Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

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.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

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-ß (TGFß) 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 TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 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. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 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.

Unraveling the role of TGFß signaling in thoracic aortic aneurysm and dissection using Fbn1 mutant mouse models.

Although abnormal TGFß 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 TGFß 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 TGFß signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFß signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFß signaling, were associated with aortic rupture. Our data indicate that TGFß signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.

The Fraser Complex Proteins (Frem1, Frem2, and Fras1) Can Form Anchoring Cords in the Absence of AMACO at the Dermal-Epidermal Junction of Mouse Skin.

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.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

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.

Lysyl hydroxylase 3-mediated post-translational modifications are required for proper biosynthesis of collagen α1α1α2(IV).

Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome.

Anchoring Cords: A Distinct Suprastructure in the Developing Skin.

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, cryptophthalmos, 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.

Elevated TGFß signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice.

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 proteins that provide structural support to tissues and modulate signaling through interactions with other extracellular 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 TGFß signaling contributes to ASD in mouse models of Gould syndrome. We show that TGFß signaling was elevated in anterior segments from Col4a1 mutant mice and that genetically reducing TGFß signaling partially prevented ASD. Notably, we identified distinct roles for TGFß1 and TGFß2 in ocular defects observed in Col4a1 mutant mice. Importantly, we show that pharmacologically promoting type IV collagen secretion or reducing TGFß signaling ameliorated ocular pathology in Col4a1 mutant mice. Overall, our findings demonstrate that altered TGFß signaling contributes to COL4A1-related ocular dysgenesis and implicate this pathway as a potential therapeutic target for the treatment of Gould syndrome.

In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial.

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.

A new murine model of Barth syndrome neutropenia links TAFAZZIN deficiency to increased ER stress-induced apoptosis.

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.