic therapy

The reaction of normal fibrous tissue adjacent to tumours subjected to photodynamic therapy (PDT) was investigated by assessment of the immunohistochemical expression of heat shock protein 47 (HSP47) and proliferating cell nuclear antigen (PCNA), as well as by immunoblot analysis of procollagen type I. PDT was administered to NR-S1 mouse squamous cell carcinoma or normal mouse skin. Each of four mice was investigated at several time points after receiving PDT. The levels of HSP47 expression were determined by computer-assisted image analysis. The expression of procollagen type I in the fibrous tissue adjacent to the tumours was examined by immunoblot analysis at intervals of 24 and 48h after PDT. The expression of HSP47 was first detected 6h post-PDT in the tumour-bearing mice, but no such expression was observed in the normal mice. It was also revealed that, after PDT, the fibroblast PCNA labeling indices at 24, 48, and 72h were significantly higher in both the tumour-bearing and the normal mice than in the control animals that did not receive PDT. Furthermore, procollagen type I was detected in the fibrous tissue adjacent to the tumours at 24 and 48h after PDT, but was not detected in the fibrous tissue adjacent to tumours of mice that did not receive PDT. Therefore, the present results suggest that PDT enhances the synthesis of collagen type I in the fibrous tissue adjacent to NR-S1 squamous cell carcinoma in mice, which contributes to the resultant encapsulation of such tumours.

Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation

Platelet-rich plasma (PRP) is widely used to promote tissue healing. However, there is no concrete evidence for the biological effects of PRP. This study evaluated the biological effects of PRP on the proliferation and differentiation of 2 human osteoblast-like cell lines. Human osteosarcoma cell lines HOS and SaOS-2 were used in this study. PRP was prepared from freshly drawn human venous blood containing a large number of platelets. The MTT assay was used to examine the effects of PRP on osteoblast viability. To evaluate the growth and differentiation, alkaline phosphatase activity was assessed and the expression of procollagen type I, osteopontin, and osteoprotegerin mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction. Further, core binding factor alpha 1 (cbfa1/Runx2/AML3/PebpalphaA), a critical regulator of osteoblast differentiation, was also determined. The administration of PRP enhanced the viability of HOS and SaOS-2 cells in a dose-dependent manner. Alkaline phosphatase activity was suppressed during the cell growth phase, but was strongly enhanced when the cells reached confluence. Semiquantitative reverse-transcription polymerase chain reaction analysis showed that PRP enhanced the levels of procollagen type I, osteopontin, osteoprotegerin, and core binding factor alpha 1 (cbfa1) mRNA. These results suggest that PRP has a favorable effect on human osteoblast-like cells, and acts both to enhance bone regeneration and as an activator in wound healing.