BACKGROUNDWe determined the diagnosis of hereditary hemorrhagic telangiectasis (HHT) in a suspected HHT family, identified ALK1 gene mutation and established a gene diagnosis method of HHT. The level of related plasma proteins (transforming growth factor beta and thrombomodulin) were also analyzed.

METHODSBleeding history and family history were collected; Dilatant nasal mucosal capillaries in proband were observed under nasal cavity endoscope; exons 3, 7, 8 of ALK1 gene in proband and her family members were amplified with polymerase chain reaction (PCR), and the PCR products were analyzed. Using enzyme-linked immunosorbent assay (ELISA), plasma TGF-beta1 and TGF-beta2 concentrations were measured. Plasma thrombomodulin (TM) level was detected by Western blotting.

RESULTSOf all family members, four had epstaxis, two had evident telangiectases on skin or mucosa. Gene screening results showed that C to T substitution at position 1231 in exon 8 of ALK1 gene (CGG-->TGG) existed in proband, her affected brother and their father. The mutation did not exist in proband's sister-in-law and nephew. Plasma TGF-beta1 concentrations in the affected HHT was 20,538, 17,194, 13,131 pg/ml, while that of normal control and unaffected family members was 15,950, 20,297, 12,836 pg/ml, respectively. Plasma TGF-beta2 in HHT patients was 14,502, 9550, 10,592 and that of normal controls 8579, 20,297, 7680 pg/ml respectively. Level of plasma TM was in HHT subjects significantly lower than in normal subjects.

CONCLUSIONSChinese HHT individuals have mutant ALK1 gene, a C1231T variation on exon 8 of ALK1 is responsible for HHT clinical phenotypes in this family. ALK1 gene analysis, together with special clinical phenotypes and family history, provides a reliable method in diagnosing HHT. In affected HHT subjects, plasma TGFbeta levels were not obviously different from those of normal subject; while plasma TM concentration was significantly lower than that in normal subjects. The significance and mechanism remain to be elucidated.

Activin Receptors, Type I ; genetics ; Activin Receptors, Type II ; Aged ; Asian Continental Ancestry Group ; genetics ; China ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Telangiectasia, Hereditary Hemorrhagic ; blood ; genetics ; Thrombomodulin ; blood ; Transforming Growth Factor beta ; blood

OBJECTIVEFibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant inherited disease caused by mutations of ACVR1 gene and can be inherited from either mother or father. FOP is characterized by the presence of malformations of the big toes and of progressive extra-skeletal ossification. Direct sequence analyses of genomic DNA have demonstrated that there is an identical single nucleotide substitution (c617G-->A, R206H) in the glycine-serine (GS) activation domain of ACVR1 gene, responsible for all affected individuals reported so far. We report a Chinese girl with typical FOP characteristics, in whom the same mutation in ACVR1 was identified.

METHODSClinical diagnosis was based on physical examination, radiological findings, and biochemical tests. For mutation detection, peripheral blood was obtained with informed consent from the patient and the parents. Genomic DNA was extracted from peripheral blood using standard method. Exon 4 of ACVR1 was amplified by polymerase chain reaction (PCR), and the PCR products were subjected to automatic DNA sequencing.

RESULTSThe affected girl is 3-year-old and showed typical clinical manifestations of FOP. She had malformations of the halluces at birth and subsequently progressive extra-skeletal ossification developed at the age of 8 - 9 months. Then, she gradually developed stiffness of the knee joint and neck but remained ambulant. Radiographic changes were observable, e.g., the extra-skeletal ossification was found at cervical spine. Her mother has congenital malformations of the halluces, but had no postnatal progressive extra-skeletal ossification. Her father and other family members are normal. With direct sequencing of the PCR products, a G to A substitution at c617 of ACVR1 (R206H) was detected in the patient only but not in her parents. Paternity analysis suggested that it is a de novo mutation.

CONCLUSIONThis is the first case reported in a Chinese patient with FOP in the mainland of China, which was confirmed by direct sequencing. Although sporadic cases of FOP have been reported in diverse geographic and ethnic group, the mutations of ACVR1 c617 (R206H) are identical up to now. The presence of mutation hot spot facilitates molecular diagnosis in clinical practice. Genetic detection is important for FOP patients to avoid misdiagnosis and further damages, including those from medical intervention.

Activin Receptors, Type I ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child, Preschool ; Female ; Humans ; Molecular Sequence Data ; Myositis Ossificans ; genetics ; Point Mutation ; Sequence Analysis, DNA

Fibrodysplasia ossificans progressiva (FOP; OMIM 135100) is a rare but extremely disabling genetic disorder of the skeletal system, and is characterized by the progressive development of ectopic ossification of skeletal muscles and subsequent joint ankylosis. The c.617G>A; p.R206H point mutation in the activin A type I receptor (ACVR1) gene has been reported to be a causative mutation of FOP. In the present study, mutation analysis of the ACVR1 gene was performed in 12 patients diagnosed or suspected to have FOP. All patients tested had a de novo heterozygous point mutation of c.617G>A; p.R206H in ACVR1. Mutation analysis confirmed a diagnosis of FOP in patients with ambiguous features, and thus, could be used for diagnostic purposes. Early confirmation through mutation analysis would allow medical professionals to advise on the avoidance of provoking events to delay catastrophic flare-ups of ectopic ossifications.
Activin Receptors, Type I/*genetics ; Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Child ; Female ; Genetic Predisposition to Disease ; Heterozygote ; Humans ; Korea ; Male ; Myositis Ossificans/*diagnosis/genetics/radiography ; Point Mutation ; Young Adult

Fibrodysplasia ossificans progressiva (FOP; OMIM 135100) is a rare but extremely disabling genetic disorder of the skeletal system, and is characterized by the progressive development of ectopic ossification of skeletal muscles and subsequent joint ankylosis. The c.617G>A; p.R206H point mutation in the activin A type I receptor (ACVR1) gene has been reported to be a causative mutation of FOP. In the present study, mutation analysis of the ACVR1 gene was performed in 12 patients diagnosed or suspected to have FOP. All patients tested had a de novo heterozygous point mutation of c.617G>A; p.R206H in ACVR1. Mutation analysis confirmed a diagnosis of FOP in patients with ambiguous features, and thus, could be used for diagnostic purposes. Early confirmation through mutation analysis would allow medical professionals to advise on the avoidance of provoking events to delay catastrophic flare-ups of ectopic ossifications.
Activin Receptors, Type I/*genetics ; Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Child ; Female ; Genetic Predisposition to Disease ; Heterozygote ; Humans ; Korea ; Male ; Myositis Ossificans/*diagnosis/genetics/radiography ; Point Mutation ; Young Adult

OBJECTIVESTo investigate the biological responses of cultured hepatic stellate cells (HSC) at different differentiated stages on exotic transforming growth factor (TGF-beta1).

METHODSHSC was isolated from rat and primarily cultured in uncoated disc for 1 d, 4 d and 7 d, when the cells were at quiescent, intermediate activated and full activated stages respectively. The cells were incubated with 10 pmol/L to 500 pmol/L TGF-beta1 for 24 h, cell proliferation was measured with [3H] TdR incorporation, alpha-smooth muscle actin (alpha-SMA) and type I collagen protein were assayed with Western blot, and total protein secretion in the culture supernatant was analyzed by [3H] proline pulse and collagenase digestion. HSC was treated with 100 pmol/L TGF- beta1 for 15 min to 90 min, and type I pro-collagen mRNA level was assayed by Northern blot.

RESULTSTGF-beta1 remarkably inhibited d1 HSC proliferation, the percentage of [3H] TdR incorporation at 10 pmol/L to 500 pmol/L TGF-beta1 was 52.8% to 16.8% of the control, q value was 5.44 to 10.37 and P<0.01 vs control. But TGF-beta1 had no influence on d4 and d7 HSC. As the cells cultivation prolonged and activated, the basal levels of alpha-SMA, type I collagen and gene expression increased gradually. TGF-beta1 increased the above protein and gene expression. The basal and TGF-beta1 stimulated total protein secretion levels at d1-d7 HSC were 804+/-274 vs 1200+/-708; 2966+/-1701 vs 6160+/-1123, t=3.84, P<0.01; 2580+/-767 vs 4583+/-1467, t=2.96, P<0.05. While d4 HSC showed the strongest response of total protein secretion and alpha-SMA expression.

CONCLUSIONSTGF-beta1 remarkably inhibited quiescent HSC proliferation, and promoted HSC collagen production at both quiescent and activated HSC. Intermediate HSC had the strongest response to TGF-beta1, while activated HSC lost the response to TGF-beta1 inhibitory growth, and TGF-beta1 exerted divergent actions on HSC as the cells activated.

Activin Receptors, Type I ; analysis ; Animals ; Cell Division ; drug effects ; Collagen Type I ; genetics ; Liver ; cytology ; drug effects ; metabolism ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; analysis ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factor beta1

Activin Receptors, Type I ; analysis ; Animals ; Arcuate Nucleus of Hypothalamus ; chemistry ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Immunohistochemistry ; Liver Regeneration ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; analysis ; Rats ; Receptors, Transforming Growth Factor beta ; analysis ; Sodium Glutamate ; Transforming Growth Factor alpha ; analysis ; genetics ; Transforming Growth Factor beta ; analysis ; genetics

OBJECTIVETo study transforming growth factor-beta1 (TGF-beta1) autoproduction in keloid fibroblasts and the regulation effect of blocking TGF-beta intracellular signaling on rhTGF-beta1 autoproduction.

METHODSKeloid fibroblasts cultured in vitro were treated with either rhTGF-beta1 (5 ng/ml) or recombinant adenovirus containing a truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects of regulating gene expression of TGF-beta1 and its receptor I and II were observed with Northern blot.

RESULTSrhTGF-beta1 up-regulated the gene expression of TGF-beta1 and receptor I, but not receptor II. Over-expression of the truncated receptor II down-regulated the gene expression of TGF-beta1 and its receptor I, but not receptor II.

CONCLUSIONSTGF-beta1 autoproduction was observed in keloid fibroblasts. Over-expression of the truncated TGFbeta receptor II decreased TGF-beta1 autoproduction via blocking TGF-beta receptor signaling.

Activin Receptors, Type I ; biosynthesis ; pharmacology ; Cells, Cultured ; Down-Regulation ; Fibroblasts ; drug effects ; metabolism ; Gene Expression ; Humans ; Keloid ; metabolism ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; genetics ; metabolism ; Receptors, Transforming Growth Factor beta ; biosynthesis ; metabolism ; Sensitivity and Specificity ; Signal Transduction ; Trans-Activators ; metabolism ; Up-Regulation

BACKGROUNDThe function of peroxisome proliferator-activated receptor gamma (PPARgamma) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARgamma signaling were investigated.

METHODSHSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy lsulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARgamma subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay.

RESULTSCurcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARgamma nuclear expression level decreased. Curcumin up-regulated PPARgamma expression and significantly inhibited the production of alpha-SMA and collagen I. PPARgamma is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFkappaB p65 protein and TGFbetaR-I protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin.

CONCLUSIONSCurcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARgamma signal transduction pathway and associated with PPARgamma nuclear translocation/redistribution.

Active Transport, Cell Nucleus ; drug effects ; Activin Receptors, Type I ; analysis ; Animals ; Apoptosis ; drug effects ; Cell Nucleus ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Curcumin ; pharmacology ; Cyclin D1 ; genetics ; Liver ; cytology ; metabolism ; Liver Cirrhosis ; drug therapy ; etiology ; Male ; Matrix Metalloproteinase 9 ; metabolism ; PPAR gamma ; physiology ; Protein-Serine-Threonine Kinases ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; analysis ; Signal Transduction ; Transcription Factor RelA ; genetics ; bcl-2-Associated X Protein ; analysis