Α1 Chain Of Type Research Articles

Genetic Analysis, Phenotypic Spectrum and Functional Study of Rare Osteogenesis Imperfecta Caused by CRTAP Variants.

Deficiency of cartilage-associated protein (CRTAP) can cause extremely rare autosomal recessive osteogenesis imperfecta (OI) type VII. We investigated the pathogenic mechanisms of CRTAP variants through functional studies on bones of patients with OI. Two nonconsanguineous families with CRTAP mutations were included and their phenotypes and genotypes were evaluated. Bone specimens were obtained from 1 patient with OI and a normal control during orthopedic surgery. The impacts of the novel variant on the CRTAP transcript were confirmed. The expression levels of CRTAP mRNA and CRTAP protein were analyzed. The quantification of prolyl 3-hydroxylation in the α1 chain of type I collagen was evaluated. Patients with OI type VII had early-onset recurrent fractures, severe osteoporosis, and bone deformities. The c.621 + 1G > A and c.1153-3C > G mutations were identified in CRTAP in the patients with OI. The c.621 + 1G > A variant was a novel mutation that could impair mRNA transcription, leading to a truncated CRTAP protein. In a patient with c.621 + 1G > A and c.1153-3C > G mutations in CRTAP, the mRNA and protein levels of CRTAP in osteoblasts were significantly decreased and the osteoid volume and osteoblast numbers were markedly reduced compared with those in the normal control individual. This was simultaneously accompanied by significantly reduced prolyl 3-hydroxylation at Pro986 in the α1 chain of type I collagen and invisible active bone formation in bone. The novel c.621 + 1G > A mutation in CRTAP expands the genotypic spectrum of type VII OI. Biallelic mutations of c.621 + 1G > A and c.1153-3C > G in CRTAP can lead to reduced CRTAP mRNA and deficient CRTAP protein in osteoblasts, which reduces 3-hydroxylation in Pro986 of the α1 chain of type I collagen and impairs bone formation, thus contributing to severe OI type VII.

Genetic parameters for productive traits and skin quality in Nile tilapia

Tilapia skin has been considered an important biomaterial in the treatment of burns and tissue reconstruction in vaginoplasty. Improvements in tilapia skin quality are of great interest to regenerative medicine. This study aimed to (i) estimate variance components and genetic parameters for growth traits, fillet yield, and skin quality; (ii) assess genetic associations between productive traits and skin thickness; and (iii) investigate the effect of groups with high (HGM) and low (LGM) genetic merit for skin thickness on the expression of genes encoding the α2 and α3 chains of type I collagen in Nile tilapia (Oreochromis niloticus). Components of variance, heritability, and genetic correlations were estimated for skin thickness (ST, measured in millimeters), fillet yield (FY, measured in percentage), and weight at harvest (WH, measured in grams). The database contained records of 1221 mean values of three measurements for ST, 1213 measurements for FY, and 3171 measurements for WH. The sample included animals from the tenth generation of selection of the Nile tilapia breeding program of the State University of Maringá, Brazil. For analysis of gene expression, skin samples were collected from fish with LGM and HGM for ST. Heritability estimates were high for WH (0.71), intermediate for ST (0.59), and moderate for FY (0.21). The repeatability coefficient of ST was high (0.66). The genetic correlation between ST and WH was high and strong (0.77), whereas that between ST and FY was not significant (−0.02). There was a significant difference (p = 0.038) in COL1A3 expression between HGM (7.38 AU) and LGM (4.88 AU) groups. The results indicated that it is possible to increase the thickness and amount of type I collagen fibers in tilapia skin through selection. Genetic correlations indicated that selection for WT positively impacts ST without influencing FY. Selection for weight gain proved to be an effective tool for the production of Nile tilapia skins more suitable for use as xenografts.

Diagnostic Value of Type IV Collagen Expression in Renal Glomeruli at Alport’s Syndrome

The Alport’s syndrome is the hereditary multisystem disease characterized by the development of the progressive nephropathy. The early diagnosis and subsequent prescription of nephroprotective therapy improves significantly the nephrological prognosis. Purpose of the Study. Determine the value of the immunohistochemical method for the Alport’s syndrome diagnosis. Material and methods. The clinical, laboratory and morphological data of 35 patients with suspected Alport’s syndrome (13 years of age [11; 16]; 18 boys and 17 girls) examined in the Nephrology Department in 2013–2019 were summarized. The study of the renal tissue included the light, immunofluorescence, electron microscopy of the kidney biopsy sample, determination of the expression of α1, α3 and α5 chains of type IV collagen in the renal glomeruli using the immunohistochemical method; the genetic testing was carried out for 26 patients. The children were divided into groups depending on the glomerular expression of α5 chain of type IV collagen: normal (group 1, n=18), decreased (group 2, n=4), negative (group 3, n=13). Results are as the following: The disorder of the expression of α5 chain was detected in ¾ (q = 0.78) patients with genetically confirmed Alport’s syndrome and in almost all children with the X-linked variant of the disease (q = 0.94). Results. Based on the genetic testing, the Alport’s syndrome was confirmed in ¼ of the children of the 1st group (the children with the heterozygous variants of COL4A3, COL4A5 genes) and in all children of the 2nd and 3rd groups (COL4A5 variants). The sensitivity/ specificity of the immunohistochemical study for the Alport’s syndrome diagnosis was 78% /100%, that of the electron microscopy – 93% /87%. The predictive value of the positive/negative result of the immunohistochemical study was 100% /66%, that of the electron microscopy – 95% / 88% compared with 100% / 88% with the combine use of two methods. Conclusion. The determination of the expression of α5 chain of type IV collagen in the renal glomeruli has the independent diagnostic value, but it is inferior to the electron microscopy in the heterozygous variants of the Alport’s syndrome. The high specificity of the immunohistochemical method makes it possible to confirm the Alport’s syndrome in the case of the change in the expression of α5 chain of type IV collagen in the renal glomeruli.

Clinical and genetic analysis in 185 Chinese probands of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a well-known heritable disorder of connective tissue characterized by skeletal fragility and low bone mass. Nearly 90% of patients with OI have disease variants in COL1A1 and COL1A2 that encode for the α1 and α2 chains of type I collagen. A retrospective analysis of 185 probands who were diagnosed with OI in Shanghai Jiao Tong University Affiliated Sixth People's Hospital from March 2005 to December 2019 was performed. A total of 140 mutations in COL1A1 and 45 mutations in COL1A2 were identified, of which 18 variations were novel. In the phenotype analysis, there were more sporadic cases than familial OI cases in China (54.6% vs. 45.4%, P < 0.001). A total of 98.9% of patients presented with a fracture history. The most common fracture sites were extremity long bones (femur, tibia-fibula and radius-ulna accounted for 36.6%, 17.1% and 11.7%, respectively). Patients with OI types III and IV, especially type III, had a higher proportion of dentinogenesis imperfecta (DI) than patients with OI type I (55% vs. 28%, P < 0.001). Interestingly, G767S and D1219N in COL1A1 and G337S in COL1A2 were the most frequent (3.52%, 2.11% and 8.89%, respectively), which seem to be hotspot mutations in the COL1A1 and COL1A2 genes in Chinese patients. This study describes the mutations in the main pathogenic genes, COL1A1 and COL1A2, and the clinical characteristics of osteogenesis imperfecta in China. Furthermore, these findings help reveal the genetic basis of Asian OI patients and contribute to genetic counselling.

Abstract 3149: An assay quantifying pro-peptides of type XI collagen (PRO-C11) in serum provides novel insight into the pathology of non-small cell lung cancer (NSCLC)

Abstract Background: The tumor microenvironment (TME) is characterized by an extensive number and activity of cancer-associated fibroblasts (CAFs). CAFs are the most abundant cell type in the TME of stroma-rich tumors and dictate patient outcome. One of the most specific CAF genes is COL11A1 which encodes the α1-chain of type XI collagen. Type XI collagen is synthesized as a pro-collagen and proteolytically cleaved by bone-morphogenetic protein 1 (BMP-1) to yield mature type XI collagen. Cleaved pro-peptides released to the circulation could serve as biomarkers of type XI collagen formation and provide novel insight into patients with cancer. We developed and evaluated an assay targeting the N-terminal pro-peptide of type XI collagen for quantifying this specific fragment in the circulation (serum) of patients with non-small cell lung cancer (NSCLC). Methods: A monoclonal antibody was raised against the C-terminal neo-epitope on the N-terminal pro-peptide of type XI collagen and employed in the development of a competitive ELISA (PRO-C11). PRO-C11 was measured in serum from 40 stage 3+4 NSCLC patients and compared to 32 age-matched healthy controls. PRO-C11 was correlated with demographic and clinical characteristics (age, BMI, smoking, metastasis, histological subtype). Results: The PRO-C11 assay is technically robust with specificity only for the BMP-1 generated type XI collagen pro-peptide neo-epitope. PRO-C11 was significantly elevated in NSCLC compared to age-matched healthy controls (p&amp;lt;0.0001). No correlation (Pearson, p&amp;gt;0.05) was seen between PRO-C11 and age, BMI, smoking status, metastasis (y/n) and histological subtype (squamous cell carcinoma vs. adenocarcinoma). Conclusion: The present study validates a new serological assay quantifying the pro-peptide of type XI collagen. The biomarker has promising potential for NSCLC and warrants further investigations into stroma-rich cancers. Further studies are needed to evaluate the diagnostic/prognostic use of PRO-C11 in NSCLC. Citation Format: Nicholas Willumsen, Stephanie N. Kehlet, Tina M. Jensen, Neel I. Nissen, Morten A. Karsdal. An assay quantifying pro-peptides of type XI collagen (PRO-C11) in serum provides novel insight into the pathology of non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3149.

Exploring new genetic variants within COL5A1 intron 4-exon 5 region and TGF-β family with risk of anterior cruciate ligament ruptures.

Variants within genes encoding structural and regulatory elements of ligaments have been associated with musculoskeletal soft tissue injury risk. The role of intron 4-exon 5 variants within the α1 chain of type V collagen (COL5A1) gene and genes of the transforming growth factor-β (TGF-β) family, TGFBR3 and TGFBI, was investigated on the risk of anterior cruciate ligament (ACL) ruptures. A case-control genetic association study was performed on 210 control (CON) and 249 participants with surgically diagnosed ruptures (ACL), of which 147 reported a noncontact mechanism of injury (NON). Whole-exome sequencing data were used to prioritize variants of potential functional relevance. Genotyping for COL5A1 (rs3922912 G>A, rs4841926 C>T, and rs3124299 C>T), TGFBR3 (rs1805113 G>A and rs1805117 T>C), and TGFBI (rs1442 G>C) was performed using Taqman SNP genotyping assays. Significant overrepresentation of the G allele of TGFBR3 rs1805113 was observed in CON vs ACL (P = .014) and NON groups (P = .021). Similar results were obtained in a female with the G allele (CON vs ACL: P = .029; CON vs NON: P = .016). The TGFBI rs1442 CC genotype was overrepresented in the female ACL vs CON (P = .013). Associations of inferred allele combinations were observed in line with the above results. COL5A1 intron 4-exon 5 genomic interval was not associated with the risk of ACL ruptures. Instead, this novel study is the first to use this approach to identify variants within the TGF-β signaling pathway to be implicated in the risk of ACL ruptures. A genetic susceptibility interval was identified to be explored in the context of extracellular matrix remodeling.

Infantile Cortical Hyperostosis (ICH) Secondary to Prostaglandin E1 Infusion in a Newborn with Complex Cyanotic Heart Disease: A Case Report

Infantile cortical hyperostosis (ICH), also known as Caffey’s disease, is a benign self-limiting condition affecting young infants. The initial symptom of the classical ICH is usually irritability, fever and soft tissue swelling affecting one or more bones. Diagnosis is usually delayed because its clinical presentation mimics many other clinical conditions such as osteomyelitis, scurvy, hypervitaminosis A, child abuse, bone tumor or even fracture. There are no laboratory tests to confirm the diagnosis. There are two types of ICH been described; the classical mild infantile form and the more severe prenatal form. Most of the cases of ICH are sporadic, but autosomal dominant and recessive patterns have been reported as well. It is linked with missense mutation in COL1A1, the gene encoding the α1 chain of type I collagen and has raised some doubts whether it is a type of collagen disorder, like osteogenesis imperfect. In this case, the newborn developed ICH due to prolonged infusion with prostaglandin E1 which was given in view of congenital cyanotic heart disease. Prostaglandin E1 infusion is used for maintaining the patency of ductus arteriosus in ductus dependent congenital heart defects in neonates and is usually administered before corrective heart surgery. ICH is mostly self-limiting and resolves within 12-24 months and usually does not require any treatment. Anti-inflammatory drugs such as NSAIDS(indomethacin and naproxen) and steroids has been used in symptomatic cases with success. The outcome is generally good with symptoms resolution usually before the age of one year. Relapses are uncommon.

Nucleotide Sequences of Porcine α1 and α2 Chains of Type I Collagen cDNA and Their Different Expression Levels in Tissues

Nucleotide Sequences of Porcine α1 and α2 Chains of Type I Collagen cDNA and Their Different Expression Levels in Tissues

The pro-α1(V) collagen gene (Col5a1) is coordinately regulated by miR-29b with core promoter in cultured cells

ABSTRACTAims: Col5a1 encodes the α1 chain of type V collagen, a quantitatively minor fibrillar collagen that is critical for the formation and function of the organs in the body. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate biological functions by binding to the 3ʹ-untranslated region (3ʹUTR) of specific target mRNA. In this study, we investigated the posttranscriptional regulation of miRNAs on the Col5a1 gene expression. Materials and Methods: We cultured osteoblasts and fibroblasts of cell lines. To examine the 3ʹUTR activity of the Col5a1 gene, chimeric plasmids constructs containing the core promoter and 3ʹUTR of Col5a1 were generated and luciferase assays were performed. We also evaluated the role of miRNA using constructs that were mutated at the putative binding sites of miRNA. In addition, we evaluated the endogenous mRNA and protein, and luciferase activity of the Col5a1 gene after miRNA overexpression/knockdown or CRISPR/Cas9-induced knockout. Results: The luciferase assay showed a decreased activity of the 3ʹUTR of Col5a1 gene. However, the expression of the mutant constructs of miRNA-binding sites was restored. The overexpression of miRNA inhibited the Col5a1 gene not only with regard to the luciferase activity and endogenous mRNA but also at the protein level. In contrast, the RNAi-mediated knockdown or CRISPR/Cas9 system increased the expression of the Col5a1 gene. Conclusion: These results provided evidence that miR-29b regulates the Col5a1 gene expression through binding to the 3ʹUTR, which might play an important role in the pathogenesis of disease related to bone metabolism and fibrogenic reactions.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.

Atypical anti-glomerular basement membrane disease superimposed on IgA nephropathy

Anti-glomerular basement membrane (anti-GBM) disease is an autoimmune disorder characterized by crescentic glomerulonephritis, pulmonary hemorrhage, and the presence of circulating anti-GBM antibodies which bind to the α3 chain of Type IV collagen found in the GBM. IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. The simultaneous occurrence of atypical anti-GBM disease and IgAN has not been reported previously. We report here two female patients who presented with oliguria, hypertension, and renal failure. Renal biopsy revealed crescentic glomerulonephritis and bright linear IgG staining along glomerular capillary walls and mesangial IgA (3+) deposits in immunofluorescence. Serology was negative for anti-GBM antibodies both by ELISA and immunoblot assays. Hence, a diagnosis of atypical anti-GBM disease with superimposed IgAN was made. Both patients were treated with hemodialysis, intravenous steroids, and cyclophosphamide with the improvement of renal function in one patient and the other progressed to end-stage renal disease.

Abstract B28: Dynamic interplay between type I collagen signaling and invasion in pancreatic cancer

Abstract The stroma of pancreatic cancer can restrict as well as promote tumor progression. Why the stroma exhibits these paradoxical functions remains unclear. One of the major components of the pancreatic cancer stroma is fibrillar type I collagen. High levels of collagen in primary tumor specimens have been associated with poor survival and resistance to therapy in pancreatic cancer patients. To determine the effects of type I collagen on cancer cell behavior, we performed intravital confocal microscopic imaging in mice. We used mice that expressed GFPtopaz fused to the α2 chain of type I collagen under control of a 3.6kb fragment of the COL1A1 promoter [α2(I)-col-GFP mice]. Live imaging of pancreatic tumors, formed after orthotopic injection of cells derived from the classical KPC mouse models, showed that type I collagen architecture in the tumors was heterogeneous. Strikingly, we observed that invasive cancer cells migrated out of the tumors on linear type I collagen. Linearization of collagen fibers through cross-linking, catalyzed by lysyl oxidases (LOXs), is inversely correlated with metastasis-free survival in breast cancer. However, in our pancreatic cancer model, inhibiting LOX promoted collagen linearization, tumor growth and metastatic spread. Elevated collagen-mediated signaling, through activation of focal adhesion kinase (FAK) and discoidin domain receptor (DDR1), was also observed in the tumors treated with lysyl oxidase inhibitor. Lysyl oxidase inhibition also promoted cell contractility as shown by elevated phosphorylated mysosin light chain 2 (p-MLC2) in vivo and by in collagen contractility assays in vitro. TGF-β signaling is a potent stimulator of collagen secretion and of invasive behavior, and it is aberrantly regulated in pancreatic cancer. We observed that lysyl oxidase inhibitor increased phosphorylated Smad3, a downstream effector of TGF-β signaling. Together, our data suggest that the influence of collagen on tumor behavior depends on its architecture. In areas of linear collagen, we find high cancer cell motility out of the tumors and this correlates with increased FAK and TGF-β signaling activity. The architecture-specific function of collagen could potentially explain the paradoxical role for the stroma in pancreatic cancer progression. Citation Format: Mario A. Shields, Pascal Maguin, Sarah L. Dallas, Mikala Egeblad.{Authors}. Dynamic interplay between type I collagen signaling and invasion in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B28.

Anti-fibrotic action of pirfenidone in Dupuytren's disease-derived fibroblasts.

BackgroundDupuytren’s disease (DD) is a complex fibro-proliferative disorder of the hand that is often progressive and eventually can cause contractures of the affected fingers. Transforming growth factor beta (TGF-β1) has been implicated as a key stimulator of myofibroblast activity and fascial contraction in DD. Pirfenidone (PFD) is an active small molecule shown to inhibit TGF-β1-mediated action in other fibrotic disorders. This study investigates the efficacy of PFD in vitro in inhibiting TGF-β1-mediated cellular functions leading to Dupuytren’s fibrosis.MethodsFibroblasts harvested from (DD) and carpal tunnel (CT)- tissues were treated with or without TGF-β1 and/or PFD and were subjected to cell migration, cell proliferation and cell contraction assays. ELISA; western blots and real time RT-PCR assays were performed to determine the levels of fibronectin; p-Smad2/Smad3; alpha-smooth muscle actin (α-SMA), α2 chain of type I collagen and α1 chain of type III collagen respectively.ResultsOur results show that PFD effectively inhibits TGF-β1-induced cell migration, proliferation and cell contractile properties of both CT- and DD-derived fibroblasts. TGF-β1−induced α-SMA mRNA and protein levels were inhibited at the higher concentration of PFD (800 μg/ml). Interestingly, TGF-β1 induction of type I and type III collagens and fibronectin was inhibited by PFD in both CT- and DD- derived fibroblasts, but the effect was more prominent in DD cells. PFD down-regulated TGF-β1-induced phosphorylation of Smad2/Smad3, a key factor in the TGF-β1 signaling pathway.ConclusionTaken together these results suggest the PFD can potentially prevent TGF-β1−induced fibroblast to myofibroblast transformation and inhibit ECM production mainly Type I- and Type III- collagen and fibronectin in DD-derived fibroblasts. Further in-vivo studies with PFD may lead to a novel therapeutic application in preventing the progression or recurrence of Dupuytren’s disease.

Epigallocatechin-3-gallate inhibits transforming-growth-factor-β1-induced collagen synthesis by suppressing early growth response-1 in human buccal mucosal fibroblasts

Transforming growth factor (TGF)-β is a key regulator in the pathogenesis of oral submucous fibrosis (OSF). Early growth response (Egr)-1 is essential for fibrotic responses to TGF-β. Because TGF-β signaling is cell-type- and context-dependent, we investigated the signaling involved in TGF-β-induced Egr-1 in primary human buccal mucosal fibroblasts (BMFs). TGF-β-induced Egr-1 and its signaling were assessed by western blotting in BMFs. Egr-1 small interfering RNA was used to define the role of Egr-1 on TGF-β-induced mRNAs of the α1- and α2-chains of type I collagen (COL1A1 and COL1A2) and acid-soluble collagen production (via Sircol collagen assay). The effects of epigallocatechin-3-gallate (EGCG) on TGF-β-induced Egr-1 protein and acid-soluble collagen were also evaluated. TGF-β1 stimulated Egr-1 production in BMFs. Pretreatment with PD98059, SP600125, SB431542, and SIS3, but not SB203580, significantly reduced TGF-β1-induced Egr-1 protein expression. Genetic targeting of Egr-1 completely inhibited TGF-β1-induced type I collagen mRNAs and collagen protein expression. EGCG fully inhibited TGF-β1-induced Egr-1 and TGF-β1-stimulated production of acid-soluble collagens. We conclude that activin receptor-like kinase (ALK)5, Smad3, extracellular signal-regulated kinase, and c-Jun N-terminal kinase are involved in the TGF-β1-induced Egr-1 protein production in BMFs. Egr-1 mediates TGF-β1-induced COL1A1 and COL1A2 mRNA expression and acid-soluble collagen production in BMFs. EGCG can block TGF-β1-induced collagen production by attenuating Egr-1 expression in BMFs. Egr-1 is a key mediator in TGF-β1-induced pathogenesis of OSF. EGCG may be useful in the prevention or treatment of OSF.

Production and Characterization of Recombinant Rat Non-Collagen Domain of &lt;i&gt;α&lt;/i&gt;3 Chain of Type IV Collagen &lt;i&gt;α&lt;/i&gt;3 (IV) NC1 Antigen

The glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney that causes kidney diseases. The reason of glomerulonephritis disease is to deposit the anti-GBM auto antibody in the glomerular basement membrane. The type IV collagen is the main component of glomerular basement membrane that has α3 chain of type (IV) collagen of non-collagenous domain which contains N-terminal 7S domain, a triple helical collagenous domain and C-terminal non-collagenous glomerular domain (NC1). The amino terminal of α3 (IV) NC1 that induces the Experimental Autoimmuno Glomerulonephritis (EAG) in rat model has been identified. The recombinant rat α3 (IV) NC1 antigen has nine amino acid spans that are consistent with antibody or T cell epitope that induces in EAG. The research is carried out on the recombinant rat α3 (IV) NC1 production, purification, quantification, and characterization. The circulation of anti-GBM antibody in glomerular basement membrane can be measured by the ELISA assay. In addition, the recombinant rat antigen is secreted in HEK293 cell supernatant that is purified by Anti-FLAG M2 monoclonal IgG antibody affinity column and characterized and quantified by SDS-PAGE gel electrophoresis and Western blotting techniques.

Gender characteristics of the clinical manifestations of cardiovascular system involvement in X-linked Alport syndrome

The paper describes a clinical case of a family, in which its 5 members (4 women and 1 man) were found to have the new missence mutation c. 3098G>A, p.(1033D) in the gene encoding the α5-chain of type IV collagen (COL4A5) responsible for the development of X-linked Alport syndrome. At the same time, all the four female carriers were observed to have had kidney injury since infancy (at the age of 3 years), an age progressive glomerular filtration rate reduction to 78 ml/min; proteinuria up to 2 g/day; hypertension, and aortic dilatation (at the level of the ring, sinuses, and ascending aorta). In this family, one man aged 33 years had died from renal failure (DNA diagnosis was not made). The second 26-year-old man with the documented mutation in the clinical picture had proteinuria up to 2.5 g/l, persistent hematuria up to 100 red blood cells in the field of vision; a glomerular filtration rate decrease to 50 ml/min; sensorineural deafness, and cardiovascular abnormalities (dilatation of the left ventricle, ring, sinuses, and ascending aorta; aortic regurgitation, 2 cm; and stable hypertension).

Altered cytoskeletal organization characterized lethal but not surviving Brtl+/- mice: insight on phenotypic variability in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl(+/-) to investigate the molecular basis of OI phenotypic variability. Brtl(+/-) resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl(+/-) mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl(+/-) lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-β signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment.

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.

Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels - a Novel Therapeutic Approach in Osteogenesis Imperfecta

Osteogenesis imperfecta, also known as “brittle bone disease”, is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3'UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3'UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis imperfecta.

Caveolin-1 Deficiency Induces Spontaneous Endothelial-to-Mesenchymal Transition in Murine Pulmonary Endothelial Cells in Vitro

It was previously demonstrated that transforming growth factor β (TGF-β) induces endothelial-to-mesenchymal transition (EndoMT) in murine lung endothelial cells (ECs) invitro. Owing to the important role of caveolin-1 (CAV1) in TGF-β receptor internalization and TGF-β signaling, the participation of CAV1 in the induction of EndoMT in murine lung ECs was investigated. Pulmonary ECs were isolated from wild-type and Cav1 knockout mice using immunomagnetic methods with sequential anti-CD31 and anti-CD102 antibody selection followed by invitro culture and treatment with TGF-β1. EndoMT was assessed by semiquantitative RT-PCR for Acta2, Col1a1, Snai1, and Snai2; by immunofluorescence for α-smooth muscle actin; and by Western blot analysis for α-smooth muscle actin, SNAIL1, SNAIL2, and the α2 chain of type I collagen. The same studies were performed in Cav1(-/-) pulmonary ECs after restoration of functional CAV1 domains using a cell-permeable CAV1 scaffolding domain peptide. Pulmonary ECs from Cav1 knockout mice displayed high levels of spontaneous Acta2, Col1A, Snai1, and Snai2 expression, which increased after TGF-β treatment. Spontaneous and TGF-β1-stimulated EndoMT were abrogated by the restoration of functional CAV1 domains using a cell-permeable peptide. The findings suggest that CAV1 regulation of EndoMT may play a role in the development of fibroproliferative vasculopathies.