Abstract
Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures.To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of several structural proteins crucial to the integrity of soft connective tissues.
Highlights
Vascular Ehlers-Danlos syndrome is a rare autosomal dominant inherited connective tissue disorder caused by mutations in the COL3A1 gene encoding the pro-α 1 chain of type III procollagen (COLLIII) [1]
To identify biological processes that were over- or under-represented in patients’ cells, we classified all up- and down-regulated genes according to the Gene Ontology (GO) categories by using the DAVID and ToppGene biological databases
Structural misfolding mutations in COLLs-encoding genes or in other extracellular matrix (ECM)-related molecules, such as COL1A1, COL1A2, COL2A1, COL6A1, COL6A2, COL6A3, and COMP, are well known to cause a dominant negative effect, leading to partial or complete cellular retention and/or degradation of mutant proteins, and normal proteins being assembled into mutant-containing multimers
Summary
Vascular Ehlers-Danlos syndrome (vEDS, OMIM#130050) is a rare autosomal dominant inherited connective tissue disorder caused by mutations in the COL3A1 gene encoding the pro-α 1 chain of type III procollagen (COLLIII) [1]. COL3A1 missense mutations altering the glycine codons together with in-frame splice mutations in the triple helical domain account for the majority of disease-causing variants and lead to misfolding of COLLIII in the endoplasmic reticulum (ER) and retention of seven eighths of misfolded procollagen trimers in the cell with consequent enlargement of ER vesicles [8,9,10]. Cold methanol fixed fibroblasts were immunoreacted with 1:100 anti-COLLIII Ab. To analyze FBNs and ELN organization into ECM, cells were grown, fixed, and immunoreacted as reported in detail previously [19]. Analyses were performed with GraphPad Prism software (GraphPad Software Inc., USA)
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