Procollagen Alpha Research Articles

Collagen hydroxylysine glycosylation: non-conventional substrates for atypical glycosyltransferase enzymes.

Collagen is a major constituent of the extracellular matrix (ECM) that confers fundamental mechanical properties to tissues. To allow proper folding in triple-helices and organization in quaternary super-structures, collagen molecules require essential post-translational modifications (PTMs), including hydroxylation of proline and lysine residues, and subsequent attachment of glycan moieties (galactose and glucose) to specific hydroxylysine residues on procollagen alpha chains. The resulting galactosyl-hydroxylysine (Gal-Hyl) and less abundant glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl) are amongst the simplest glycosylation patterns found in nature and are essential for collagen and ECM homeostasis. These collagen PTMs depend on the activity of specialized glycosyltransferase enzymes. Although their biochemical reactions have been widely studied, several key biological questions about the possible functions of these essential PTMs are still missing. In addition, the lack of three-dimensional structures of collagen glycosyltransferase enzymes hinders our understanding of the catalytic mechanisms producing this modification, as well as the impact of genetic mutations causing severe connective tissue pathologies. In this mini-review, we summarize the current knowledge on the biochemical features of the enzymes involved in the production of collagen glycosylations and the current state-of-the-art methods for the identification and characterization of this important PTM.

Age-Related Changes of Procollagen Alpha Polypeptide in Vascular Remodeling in Rat Vascular Smooth Muscle Cell

The current study revealed that increased synthesis and secretion of collagen types I and III play major roles in arterial wall remodeling, aneurysm formation, and atherosclerotic cap stability. The aim is to investigate the age-related changes of the procollagen alpha polypeptide gene mRNA and protein expression in the vascular smooth muscle cells (VSMCs) in rats, as well as the possible underlying mechanisms. We tested in vitro culture of VSMC from the thoracoabdominal aorta in neonate and 9-month-old healthy male Wistar rats; procollagen alpha polypeptide mRNA and procollagen alpha polypeptide protein expression were detected, using RT-PCR, VG staining, Western blot and ELISA methods. Semi-quantitative analysis displayed that, in the real-time reverse transcription polymerase chain reaction (RT-PCR), the type I collagen α polypeptide chain mRNA increased in the adult group, but not significantly (P = 0.05). Further, there was no significant difference between the two groups of type III collagen α polypeptide chain mRNA (P > 0.05). Both the type I and type III procollagen alpha polypeptide protein expression were increased significantly in the older group as compared with the young group (P < 0.05). This phenomenon mainly lies in the fact that the regulatory pathway on age-related changes of procollagen alpha polypeptides may be one of the molecular mechanisms in vascular remodeling during aging.

Inducible knockdown of procollagen I protects mice from liver fibrosis and leads to dysregulated matrix genes and attenuated inflammation

Organ fibrosis is characterized by a chronic wound-healing response, with excess deposition of extracellular matrix components. Here, collagen type I represents the most abundant scar component and a primary target for antifibrotic therapies. Liver fibrosis can progress to cirrhosis and primary liver cancer, which are the major causes of liver related morbidity and mortality. However, a (pro-)collagen type I specific therapy remains difficult and its therapeutic abrogation may incur unwanted side effects. We therefore designed tetracycline-regulated procollagen alpha1(I) short hairpin (sh)RNA expressing mice that permit a highly efficient inducible knockdown of the procollagen alpha1(I) gene in activated (myo-)fibroblasts, to study the effect of induced procollagen type I deficiency. Transgenic mice were generated using recombinase-mediated integration in embryonic stem cells or zinc-finger nuclease-aided genomic targeting combined with miR30-shRNA technology. Liver fibrosis was induced in transgenic mice by carbon tetrachloride, either without or with doxycycline supplementation. Doxycycline treated mice showed an 80–90% suppression of procollagen alpha1(I) transcription and a 40–50% reduction in hepatic collagen accumulation. Procollagen alpha1(I) knockdown also downregulated procollagens type III, IV and VI and other fibrosis related parameters. Moreover, this was associated with an attenuation of chronic inflammation, suggesting that collagen type I serves not only as major scar component, but also as modulator of other collagens and promoter of chronic inflammation.

The role of procollagen alpha 1 type 1 (Col1A1) on invasion and cellular signaling in glioma cells.

e23165 Background: We have previously shown that Procollagen alpha 1 type 1 (Col1A1) is found more in low and intermediate grade glioma and less often in glioblastoma(GBM) (Cancer Invest. 23:577, 2005). We now investigate their role in cellular function. Methods: 4 glioma cell lines: Glioma 1 and U118 express high amount of Col1A1 ( Col1A1+) ; A172 and SW1783 express insignificant amount of Col1A1 ( Col1A1 - ) as the model . All four cell lines express SPARC. Scratch, transwell, and metrigel assay were used to study migration and invasion. Cell cycles were analyzed by flowcytometry. Results: U118 has the highest amount of SPARC followed by A172, SW1783 and Glioma 1. Thus it does not appear to have any relationship between these two proteins which are known as binding partner. Glioma 1 showed the least invasion and migration followed by U118, SW1783 and A172. Thus, Col1A1 expression appear to correlate with invasiveness. To further confirm this, we have silence Col1A1 in Glioma 1 and U118 using both siRNA and shRNA. All clones exhibit more migration and invasion. However, it does not affect both intracellular and extracellular levels of SPARC. Silencing Col1A1 results in increasing G2M arrest; 11% in U118 and 6% in Glioma 1. However it does not affect cellular proliferation. To further verify this, we have overexpressed Col1A1 in A172 and SW1783 using plasmid containing Col1A1 and DDK tag. These Col1A1 (+) A172 and SW1783 transfectants exhibit less migration and invasion. However, there is no effect on SPARC levels. These Col1A1 positive cells exhibit 12% increase in Go/G1 arrest and decrease in proliferation. A limited protein array also showed that silencing Col1A1 increase in STAT3, 5 and 6 and AKT levels. Interestingly, a difference in sensitivity to STAT3/5 inhibitors also noted in parental and their Col1A1 knock down transfectants. Conclusions: our results support the role of Col1A1 in glioma cell invasiveness, and hence confirm our previous data which showed that Col1A1 is found more in low grade and intermediate grade glioma. Thus, Col1A1 could be an additional useful marker to assess the aggressiveness of GBM beside histopathological grading. Col1A1 may also play a role in cellular signaling pathway.

Abstract 3712: The relationship of Procollagen alpha 1 type 1 (Col1A1) / DDR2 signaling in malignant glioma and sensitivity to STAT 3/5 inhibitor

Abstract We have previously shown that Procollagen alpha 1 type 1 (Col1A1) is found in low and intermediate grade glioma and in less aggressive glioblastoma multiforme (GBM) (Cancer Invest. 23:577, 2005). However, these tumor cells are more prone to ER stress -inducing agents such as Befeldine (BFA) which block the transport of Procollagen to the cell surface. We also found that GBM cells which possess Col1A1 also express DDR2. The role of Col1A1/ DDR2 signaling in glioma is not known. We have used three GBM cell lines: Glioma1 established in our laboratory from a patient who progressed from grade 3 to grade 4, U-118, A-172 and U-373 to study this signaling pathway. Both Glioma1 and U-118 express Col1A1 and DDR1 and 2. A-172 and U-373 do not express Col1A1 but express both DDR2. U-373 does not express HSP47 which is an essential protein to fold collagen. In order to define the function of DDR2/ Col1A1 in GBM, we have used knock down COL1A1 and DDR2. Silencing Col1A1 leads to a significant increase in invasiveness by Matrigel assay which indicated that Col1A1 is important in preventing invasion in GBM and hence is found more in low and intermediate grade glioma. However, it has minimal effect on cell cycle or cell proliferation. Next, we have silenced DDR2 in all 4 cell lines and studied the biochemical changes. We found that silencing DDR2 does not affect cell proliferation, cell death or cell cycle. However, it does affect the sensitivity to BFA. In cell lines which possess Col1A1, the cell viability increased by 20 -30% (p &amp;lt;0.05). In contrast, cell lines which do not express Col1A1, the cell viability decreased by 25-30% (p&amp;lt;0.01). However, there is no difference in sensitivity to the DNA damaging agent cisplatin. Thus, DDR2 may function differently depending on the presence of Col1A1. To explore this further, we have performed a limited phoshoprotein kinase array in Glioma 1 with siCol1A1 or siDDR2. We found increase in STAT3, 5 and STAT6 upon silencing either Col1A1 or DDR2 which is further confirmed by immunoblot in Glioma 1 for pSTAT 3 and 5. We then tested the antitumor effect of STAT3/5 inhibitor (SH4-54) a benzoic acid based inhibitor (provided by Dr. James Turkson) which interferes with the SH2 and DNA binding domains as well as tyr705 phosphorylation in Glioma1 and U-118 w/wo SiCol1A1. At 0.5 uM the cell viability in Glioma 1 is 55.65 + 1.35 and 74.2 + 4.6 in U-118 while SiCol1A1 it decreased to 44.5 + 3.53% in Glioma 1 and 58.2+ 1.9, respectively, with minimal activity in A-172 which does not possess Col1A1. In contrast, there is no effect with another commercially available STAT 3 inhibitor S31-201 which only interferes with DNA binding domain. Overall, our data suggest that Col1A1/DDR2/ STAT signaling may be important in certain GBM cell lines and can be exploited for future treatment in brain tumor (Supported by Wanfang Hospital-Taipei Medical University Fellowship and VA Research Fund). Citation Format: Shumei Chen, Chunjing Wu, Ying-Ying Li, Medhi Wangpaichitr, Ronald J. Benveniste, James Turkson, Niramol Savaraj, Lynn G. Feun. The relationship of Procollagen alpha 1 type 1 (Col1A1) / DDR2 signaling in malignant glioma and sensitivity to STAT 3/5 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3712.

Angiotensin I‐converting enzyme inhibitory peptides FQPSF and LKYPI identified in Bacillus subtilis A26 hydrolysate of thornback ray muscle

SummaryAngiotensin I‐converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC50 of 0.83 mg mL−1. To identify peptides responsible for this activity, the extract was eluted through size‐exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC50 of 0.42 and 0.51 mg mL−1, respectively. These fractions were analysed by nano‐liquid chromatography coupled to tandem mass spectrometry (nLC‐MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC50 of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides.

Pathophysiology and therapeutic options in osteogenesis imperfecta: an update

Pathophysiology and therapeutic options in osteogenesis imperfecta: an update Evelise Brizola,1 Temis M Félix,2 Jay R Shapiro3 1Bone and Osteogenesis Imperfecta Department, Kennedy Krieger Institute, Johns Hopkins School of Medicine, Baltimore, MD, USA; 2Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil; 3Osteoporosis and Metabolic Bone Disorders Center, Bethesda, MD, USAAbstract: Osteogenesis imperfecta (OI) is a rare, heritable systemic disorder of bone and connective tissue, which in almost 90% of cases is due to mutations affecting the normal synthesis of type I collagen. In 1979, four OI phenotypes were categorized which where inherited as autosomal dominant characteristics. Individuals with OI present both genetic and phenotypic variabilities. Major characteristics of OI are bone fragility, blue sclerae, dentinogenesis imperfecta, short stature, scoliosis, and joint hyperextensibility. Both autosomal dominant and recessive inheritance are now recognized. Advances in molecular diagnosis have led to a major expansion in our understanding of the genetic basis for different OI phenotypes. To date, sequence variants in 17 genes are described as causative of OI. These genes regulate the synthesis of type I collagen pro-alpha polypeptide chains, proteins involved in type I collagen processing in the endoplasmic reticulum and proteins involved in osteoblast function. These new genetic associations have also led to uncertainty with regard to the current classification of OI phenotypes. Bisphosphonates have been widely used to improve bone mass and decrease fractures in both children and adults with OI. While effective in many but not all children when administered for 2–4 years, bisphosphonates have not proven effective in adults with OI. Studies are limited for treatment of adults with teriparatide and denosumab. Advances have been reported in the surgical management of OI. Although the role of physical therapy in the management of children and adults was previously described, this important treatment modality is significantly underutilized. Keywords: osteogenesis imperfecta, sequence variants, collagen, bisphosphonates, bone, treatment

Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis.

The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells,a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

Abstract 43: The role of discoidin domain receptor 2 (DDR2) and its relationship with procollagen alpha 1 type 1(Col1A1) in malignant glioma

Abstract We have previously shown that Col1A1 expression is found more frequently in low to intermediate grade glioma than high grade glioma(Cancer invest. 23:577,2005). On the other hand, its receptor DDR1 is found in high grade glioma and often signifies poor prognosis. We hypothesize that DDR2 which also binds to Col1A1 may play a role in mediating this complex relationship. We have investigated this relationship using 3 cell lines (2 cell lines, Glioma 1 and U-118, both express both Col1A1 and DDR1&amp;2, A-172 which does not express Col1A1 but does express both DDR1&amp;2). All cell lines were maintained in EMEM with 10% FCS. Treatment with Befeldin A (BFA), which blocks Col1a1 to the cell surface, results in ER stress with increase in GRP78 and growth inhibition in all three cell lines. U-118 was the most sensitive (ID50 of 9 ng/ml for U- 118, Glioma 1 (13ng/ml), and A-172 (24ng/ml)). Silencing Col1A1 resulted in decreased DDR1 &amp; 2 in Glioma 1 and U-118 and decreased sensitivity to BFA by 20% for Glioma 1 and 10% in U-118. Silencing Col1 A1 resulted in G2M arrest and decrease in ERK1/2, JNK, and c-Jun, but increase in PDGF, CREB and P70 by protein array analysis and confirmed by Western blot. We further investigated the biological effect of silencing either DDR1 (primary DDR1a which is the major isoform found in Glioma 1 and DDR2). We found that silencing DDR 1 had no effect on DDR2 expression and vice versa. Interestingly, in contrast to silencing Col1A1, silencing either DDR1 or 2 had no effect on Col1A1 expression in Glioma 1 and U-118 and also no effect on cell cycle or cellular proliferation. In addition, silencing DDR1 or 2 led to decreased sensitivity to BFA for both Glioma and U-118 with increase in ID50 by 35-50% respectively. Furthermore, we have also found an increase in p-AKT, p-38, JNK and p-Jun after 48-hr post silencing of DDR2 while silencing DDR1 had minor effect. However, these proteins decreased after BFA treatment. In contrast, silencing DDR1 or DDR2 led to increased sensitivity to BFA in A-172 cells with reduction of ID50 by 20%. In addition, a decrease in p-AKT, p-38 and ERK were also seen. Silencing DDR1 or DDR2 also showed significant reduction in invasion in A-172 cell line, while minimal changes were seen in Glioma 1 and U-118. Since DDR1 has multiple subfamilies, while DDR1a is the most common one in brain tumor, it is possible that silencing one subfamily may upregulate the other subfamily. However, DDR2 has only one family and may explain the more profound effect in silencing DDR2. Taken together, our data suggest that the function of DDR2 is different among cell lines, and may be dictated by the presence of Col1A1 which is present in certain malignant glioma. We are currently investigating the possible mechanism(s) involved. Future targeted therapy in malignant glioma will need to take into consideration the different functions of DDR2. (Supported by Wanfang Hospital-Taipei medical university fellowship award and VA research grant) Citation Format: Shumei Chen, Chunjing Wu, Ying Ying Li, Medhi Wangpaichitr, Lynn G. Feun, Ronald Benveniste, Vy Dinh, Niramol Savaraj. The role of discoidin domain receptor 2 (DDR2) and its relationship with procollagen alpha 1 type 1(Col1A1) in malignant glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 43. doi:10.1158/1538-7445.AM2015-43

Severe osteogenesis imperfecta caused by double glycine substitutions near the amino‐terminal triple helical region in COL1A2

Most cases of osteogenesis imperfecta (OI) are caused by heterozygous mutations in COL1A1 or COL1A2, the genes encoding the two type I procollagen alpha chains, proα1 (I) and proα2 (I). We report on a unique case of severe OI, a long term survivor of lethal type II OI, rather than progressively deforming type III, due to double substitutions of glycine residues in COL1A2 (p.Gly208Glu and p.Gly235Asp), located on the same allele. To the best of our knowledge, this is the first example of a patient with double COL1A2 glycine substitution mutations on the same allele. We show for the first time that double COL1A2 glycine substitution mutations located near the amino-terminal triple helical region, which individually are likely to result in mild OI, cause severe OI in combination.

Abstract 4192: Exploiting discoidin domain receptor (DDR) and collagen signaling as a new approach to treat human malignant gliomas

Abstract Discoidin domain receptor (DDR) belongs to the receptor tyrosine kinase family l and is activated by binding to its ligand (collagen). Once activated, DDR undergoes autophosphorylation and triggers cellular signaling for proliferation and invasion. While studies have shown that high grade glioma expressed high levels of DDR1 and signifies poor clinical outcome, the underlying mechanism(s) is not known. We attempted to exploit this pathway for future treatment. DDR comprises of 2 members (DDR1 and DDR2). DDR1 is composed of five isoforms, i.e. a,b,c,d, and e. (d and e are inactive). We have studied the expression of both DDR1 (a,b,c) and DDR2 in 6 glioma cell lines (U118, Glioma1, SW1783, U87, A172, and U373). All 6 cell lines express all three subtypes of DDR1 as well as DDR2 by RT-PCR . DDR1 protein was also detected in all 6 cell lines. Knockdown DDR1 inhibited glioma cell migration by scratch assay and deactivated MMP9 by zymography analysis, which was best seen in Glioma1, U-118, and much less in A-172 and U-373. Knock down DDR1 also decreased AKT, ERK, and Bcl-xl which suggests positive regulation of these prosurvival proteins by DDR1. Since there is no inhibitor of DDR1, we have used Brefeldin A (BFA), which can block procollagen (ligand for DDR1 and DDR2) secretion. Treatment with BFA results in decreased DDR 1a,b and c mRNA in U-118, Glioma SW1783 and U-87 and no change in A-172 and U373. A decrease in DDR2 also was seen in U-118 and SW1783 and no change was seen in the other 4 cell lines. The ID50 of BFA ranged from 6 to 13 ng/ml in U-118, Glioma1 SW1783 and U-87 and 23 ng/ml in A-172. Treatment with BFA also inhibited cell migration (scratch assay) in Glioma 1, U-118 and SW1783 but not in U-373 and A-172. A-172 and U-373 did not possess procollagen alpha 1 type 1 which may explain the insensitivity to BFA. Silencing DDR1 does not significantly alter the growth inhibitory effect of Glioma1 U-118 or A-172. This may be due to the compensatory increase of DDR2. Thus, our data showed that glioma cells possess all DDR 1 subtypes as well as DDR2. Silencing DDR1 results in decreased AKT, ERK and antiapoptotic protein Bcl-xl and migration, suggesting its role in cellular proliferation and invasion. However, decreasing the available ligand (procollagen) from exiting the cells only affected DDR1 and DDR2 expression in certain cell lines (U-118 , SW1783). Interestingly, these 2 cell lines are also very sensitive to BFA (ID50 of 6 ng/mL) and possess high levels of procollagen type1 (both alpha1 and alpha 2 ) which are the ligands for both DDR1 and DDR2, while the cell line which had the least effect (A-172) did not express procollagen alpha 1 type 1. Overall, the presence of DDR family in malignant gliomas may not indicate that this is a major pathway for proliferation /survival and invasion . However, the presence of both ligand(s) and receptor(s) are important for future application to treat malignant gliomas targeting this pathway. Citation Format: Shumei Chen, Chunjing Wu, Min You, Minh Tran, Medhi Wangpaichitr, Ying Ying Li, Ronald J. Benveniste, Macus T. Kuo, Niramol Savaraj, Lynn G. Feun. Exploiting discoidin domain receptor (DDR) and collagen signaling as a new approach to treat human malignant gliomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4192. doi:10.1158/1538-7445.AM2014-4192

The Effects of Age and the Expression of SPARC on Extracellular Matrix Production by Cardiac Fibroblasts in 3-D Cultures

Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.

Identification of three novel mutations in the COL2A1 gene in four unrelated Chinese families with spondyloepiphyseal dysplasia congenita

IntroductionSpondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. The condition occurs through a mutation in the COL2A1 gene that encodes the type II procollagen alpha1 chain (proalpha1 (II)). Method and ResultsWe investigated nine affected individuals from four unrelated Chinese families with SEDC. We screened for COL2A1 gene mutations, and identified found four missense mutations (G447A, G456A, R789C and G1152D). The G447A, G456A and G1152D mutations are novel and the R789C mutation has been reported previously in several other studies with a strikingly similar phenotype. ConclusionsOur study extends the mutation spectrum of SEDC and is helpful in early molecular diagnoses of SEDC.

Flutamide inhibits nifedipine- and interleukin-1β-induced collagen overproduction in gingival fibroblasts

To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin-1 beta- and nifedipine-induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined. Gingival fibroblasts from healthy subjects and patients with dihydropyridine-induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin-1 beta or both. The mRNA expression was examined using real-time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot. Interleukin-1 beta was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen alpha1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen alpha1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin-1 beta. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue. The data suggest that both nifedipine and interleukin-1 beta play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine-induced overgrowth cells.

Treatment with L-Valine Ameliorates Liver Fibrosis and Restores Thrombopoiesis in Rats Exposed to Carbon Tetrachloride

It has been reported that treatment with branched chain amino acids (BCAAs) increases the survival rates in cirrhotic patients. In this study, we investigated the effect of L-valine, one of BCAAs, on liver fibrosis in rat. To induce liver fibrosis, male Wistar rats were injected carbon tetrachloride (CCl(4)) intraperitoneally (2.0 mL/kg) twice a week for 12 weeks. The rats (seven to fifteen rats for each group) were then administered 1.688 g/kg/day of L-valine intravenously for 7 days or 10% amino acid preparation that provided the same amount of nitrogen. Seven days after the last administration, blood platelet counts and bone marrow megakaryocyte counts were significantly higher in the valine group than in the control group (131.2 +/- 38.3 vs. 106.3 +/- 14.5 x 10(4)/microL, p = 0.04; 18.0 +/- 2.1 vs. 13.5 +/- 2.2 per field, p < 0.01, respectively). Importantly, the mRNA level of thrombopoietin, a key regulator of thrombopoiesis, was significantly higher in the liver of the valine group than the control group. Furthermore, hepatic fibrosis was significantly reduced in the valine group, and the mRNA levels of factors associated with liver fibrosis such as procollagen alpha1(III), transforming growth factor-beta1 and connective tissue growth factor were significantly lower in the liver of the valine group 10 days after the last administration. These results indicate that L-valine treatment ameliorates liver fibrosis and restores thrombopoiesis in rats exposed to CCl(4). Therefore, L-valine supplementation may be helpful for patients with liver cirrhosis.

Reduction of Advanced Liver Fibrosis by Short-Term Targeted Delivery of An Angiotensin Receptor Blocker to Hepatic Stellate Cells in Rats

There is no effective therapy for advanced liver fibrosis. Angiotensin type 1 (AT1) receptor blockers attenuate liver fibrogenesis, yet their efficacy in reversing advanced fibrosis is unknown. We investigated whether the specific delivery of an AT1 receptor blocker to activated hepatic stellate cells (HSCs) reduces established liver fibrosis. We used a platinum-based linker to develop a conjugate of the AT1 receptor blocker losartan and the HSC-selective drug carrier mannose-6-phosphate modified human serum albumin (losartan-M6PHSA). An average of seven losartan molecules were successfully coupled to M6PHSA. Rats with advanced liver fibrosis due to prolonged bile duct ligation or carbon tetrachloride administration were treated with daily doses of saline, losartan-M6PHSA, M6PHSA or oral losartan during 3 days. Computer-based morphometric quantification of inflammatory cells (CD43), myofibroblasts (smooth muscle alpha-actin [alpha-SMA]) and collagen deposition (Sirius red and hydroxyproline content) were measured. Hepatic expression of procollagen alpha2(I) and genes involved in fibrogenesis was assessed by quantitative polymerase chain reaction. Losartan-M6PHSA accumulated in the fibrotic livers and colocalized with HSCs, as assessed by immunostaining of anti-HSA and anti-alpha-SMA. Losartan-M6PHSA, but not oral losartan, reduced collagen deposition, accumulation of myofibroblasts, inflammation and procollagen alpha2(I) gene expression. Losartan-M6PHSA did not affect metalloproteinase type 2 and 9 activity and did not cause apoptosis of activated HSCs. Short-term treatment with HSC-targeted losartan markedly reduces advanced liver fibrosis. This approach may provide a novel means to treat chronic liver diseases.

Transcriptional characteristics of type I procollagen gene of fibroblast clones from patients with systemic sclerosis.

Objective To study the mechanisms underlying high collagen synthesis of systemic scle-rosis skin fibroblasts. Methods The mRNA expression levels 6f procollagen alpha 1 type 1 (COL1A1) were detected by in situ hybridization to select heterogeneic fibroblast clones, and monoclonal fibroblasts were divided into three groups according to their mRNA levels of COL1A1. Meanwhile, five deletion con-structs containing portions of human procollagen gene promoter were ligated into plasmids and used to trans-fect the three groups of fibroblast clones. The transcription of COL1A1 in these fibroblasts was assessed by transient transfection and luciferase assay. Re.milts Fibroblast clones from both patients and normal controls showed a heterogeneity in the mRNA expression of COL1A1. Patients with systemic sclerosis had a larger proportion of fibroblasts expressing high level of COL1A1mRNA than did normal controls (50% vs 12.5%). A significant increase was also observed in the mean mRNA levels of COL1A1 in fibroblast clones from patients with systemic sclerosis compared with those from normal controls (6.64 ± 2.48 vs 3.69 ± 2.01, t = 3.690, P < 0.01 ). Maximal transcriptional activity was observed with a construct containing COL1A1 pro-moter from -174 bp to +42 bp in all fibroblast clones. The activities of COL1A1 promoter positively corre-lated with the mRNA levels of this gene. Conclusions COL1A1 gene might be activated at the transcrip-tional level in systemic sclerosis fibroblast clones with a high rate of collagen synthesis. Key words: Scleroderma; systemic; Fibroblasts; Collagen type Ⅰ; Plasmids

Influence of a commercial tattoo ink on protein production in human fibroblasts

Tattooing is an ancient art and is still widely practiced all over the world. Since the biocompatibility of tattoo dyes has not been well researched, we studied the toxicity of a commercial tattoo ink, commonly used in tattoo lab and esthetic centers, on human fibroblasts. To test cell viability, MTT assays were carried out and scanning electron microscopy to visualize changes in the cell surface after the dye exposure was performed. A possible influence of the pigment on the expression of procollagen alpha1 type I protein was visualized by western blotting analysis. The results showed a reduction in cell viability, and electron microscopy demonstrated an unmodified cell surface completely covered by pigment particles. Western blotting analysis demonstrated a clear interference of the pigment on the expression of procollagen alpha1 type I protein. These data demonstrated that the commercial tattoo dye has a time-dependent effect on protein expression. A possible connection of the influence of the tattoo ink with clinical effects is discussed.

Chronic Alcohol Feeding Stimulates the Formation of Mutagenic Etheno-Adducts in a Rodent model of NASH Via Induction of Cytochrome P450 2E1

Background and Aims: Chronic alcohol consumption may cause liver fibrosis and hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD). Synergistic effects of insulin resistance and alcohol triggering fibrogenesis and HCC are implicated. Formation of mutagenic etheno adducts binding to adenosin (EdA) generated via induced CYP2E1 has been suggested as a key mechanisms of hepatocarcinogenesis. Methods: Obese Zucker rats and lean littermates (4 groups) received Lieber-De Carli liquid diets containing either ethanol or carbohydrates for 3 months. Fibrosis and steatosis were quantified by Sirius Red and Oil Red O, respectively. EdA immunohistology was performed using a monoclonal antibody, and fibrosis-related gene and protein expression and hydroxyproline quantified. CYP2E1 and 4A2 were assayed at transcription and protein level. Results: Obese rats had more steatosis than lean animals but showed no aggravating impact of chronic alcohol intake. Histology and liver hydroxyproline showed similar extent of fibrosis in all groups. Procollagen alpha1(I), alpha smooth muscle actin, and TGFbeta-1 gene expression were not different. CYP2E1 protein was induced by 35% in both groups of obese rats, while corresponding mRNA levels were upregulated 3.5-fold in alcoholic lean rats. CYP4A2 was lower in obese rats, but upregulated 2-fold in those fed alcohol. In alcoholic obese and lean rats hepatocyte nuclei stained positive for EdA in 36% and 23%, respectively, but only 14% in obese rats without alcohol. EdA immunostaining was absent in lean non-alcoholic rats. EdA labelling correlated positively with CYP2E1 and 4-HNE immunostaining. Conclusions: Chronic alcohol feeding in obese, insulin-resistant rats exerts no effect on liver fibrosis formation, but potentiates the formation of mutagenic EdA possibly due to CYP2E1 induction. This observation may explain the increased risk of developing HCC in patients with NAFLD who chronically drink alcohol.

Role of the receptor for advanced glycation end products inhepatic fbrosis

To study the role of advanced glycation end products (AGE) and their specific receptor (RAGE) in the pathogenesis of liver fibrogenesis. In vitro RAGE expression and extracellular matrix-related gene expression in both rat and human hepatic stellate cells (HSC) were measured after stimulation with the two RAGE ligands, advanced glycation end product-bovine serum albumin (AGE-BSA) and N(epsilon)-(carboxymethyl) lysine (CML)-BSA, or with tumor necrosis factor-alpha (TNF-alpha). In vivo RAGE expression was examined in models of hepatic fibrosis induced by bile duct ligation or thioacetamide. The effects of AGE-BSA and CML-BSA on HSC proliferation, signal transduction and profibrogenic gene expression were studied in vitro. In hepatic fibrosis, RAGE expression was enhanced in activated HSC, and also in endothelial cells, inflammatory cells and activated bile duct epithelia. HSC expressed RAGE which was upregulated after stimulation with AGE-BSA, CML-BSA, and TNF-alpha. RAGE stimulation with AGE-BSA and CML-BSA did not alter HSC proliferation, apoptosis, fibrogenic signal transduction and fibrosis- or fibrolysis-related gene expression, except for marginal upregulation of procollagen alpha1(I) mRNA by AGE-BSA. Despite upregulation of RAGE in activated HSC, RAGE stimulation by AGE does not alter their fibrogenic activation. Therefore, RAGE does not contribute directly to hepatic fibrogenesis.