We evaluated the efficacy of intravenous PTR-01, a recombinant type VII collagen (C7) expressed in Chinese Hamster Ovary (CHO) cells, in a Phase 2 trial involving six patients with recessive dystrophic epidermolysis bullosa (RDEB). Results showed that PTR-01 was well-tolerated and improved wound healing; however, no anchoring fibrils (AFs) were detected at the basement membrane zone despite C7 deposition at the dermal-epidermal junction. To investigate this, we compared PTR-01 to fibroblast-derived wild-type C7. Non-reducing western blots showed additional low-molecular-weight bands in PTR-01, indicating reduced stability. Under reducing conditions, PTR-01 appeared as a doublet, with domain-specific antibodies revealing proteolytic maturation at the C-terminus in the NC2 domain. Proteomic analysis demonstrated altered glycosylation and a mixture of 40% wild-type C7 and 60% C7 containing a D1033Y substitution in the FN type III domain of NC1. This variant exhibited dominant-negative effects, reducing thermal stability and impairing binding to laminin-332 and collagen IV. These findings indicate that systemic C7 delivery to the skin is feasible and clinically beneficial in RDEB but suggest that impaired ligand binding, lower stability, and premature NC2 maturation hinder AF assembly. We have identified CHO clones that express only wild-type C7, which we plan to advance as an improved alternative to PTR-01.

Type VII collagen (C7) assembles into anchoring fibrils (AF) which are basement membrane zone (BMZ) structures that reinforce dermal-epidermal cohesion. Mutations in COL7A1, which encodes C7, cause dystrophic epidermolysis bullosa (DEB), a blistering disorder characterized by skin fragility. C7 consists of a central collagenous domain flanked by non-collagenous NC1 and NC2 regions. During AF assembly, C-proteinase-mediated cleavage of the NC2 domain enables anti-parallel dimer formation, followed by C7 lateral association into mature, banded AFs that loop through the dermis to anchor the epidermis. Here, we report two DEB patients expressing near full-length C7 truncated just proximal to the C-proteinase cleavage site (amino acids 2814-2843), which precludes anti-parallel dimerization. Despite this, electron microscopy revealed abundant, banded AFs, consistent with lateral association, but the fibrils failed to loop and reinsert into the BMZ. These findings demonstrate that C7 lateral association can occur independently of anti-parallel dimer formation. This work also highlights the importance of AF looping in maintaining skin integrity and how specific COL7A1 mutations may impact fibril architecture and clinical phenotype.

Osteoarthritis (OA) is a complex disease associated with genetic, biological, and mechanical risk factors that act, in part, to alter chondrocyte homeostasis. Our recent exome sequencing studies identified a damaging genetic variant in COL6A3, a monomeric unit of collagen type VI and a distinguishing component of the pericellular matrix (PCM) of articular cartilage, a transducer of mechanical and biochemical signals for the chondrocyte. To study the effect of this genetic variant, human induced pluripotent stem cell (hiPSC)-derived chondrocytes, genetically edited to harbor the COL6A3 mutation, were used as an in vitro model to investigate chondrocyte mechanobiology and pathobiology. The COL6A3 variant resulted in lower PCM elastic modulus and reduced expression of key matrix proteins, suggesting altered PCM structural composition and mechanical properties. Functional analyses revealed altered mechanotransduction, characterized by heightened osmotically-induced calcium signaling, consistent with reduced PCM modulus, and reduced anabolic response to TRPV4 activation, both at the transcriptional level and in matrix biosynthesis. RNA-sequencing identified dysregulated pathways and aberrant TRPV4 signaling in mutant chondrocytes following mechanical loading. The presence of the COL6A3 variant also resulted in disrupted circadian rhythms, with increased BMAL1 expression and a significant phase shift, suggesting that PCM properties influence the circadian clock. Finally, COL6A3 mutant chondrocytes exhibited an exacerbated catabolic response to interleukin-1, an inflammatory cytokine implicated in OA. Our study demonstrates the utility of human iPSCs for studying the pathophysiology of specific OA risk alleles. These findings highlight the impact of the COL6A3 variant on chondrocyte physiology and support targeting mechanotransduction signaling pathways as a potential strategy for OA intervention.

Clinical presentation and operative outcomes in vascular Ehlers-Danlos syndrome (VEDS) are heterogeneous. We present two patients with genetically confirmed VEDS who exhibited markedly different iliac pathology phenotypes. One ruptured a common iliac artery at a near-normal diameter and the other tolerated a large aortoiliac aneurysm without rupture. The Aortic and Arterial Vulnerability Spectrum is a conceptual framework that characterizes aortic and arterial failure (dissection/rupture) along a continuum of underlying tissue vulnerability, integrating extracellular matrix (ECM) ultrastructure and clinical phenotypes beyond traditional diameter-based models. Using ultrastructural analysis of skin biopsy as a window into arterial integrity, we demonstrate distinct differences in ECM organization that paralleled the vascular phenotypes. Viewed through the Aortic and Arterial Vulnerability Spectrum, these cases show how genotype-driven vulnerability shapes surgical phenotype in VEDS and highlight ultrastructural skin analysis as a tool for ECM assessment and possibly biologic risk stratification.

The collagen IV scaffold serves as a fundamental structural unit of the basement membrane (BM). Understanding its structure, assembly, and function is essential for tissue engineering, the design of organoid models, and developing therapies for diseases such as Alport syndrome, Gould syndrome, psoriasis, eye abnormalities, hearing loss, and others, where collagen IV is required for structural integrity and functionality of the BM. The collagen IV molecule is a 400 nm long heterotrimer, comprising non-collagenous 1 (NC1), collagenous, and 7S domains. The assembly of the collagen IV scaffold involves oligomerization of the C-terminal NC1 and the N-terminal 7S domains, along with lateral associations within the collagenous domain. However, the detailed architecture and assembly mechanisms of the collagen IV scaffold remain unclear. Here, we investigated the potency and mechanism of recombinant single-chain NC1 trimer incorporation into the collagen IV scaffold. We discovered that the NC1 trimer influences the overall assembly of the basement membrane by affecting the quality of the developing collagen IV scaffold in a dose-dependent manner, without impacting already established scaffolds. This interference occurs through the hexamerization of supplemented NC1 trimers with endogenous NC1 domains, as the NC1 trimer becomes sulfilimine crosslinked with the existing chains. Overall, the single-chain NC1 trimer of collagen IV is crucial for developing novel extracellular therapies in two main ways: (1) facilitating the delivery and incorporation of functional replacements like collagen IV fragments and (2) inhibiting the formation of new basement membranes in conditions such as tumor growth and detrimental vascularization.

Mutations in the collagen-modifying enzyme lysyl hydroxylase 1 (LH1) cause Warmblood Fragile Foal Syndrome (WFFS) in horses. We investigated the impact of this mutation on collagen structure and function. Our results show that LH1 deficiency leads to reduced lysine hydroxylation, altered collagen fibril organization, and tissue abnormalities resembling human Ehlers-Danlos syndrome. These findings highlight the critical role of LH1 in collagen biosynthesis and provide insights into the pathogenesis of WFFS.

Supramolecular extracellular matrix (ECM) networks play an essential role in skin architecture and function. Elastin microfibril interface-located proteins (EMILINs) comprise a family of three extracellular glycoproteins that serve as essential structural components of the elastin/fibrillin microfibril network, and exert crucial functions in cellular signaling. Little is known about the structural nature of EMILIN networks in skin. We therefore investigated the spatiotemporal localization of EMILIN-1, -2, -3 in human skin induced by aging, UV-exposure, fibrosis, and connective tissue disorder. Confocal immunofluorescence and immunogold electron microscopy analysis identified all EMILINs as components of elastic fibers and elastin-free oxytalan fibers inserted into the basement membrane (BM). Further, our ultrastructural analysis demonstrates cellular contacts of dermally localized EMILIN-1 positive fibers across the BM with the surface of basal keratinocytes. Analysis of skin biopsies and fibroblast cultures from fibrillin-1 deficient Marfan patients revealed that EMILINs require intact fibrillin-1 as deposition scaffold. In patients with scleroderma and the bleomycin-induced murine fibrosis model EMILIN-2 was upregulated. EMILIN-3 localizes to the tips of candelabra-like oxytalan fibers, and to specialized BMs engulfing hair follicles and sebaceous glands. Our data identify EMILINs as important markers to monitor rearrangements of the dermal ECM architecture induced by aging and pathological conditions.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-β (TGFβ) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFβ signaling in tendon prompted us to utilize TGFβ1 to induce tendon-like structures in 3D tendon constructs. TGFβ1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFβ1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFβ1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Although abnormal TGFβ signaling is observed in several heritable forms of thoracic aortic aneurysms and dissections including Marfan syndrome, its precise role in aortic disease progression is still disputed. Using a mouse genetic approach and quantitative isobaric labeling proteomics, we sought to elucidate the role of TGFβ signaling in three Fbn1 mutant mouse models representing a range of aortic disease from microdissection (without aneurysm) to aneurysm (without rupture) to aneurysm and rupture. Results indicated that reduced TGFβ signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFβ signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFβ signaling, were associated with aortic rupture. Our data indicate that TGFβ signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.

AMACO (VWA2 protein), secreted by epithelial cells, is strongly expressed at basement membranes when budding or invagination occurs in embryos. In skin, AMACO associates with proteins of the Fraser complex, which form anchoring cords. These, during development, temporally stabilize the dermal–epidermal junction, pending the formation of collagen VII-containing anchoring fibrils. Fraser syndrome in humans results if any of the core members of the Fraser complex (Fras1, Frem1, Frem2) are mutated. Fraser syndrome is characterized by subepidermal blistering, cryptophthalmos, and syndactyly. In an attempt to determine AMACO function, we generated and characterized AMACO-deficient mice. In contrast to Fraser complex mutant mice, AMACO-deficient animals lack an obvious phenotype. The mutually interdependent basement membrane deposition of the Fraser complex proteins, and the formation of anchoring cords, are not affected. Furthermore, hair follicle development in newborn AMACO-deficient mice showed no gross aberration. Surprisingly, it appears that, while AMACO is a component of the anchoring cords, it is not essential for their formation or function.

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1 , the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain–severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

Ocular anterior segment dysgenesis (ASD) refers to a collection of developmental disorders affecting the anterior structures of the eye. Although a number of genes have been implicated in the etiology of ASD, the underlying pathogenetic mechanisms remain unclear. Mutations in genes encoding collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome, a multi-system disorder that often includes ocular manifestations such as ASD and glaucoma. COL4A1 and COL4A2 are abundant basement membrane pro-teins that provide structural support to tissues and modulate signaling through interactions with other extracel-lular matrix proteins, growth factors, and cell surface receptors. In this study, we used a combination of histological, molecular, genetic and pharmacological approaches to demonstrate that altered TGF13 signaling contributes to ASD in mouse models of Gould syndrome. We show that TGF13 signaling was elevated in ante-rior segments from Col4a1 mutant mice and that genetically reducing TGF13 signaling partially prevented ASD. Notably, we identified distinct roles for TGF131 and TGF132 in ocular defects observed in Col4a1 mutant mice. Importantly, we show that pharmacologically promoting type IV collagen secretion or reducing TGF13 signaling ameliorated ocular pathology in Col4a1 mutant mice. Overall, our findings demonstrate that altered TGF13 signaling contributes to COL4A1-related ocular dysgenesis and implicate this pathway as a potential therapeutic target for the treatment of Gould syndrome. (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mouse and zebrafish hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 in basement membrane integrity. We show that HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately ten-fold lower affinity and in a competitive manner. Furthermore, immunofluorescence and immunogold labelling revealed that HMCN1/Hmcn1 is localized close to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as the loss of Laminin-α5 (Lama5) or Hmcn1. Finally, combined partial loss-of-function studies indicated that nid2a genetically interacts with both hmcn1 and lama5 . Together, these findings suggest that despite their mutually exclusive physical binding, hemicentins, nidogens, and laminins tightly cooperate and support each other during formation, maintenance, and function of basement membranes to confer tissue linkage.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

AMACO (VWA2 protein) is a basement membrane-associated protein secreted by epithelial cells. It is strongly expressed when invagination or budding occurs during development. AMACO associates with the Fraser complex, which when mutated causes Fraser syndrome, characterized by subepidermal blistering, cryptoph-thalmos, and syndactyly. The core Fraser complex proteins FRAS1, FREM1, and FREM2 localize at the dermal-epidermal junction and mediate adhesion to the underlying dermis during embryonic development. Earlier transmission electron microscopy studies of adult mouse skin showed clustered AMACO deposition below the lamina densa. In this study, we report a distinct cord-like suprastructure in the neonate dermis to which AMACO- and Fraser complex-associated proteins contribute. We propose anchoring cords to designate the suprastructure. Anchoring cords have a diameter of 60 nm when immunolabeled, originate from the basement membrane, and extend several microns into the dermis. In normal skin, they are evident after immunogold electron microscopy and are strikingly appreciated in thicker sections. In recessive dystrophic epidermolysis bullosa skin, they are directly visible where collagen VII anchoring fibrils are ablated. Immunofluorescence and coimmunoprecipitation of skin extracts identify a direct interaction of FREM2 and AMACO.