Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome

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.

Targetable cellular signaling events mediate vascular pathology in vascular Ehlers-Danlos syndrome

“…ER stress and consequent autophagy due to abnormal collagen secretion has also been proposed as a possible pathogenic mechanism by which alterations in collagen III biosynthesis may lead to altered tissue mechanics and integrity (16,26). Fibroblasts in skin biopsies from individuals with vEDS demonstrate marked rough ER distention (26) and transcriptomic analysis of skin fibroblasts with COL3A1 mutations demonstrated changes in expression of several genes involved in ER homeostasis (39). However, rough ER distention has not been observed in arteries from humans or animal models with vEDS (16).…”

Multifaced Roles of the αvβ3 Integrin in Ehlers–Danlos and Arterial Tortuosity Syndromes’ Dermal Fibroblasts

“…The skin is one of the connective tissues affected in all EDS types as well as in other HCTDs, and dermal fibroblasts have been shown to represent an excellent in vitro cell model to study ECM organization and molecular mechanisms involved in the pathophysiology of several HCTDs [ 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 ]. In vitro grown human dermal fibroblasts synthesize and secrete several ECM structural proteins that are deposited on the substrate and organized in a network covering the cell layer.…”

“…In vitro grown human dermal fibroblasts synthesize and secrete several ECM structural proteins that are deposited on the substrate and organized in a network covering the cell layer. In particular, three days after seeding, control fibroblasts organize the ECM of FN (FN–ECM), with a predominant deposition of the EDA + FN variant, COLLIII, COLLV, and rare fibrils of COLLI ([ 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 ], and Table 7 ). The main COLLs receptor expressed by control cells is the α2β1 integrin, whereas FN is preferentially bound to the α5β1 integrin [ 102 , 103 , 104 ].…”

“…This phenotype is also observed in dermal fibroblasts from patients with ATS but not in LDS- TGFBR1 , MFS, and OI cells, which organize an abundant FN–ECM and express the α5β1 integrin and not αvβ3 ([ 105 , 114 ], and Table 7 ). EDS fibroblasts also show a reduced/absent deposition into the ECM of COLLI and COLLIII, in association with a variable organization of COLLV ([ 102 , 103 , 104 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 ], and Table 7 ). A similar disorganization of the COLLs–ECM is also observed in LDS- TGFBR1 , OI, and ATS cells but not in MFS fibroblasts ([ 105 ], and Table 7 ).…”

“…A similar disorganization of the COLLs–ECM is also observed in LDS- TGFBR1 , OI, and ATS cells but not in MFS fibroblasts ([ 105 ], and Table 7 ). The abnormal COLLs–ECM depositions observed in all EDS types as well as in OI and ATS cells is associated with the loss of the canonical COLLs α2β1 integrin receptor’s expression ([ 102 , 103 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 ], and Table 7 ). For the genetically defined forms of EDS as well as for OI, the abnormal COLLs–ECM organization and the consequent loss of the α2β1 integrin is easily explained by the underlying molecular defects that include not only anomalies of the collagen primary structure (cEDS, vEDS, and OI), collagen processing (dEDS, aEDS), folding and cross-linking (kEDS), but also defects in glycosaminoglycan biosynthesis (spEDS, mcEDS) that are known to impact COLLs fibril formation and deposition [ 9 ].…”