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Key features of diabetic nephropathy include the accumulation of extracellular matrix proteins. In recent studies, increased expression of type VIII collagen in the glomeruli and tubulointerstitium of diabetic kidneys has been noted. The objectives of this study were to assess whether type VIII collagen affects the development of diabetic nephropathy and to determine type VIII collagen-dependent pathways in diabetic nephropathy in the mouse model of streptozotocin (STZ)-induced diabetes.
Several zebrafish mutants identified in large-scale forward genetic screens exhibit notochord distortion. We now report the cloning and further characterization of one such mutant, gulliver(m208) (gul(m208)). The notochord defect in gul(m208) mutants is exacerbated under conditions of copper depletion or lysyl oxidase cuproenzyme inhibition that are without a notochord effect on wild-type embryos. The gul(m208) phenotype results from a missense mutation in the gene encoding Col8a1, a lysyl oxidase substrate, and morpholino knockdown of col8a1 recapitulates the notochord distortion observed in gul(m208) mutants. Of interest, the amino acid mutated in gul(m208) Col8a1 is highly conserved, and the equivalent substitution in a closely related human protein, COL10A1, causes Schmid metaphyseal chondrodysplasia. Taken together, the data identify a new protein essential for notochord morphogenesis, extend our understanding of gene-nutrient interactions in early development, and suggest that human mutations in COL8A1 may cause structural birth defects.
Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions in vivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.
Arterial stiffening is a significant predictor of cardiovascular disease development and mortality. In elastic arteries, stiffening refers to the loss and fragmentation of elastic fibers, with a progressive increase in collagen fibers. Type VIII collagen (Col-8) is highly expressed developmentally, and then once again dramatically upregulated in aged and diseased vessels characterized by arterial stiffening. Yet its biophysical impact on the vessel wall remains unknown. The purpose of this study was to test the hypothesis that Col-8 functions as a matrix scaffold to maintain vessel integrity during extracellular matrix (ECM) development. These changes are predicted to persist into the adult vasculature, and we have tested this in our investigation. Through our in vivo and in vitro studies, we have determined a novel interaction between Col-8 and elastin. Mice deficient in Col-8 (Col8-/-) had reduced baseline blood pressure and increased arterial compliance, indicating an enhanced Windkessel effect in conducting arteries. Differences in both the ECM composition and VSMC activity resulted in Col8-/- carotid arteries that displayed increased crosslinked elastin and functional distensibility, but enhanced catecholamine-induced VSMC contractility. In vitro studies revealed that the absence of Col-8 dramatically increased tropoelastin mRNA and elastic fiber deposition in the ECM, which was decreased with exogenous Col-8 treatment. These findings suggest a causative role for Col-8 in reducing mRNA levels of tropoelastin and the presence of elastic fibers in the matrix. Moreover, we also found that Col-8 and elastin have opposing effects on VSMC phenotype, the former promoting a synthetic phenotype, whereas the latter confers quiescence. These studies further our understanding of Col-8 function and open a promising new area of investigation related to elastin biology.
Glioblastoma (GBM) is one of the most lethal tumor of all human cancers. Due to its poor response to chemotherapy and radiotherapy as well as its high rate of recurrence after treatment, the treatment is still undesired. The identification of potential related genes and bio-markers in the development of GBM could provide some new targets for the treatment of GBM. Our purpose in this study was to evaluate the mission of COL8A2 in GBM. Combined with TCGA, Oncomine databases, CGGA, GEPIA website and qRT-PCR analyses, we found that COL8A2 was up-regulated both in GBM tissues and cells compared to the controls. Moreover, the high COL8A2 expression was associated with the shorter overall survival of patients with GBM. The expression of COL8A2 was also positively correlated with metastasis-associated genes including vimentin, snail, slug, MMP2 and MMP7 according to GEPIA website. Knockdown of COL8A2 could suppress the cell proliferation, cell migration and invasion, whereas the overexpression of COL8A2 significantly expedited these processes. What's more, the outcome of western blot analysis manifested that COL8A2 could induced the expression of vimentin, snail, slug, MMP2 and MMP7. Taken together, COL8A2 activated cell proliferation, cell migration and invasion via raising the relative expression of EMT-related proteins in GBM. Therefore, our investigation suggests the oncogenic role of COL8A2 in GBM and provides a potential application of COL8A2 for GBM therapy.
The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.
Osteogenesis imperfecta (OI) is a heritable disorder of bone metabolism characterized by multiple fractures with minimal trauma. Autosomal recessive OI type VIII is associated with biallelic pathogenic variants in P3H1 and classically characterized by skeletal anomalies in addition to significant bone fragility, sometimes presenting with in utero fractures and/or neonatal lethality. P3H1 encodes a collagen prolyl hydroxylase that critically 3-hydroxylates proline residue 986 on the α chain of collagen types I and II to achieve proper folding and assembly of mature collagen and is present in a complex with CRTAP and CypB. Most individuals with OI type VIII have had biallelic predicted loss-of-function variants leading to reduced or absent levels of P3H1 mRNA. The reported missense variants have all fallen in the catalytic domain of the protein and are thought to be associated with a milder phenotype. Here, we describe an infant presenting with five long bone fractures in the first year of life found to have a novel missense variant in trans with a nonsense variant in P3H1 without any other bony anomalies on imaging. We hypothesize that missense variants in the catalytic domain of P3H1 lead to decreased but not absent hydroxylation of Pro986, with preserved KDEL retention signal and complex stability, causing an attenuated phenotype.
Background Age-associated aortic remodeling includes a marked increase in intimal medial thickness (IMT), associated with signs of inflammation. Although aortic wall milk fat globule-epidermal growth factor VIII (MFG-E8) increases with age, and is associated with aortic inflammation, it is not known whether MFG-E8 is required for the age-associated increase in aortic IMT. Here, we tested whether MFG-E8 is required for the age-associated increase in aortic IMT. Methods and Results To determine the role of MFG-E8 in the age-associated increase of IMT, we compared aortic remodeling in adult (20-week) and aged (96-week) MFG-E8 (-/-) knockout and age matched wild-type (WT) littermate mice. The average aortic IMT increased with age in the WT from 50±10 to 70±20 μm (P<0.0001) but did not significantly increase with age in MFG-E8 knockout mice. Because angiotensin II signaling is implicated as a driver of age-associated increase in IMT, we infused 30-week-old MFG-E8 knockout and age-matched littermate WT mice with angiotensin II or saline via osmotic mini-pumps to determine whether MFG-E8 is required for angiotensin II-induced aortic remodeling. (1) In WT mice, angiotensin II infusion substantially increased IMT, elastic lamina degradation, collagen deposition, and the proliferation of vascular smooth muscle cells; in contrast, these effects were significantly reduced in MFG-E8 KO mice; (2) On a molecular level, angiotensin II treatment significantly increased the activation and expression of matrix metalloproteinase type 2, transforming growth factor beta 1, and its downstream signaling molecule phosphorylated mother against decapentaplegic homolog 2, and collagen type I production in WT mice; however, in the MFG-E8 knockout mice, these molecular effects were significantly reduced; and (3) in WT mice, angiotensin II increased levels of aortic inflammatory markers phosphorylated nuclear factor-kappa beta p65, monocyte chemoattractant protein 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 molecular expression, while in contrast, these inflammatory markers did not change in knockout mice. Conclusions Thus, MFG-E8 is required for both age-associated proinflammatory aortic remodeling and also for the angiotensin II-dependent induction in younger mice of an aortic inflammatory phenotype observed in advanced age. Targeting MFG-E8 would be a novel molecular approach to curb adverse arterial remodeling.
Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.
Skin wounds remain a significant problem for the healthcare system, affecting the clinical outcome, patients' quality of life, and financial costs. Reduced wound healing times would improve clinical, economic, and social aspects for both patients and the healthcare system. Skin wound healing has been studied for years, but effective therapy that leads to accelerated wound healing remains to be discovered. This study aimed to evaluate the potential of MELK silencing to accelerate wound healing. A vectorless, transient knockdown of the MELK gene using siRNA was performed in a murine skin wound model. The wound size, total collagen, type 3 collagen, vessel size, vessel number, cell proliferation, cell apoptosis, number of mast cells, and immune infiltration by CD45, CD11b, CD45, and CD8a cells were evaluated. We observed that treatment with MELK siRNA leads to significantly faster wound closing associated with increased collagen deposition.
Excessive accumulation of collagen is often used to assess the development of fibrosis. This study aims to identify collagen genes that define fibrosis in the conjunctiva following glaucoma filtration surgery (GFS). Using the mouse model of GFS, we have identified collagen transcripts that were upregulated in the fibrotic phase of wound healing via RNA-seq. The collagen transcripts that were increased the most were encoded by Col8a1, Col11a1 and Col8a2 Further analysis of the Col8a1, Col11a1 and Col8a2 transcripts revealed their increase by 67-, 54- and 18-fold, respectively, in the fibrotic phase, compared with 12-fold for Col1a1, the most commonly evaluated collagen gene for fibrosis. However, only type I collagen was significantly upregulated at the protein level in the fibrotic phase. Type VIII and type I collagens colocalized in fibrous structures and in ACTA2-positive pericytes, and appeared to compensate for each other in expression levels. Type XI collagen showed low colocalization with both type VIII and type I collagens but can be found in association with macrophages. Furthermore, we show that both mouse and human conjunctival fibroblasts expressed elevated levels of the most highly expressed collagen genes in response to TGFβ2 treatment. Importantly, conjunctival tissues from individuals whose GF surgeries have failed due to scarring showed 3.60- and 2.78-fold increases in type VIII and I collagen transcripts, respectively, compared with those from individuals with no prior surgeries. These data demonstrate that distinct collagen transcripts are expressed at high levels in the conjunctiva after surgery and their unique expression profiles may imply differential influences on the fibrotic outcome.
Coenzyme Q10 (CoQ10) is a well-known antioxidant and serves as an essential carrier for electron transport and proton translocation in the mitochondrial respiratory chain. CoQ10 has been widely commercially available in Japan as a dietary and health supple-ment since 2001 and it is used for the prevention of lifestyle-related diseases induced by aging. Recently, it was stated that for Japan, which is facing an aging society, CoQ10 has been used in many skincare products. However, the physiological actions of CoQ10 in skin fibroblasts are not fully understood. In this study, we examined the effect of CoQ10 on cultured human skin fibroblast. In this study, CoQ10 treatment increased intracellular CoQ10 level and promoted proliferation of fibroblasts. In addition, CoQ10 increased mRNA expression of type I, IV, VII collagen, elastin, and HSP47, whereas CoQ10 has little effect on mRNA of type II and VIII MMP. These results suggested that CoQ10 has the efficacy that it increases collagen production in skin, thereby there is possible of the anti-aging by CoQ10 in Japan which reached an aging society, so that it might be based on new physiological function by CoQ10.
We examined changes in the ultrastructure and localization of major extracellular matrix components, including 5 types of collagen (type I, III, IV, VI, and VIII), laminin, fibronectin, and heparan sulfate proteoglycan in Descemet's membrane of the cornea of diabetic GK rats. In the cornea of diabetic GK rats, more long-spacing collagen fibrils were observed in Descemet's membrane than in the membrane of the nondiabetic Wistar rats. Both GK and Wistar rats showed an age-dependent increase in the density of the long-spacing collagen. Immunoelectron microscopy showed that type VIII collagen was localized in the internodal region of the long-spacing collagen, which was not labelled by any of the other antibodies used. The antidiabetic agents nateglinide and glibenclamide significantly suppressed the formation of the long-spacing collagen in the diabetic rats. Long-spacing collagen would thus be a useful indicator for studying diabetic changes in the cornea and the effect of antidiabetic agents.
Osteogenesis imperfecta (OI) types VII, VIII and IX, caused by recessive mutations in cartilage-associated protein (CRTAP), prolyl-3-hydroxylase 1 (P3H1) and cyclophilin B (PPIB), respectively, are characterized by the synthesis of overmodified collagen. The genes encode for the components of the endoplasmic reticulum (ER) complex responsible for the 3-hydroxylation of specific proline residues in type I collagen. Our study dissects the effects of mutations in the proteins of the complex on cellular homeostasis, using primary fibroblasts from seven recessive OI patients. In all cell lines, the intracellular retention of overmodified type I collagen molecules causes ER enlargement associated with the presence of protein aggregates, activation of the PERK branch of the unfolded protein response and apoptotic death. The administration of 4-phenylbutyrate (4-PBA) alleviates cellular stress by restoring ER cisternae size, and normalizing the phosphorylated PERK (p-PERK):PERK ratio and the expression of apoptotic marker. The drug also has a stimulatory effect on autophagy. We proved that the rescue of cellular homeostasis following 4-PBA treatment is associated with its chaperone activity, since it increases protein secretion, restoring ER proteostasis and reducing PERK activation and cell survival also in the presence of pharmacological inhibition of autophagy. Our results provide a novel insight into the mechanism of 4-PBA action and demonstrate that intracellular stress in recessive OI can be alleviated by 4-PBA therapy, similarly to what we recently reported for dominant OI, thus allowing a common target for OI forms characterized by overmodified collagen.This article has an associated First Person interview with the first author of the paper.
Type 2B von Willebrand disease (VWD) is characterized by an increased binding affinity of von Willebrand factor (VWF) to platelet glycoprotein Ib. This can lead to clearance of high-molecular-weight (HMW) multimers and thrombocytopenia with a resulting moderate-severe bleeding phenotype. Rondoraptivon pegol (BT200) is a pegylated aptamer binding to the A1 domain of VWF with a novel mechanism of action: it enhances VWF/factor VIII (FVIII) levels by decreasing their clearance. To study the potential benefit of rondoraptivon pegol in patients with type 2B VWD, we conducted a prospective phase 2 trial. Patients with type 2B VWD received 3 mg rondoraptivon pegol subcutaneously on study days 1, 4, and 7, followed by 6 to 9 mg every week until day 28. Five patients (male:female ratio = 3:2) were included. Rondoraptivon pegol rapidly tripled platelet counts from a median of 60 to 179 × 10E9/L (P < .001). Circulating VWF antigen increased from a median of 64% to 143%, which doubled FVIII activity levels from 67% to 134%. In all thrombocytopenic patients, plasma levels of VWF:GPIbM normalized, VWF ristocetin cofactor and VWF collagen-binding activity increased, and HMW multimers appeared. These pronounced improvements reversed during washout of the drug, thus demonstrating causality. The A1 domain binding aptamer directly corrects the underlying defect of type 2B VWD, thus providing a novel potential option for prophylaxis and treatment of patients with this VWD type. These data provide the basis for a phase 2b/3 trial in such patients. This trial was registered at www.clinicaltrials.gov as #NCT04677803.
Fuchs' endothelial corneal dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.
One approach to identify genes that contribute to common complex ocular disorders such as primary open angle glaucoma (POAG) is to study the genetic determinates of endophenotypes that are defined by underlying pre-disposing heritable quantitative traits such as central corneal thickness (CCT). Collagen VIII is a major component of Descemet's membrane and studies in mice have indicated that targeted inactivation of the genes encoding the collagen type 8 alpha1 (Col8a1) and collagen type 8 alpha2 (Col8a2) subunits (COL8A1 and COL8A2) results in thinning of the corneal stroma and of Descemet's membrane. The purpose of this study is to evaluate COL8A1 and COL8A2 as candidate genes for thin CCT in human POAG patients.
Antiangiogenic therapies are considered promising for the treatment of glioblastoma (GB). The non-collagenous C-terminal globular NC1 domain of type VIII collagen a1 chain, Vastatin, is an endogenous antiangiogenic polypeptide. Sustained enhanced expression of Vastatin was shown to inhibit tumour growth and metastasis in murine hepatocellular carcinoma models. In this study, we further explored the efficacy of Vastatin in the treatment of GB xenografts.
Phage display technology utilises peptide and antibody libraries with very high diversities to select ligands with specific binding properties. The production of such libraries can be labour intensive and technically challenging and whilst there are commercial sources of libraries, the exploitation of the resulting binders is constrained by ownership of the libraries. Here, a peptide library of ~ 1 × 109 variants for display on gene VIII was produced alongside three VHH antibody libraries with similar diversity, where 12mer, 16mer or 21mer CDR3s were introduced into the highly stable cAbBCII10 scaffold displayed on gene III. The cloning strategy used a simple whole-plasmid PCR method and type IIS restriction enzyme assembly that facilitate the seamless insertion of diversity into any suitable phage coat protein or antibody scaffold. This method reproducibly produced 1 × 109 variants from just 10 transformations and the four libraries had relatively low bias with 82 to 86% of all sequences present as single copies. The functionality of both peptide and antibody libraries were demonstrated by selection of ligands with specific binding properties by biopanning. The peptide library was used to epitope map a monoclonal antibody. The VHH libraries were pooled and used to select an antibody to recombinant human collagen type 1.
Osteogenesis imperfecta (OI) is a collagen-related disorder associated to dominant, recessive or X-linked transmission, mainly caused by mutations in type I collagen genes or in genes involved in type I collagen metabolism. Among the recessive forms, OI types VII, VIII, and IX are due to mutations in CRTAP, P3H1, and PPIB genes, respectively. They code for the three components of the endoplasmic reticulum complex that catalyzes 3-hydroxylation of type I collagen α1Pro986. Under-hydroxylation of this residue leads to collagen structural abnormalities and results in moderate to lethal OI phenotype, despite the exact molecular mechanisms are still not completely clear. To shed light on these recessive forms, primary fibroblasts from OI patients with mutations in CRTAP (n=3), P3H1 (n=3), PPIB (n=1) genes and from controls (n=4) were investigated by a functional proteomic approach. Cytoskeleton and nucleoskeleton asset, protein fate, and metabolism were delineated as mainly affected. While western blot experiments confirmed altered expression of lamin A/C and cofilin-1, immunofluorescence analysis using antibody against lamin A/C and phalloidin showed an aberrant organization of nucleus and cytoskeleton. This is the first report describing an altered organization of intracellular structural proteins in recessive OI and pointing them as possible novel target for OI treatment.
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