Decellularized extracellular matrix materials have been widely studied for tissue engineering and regenerative medicine (TERM) applications, largely because of their intrinsic bioactivity and immunomodulatory potentials. These properties confer decellularized extracellular matrix biomaterials a biological advantage over other biomaterials, especially synthetic ones, leading to several successful applications in TERM. While the complex composition of decellularized materials is well known and thought to play a role in providing the regenerative advantage, the fine mechanisms laying behind their bioactivity and immunomodulation were not fully understood yet. In the last decade, researchers have discovered a novel component in decellularized extracellular matrix materials: the matrix bound nanovesicle (MBV). This newly described type of extracellular vesicle is characterized by a tight relation to the extracellular matrix, differently from other liquid phase vesicles, and presents a unique tissue specific cargo, thought to be secreted by cells for specific cell signalling purposes. Although other extracellular vesicles subtypes have been extensively studied in past years, MBVs are different in many ways, making this research field noticeably young. Major bioactivity and immune modulating ability are key features of MBVs that were evident right from the first research works. However, to understand how MBVs can recapitulate and confer decellularized biomaterials with their signature biological performance, they are being characterized in depth. In particular, their rich and varied cargo is being explored, which has shown to play a fundamental role in MBVs' biological potential. This discovery not only revolutionized the look on decellularized extracellular matrix materials, but it also opened the way for research on a novel type of biomaterial, with plenty potential in therapeutical and regenerative applications. This review presents in detail what has been discovered up to now on MBVs, their properties, biological roles, and potential in TERM.

Basement membranes (BM) are thin, nanoporous sheets of specialized extracellular matrix (ECM) that line epithelial tissues. They are dynamic structures that serve multiple key functions, as evidenced by numerous diseases, including cancer progression, that are associated with their alterations. Our understanding of the BM and its communication with adjoining epithelial cells remains highly fragmented due to the BM's complex molecular architecture, the lack of molecular tools, limitations in utilizing high-resolution imaging techniques to BMs assembled on tissues, and the difficulty of assessing their functional contributions in vivo. Here, by combining multiple -omics analyses and advanced microscopy methodologies, we characterized the BM from two normal human mammary epithelial cell lines, MCF10 and HMLE, grown as spheroids in 3D matrices. Our findings indicate that the spheroids autonomously assemble a BM exhibiting all the molecular, structural, and biophysical characteristics of physiological BM. Using these minimalist model systems, we provide evidence that collagen IV, laminins, perlecan, and hemidesmosomes all overlap in a shared porous lattice. Next, we demonstrate that the invasion-promoting PSD4/EFA6B knockout, found in patients with breast cancer, decreases the expression of BM components and their assembly on the spheroid surface. We then show that invasive spheroids develop enlarged pores in the BM via filopodia-like plasma membrane extensions, which further expand in a protease-dependent manner, thereby facilitating the passage of invasive cells.

Fibrin is a biocompatible hydrogel that is widely used as a surgical sealant and as a scaffold for in vitro cell culture. Here, we engineered a heterotrimeric chimera between fibrinogen and laminin-511 by connecting the N-terminal self-polymerization domain of fibrinogen with the C-terminal integrin-binding domain of laminin-511 via their coiled-coil regions. The resulting chimeric protein, designated Chimera-511, binds to fibrinogen in a thrombin-dependent manner and exerts integrin-binding activity in a fibrin(ogen)-bound form. Chimera-511 co-polymerizes with fibrinogen to form a fibrin gel endowed with the potent integrin-binding activity of laminin-511, thereby enabling robust three-dimensional proliferation of human induced pluripotent stem cells while maintaining their pluripotency marker expression and trilineage differentiation potential. This functionalized, biodegradable fibrin gel provides a defined and clinically compatible three-dimensional scaffold for stem cell culture, with potential applications in both basic research and regenerative medicine.

UDP-glucose 6-dehydrogenase (UGDH) is an essential enzyme that catalyzes the oxidation of UDP-glucose to UDP-glucuronate. UGDH is elevated in multiple cancers, including prostate cancer, and is functionally implicated in castration resistant recurrence. UGDH is composed of three dimeric units that associate stably as a hexamer in cellular conditions. The dynamic reorganization of noncovalent interactions at the dimer contact interfaces is essential for UGDH activity. In this study, we examined the functional relevance of a putative AGC kinase motif located at the dimer-dimer interface. We demonstrated that UGDH is phosphorylated in LNCaP cells, specifically at serine 316, by RSK2, p70S6K, and SGK1. To determine the functional implications of UGDH S316 phosphorylation, we generated and characterized phosphomimetic (S316D) and phosphodeficient (S316A) point mutations. Intrinsic properties of the purified recombinant proteins were only modestly affected by the substitutions. The stable overexpression of UGDH S316D in LNCaP cells significantly increased the rate of N- and O-glycan synthesis, as well as the production of hyaluronan and sulfated glycosaminoglycans, while reducing DHT glucuronidation, resulting in significant increases in growth of tumor spheroids, cell proliferation and motility, and resistance to enzalutamide. In contrast, UGDH S316A expression reduced the production of glycans and glycosaminoglycans, restored DHT glucuronidation, and impaired growth and motility. Overall, our results support UGDH phosphorylation as a point of control for intracellular and cell surface glycan production, thereby regulating cell proliferation, anchorage dependence, motility, and tumor cell therapeutic resistance.

A subset of rheumatoid arthritis (RA) is the production of autoantibodies, including antibodies to citrullinated proteins (ACPA) and antibodies to type II collagen (AC2A). Type II collagen (COL2) is the major protein in joint cartilage and is a target of arthritogenic autoantibodies. We could confirm that sera from RA patients react with both citrullinated and native triple-helical COL2 epitopes. We examined the modulation of COL2 processing by matrix metalloproteinase 13 (MMP-13), the main collagenase responsible for degradation of articular cartilage. Anti-COL2 antibodies (AC2A) targeting the C1 epitope (residues 359-363) partially inhibited intact COL2 and fragment hydrolysis, resulting in two distinct fragments in the 25-30 kDa range. The AC2A targeting the E10 epitope (residues 777-783, the region where MMP-13 initially cleaves COL2) partially inhibited intact COL2 and fragment hydrolysis, resulting in a distinct fragment of ∼30 kDa. The AC2As targeting the F4 epitope (residues 932-936) partially inhibited collagen fragment hydrolysis, resulting in four distinct fragments in the 20-30 kDa range. Sequencing of isolated fragments revealed 14 terminated cleavage sites. Citrullination of the COL2 cleavage site reduced MMP-13 efficiency, which should further exacerbate fragment production rather than complete digestion. The results indicated that, under normal maintenance, MMP-13 cleaves COL2 initially at the 775-776 bond, followed by further digestion of COL2 fragments. Citrullination slows the initial processing of COL2 by MMP-13. In concert, AC2As inhibit the action of MMP-13 at different stages, resulting in production of collagen fragments differing in composition encountered under normal circumstances. The abnormal COL2 fragments could activate the immune system to be more pathogenic or regulatory as well as modify chondrocyte functions, and thereby play a role in the initiation of RA.

FAM20C is part of the FAM20 family and is crucial for phosphorylating secreted proteins. It plays roles in various biological processes, including cellular calcium regulation and cardiovascular function. Pathogenic variants of FAM20C cause Raine syndrome, resulting in osteosclerotic dysplasia or hypophosphatemic rickets. Its paralog, FAM20A, is a secreted pseudokinase needed for optimal FAM20C activity. Mutations in FAM20A cause Enamel-Renal Syndrome. Common features in both syndromes include Amelogenesis Imperfecta, gingival fibromatosis and ectopic mineralization. We summarize current knowledge about the activity, interactions and regulation of FAM20C and FAM20A, with a focus on the possible role of bioactive lipids like sphingosine in activating FAM20C. We highlight the involvement of FAM20A and FAM20C in gingival fibromatosis, a fibrocalcifying disorder directly linked to the dysfunction of these proteins and the underlying molecular mechanisms. Additionally, we provide an overview of how FAM20C and FAM20A influence calcium homeostasis and mineralization. Since FAM20C-mediated phosphorylation is crucial in oral health, we detail how specific substrates such as osteopontin, periostin, or fetuin contribute to normal and ectopic mineralization and periodontal health. Although many questions about the roles of FAM20A and FAM20C in both oral and systemic diseases remain unresolved, targeting their activities could offer promising therapeutic options.

At its best, it is exhilarating to make unexpected discoveries when addressing carefully formed scientific hypotheses. This review depicts my scientific journey in the field of extracellular matrix biology, and more specifically in collagen research, starting in 1978 and continuing with exciting findings up to the present day. While recounting my early work on the enzymes of collagen biosynthesis, the focus will be on our discoveries of new types of nonfibrillar collagen: type XIII collagen, belonging to the MACIT subgroup among the collagen family of proteins, and types XV and XVIII collagens, constituting the multiplexin subgroup. We have investigated these collagens through molecular biological approaches in order to define their primary structures, and through biochemical and cell biological work to understand their special molecular properties. Furthermore, the generation of many mouse models has led us to exciting studies of the roles of these collagens in adipose tissue, bone, eye, heart, kidney, liver, peripheral nerves, skin, and cancer models, although it has of course also been rather daunting in terms of choosing the correct approach for each tissue. The work on animal models has nevertheless resulted in a broad understanding of the in vivo significance of these collagens, forming a fruitful basis for studying their relevance to human diseases, including malignant processes. Our conclusions have been that these collagens can contribute to the stability of the extracellular matrix and tissue structures, e.g., the basement membrane and the adjacent fibrillar matrix in the case of the multiplexins and the motor synapse in the case of the MACIT type XIII collagen, and more unexpectedly, that they possess major roles as extrinsic regulators of the fates and functions of cells.

The basement membrane (BM), a specialized extracellular matrix (ECM), provides structural support for epithelial, endothelial, and other parenchymal cells. Once considered a static scaffold, the BM is now recognized as a dynamic and complex nanostructure composed of a diversity of molecules that actively regulate cell behavior and tissue organization. Its molecular composition, assembly, and remodeling are precisely controlled in a tissue- and stage-specific manner, contributing to the regulation of local and global mechanical properties and biochemical signaling. Understanding BM structure and function requires integrated approaches across biological scales-from nanoscale molecular interactions to tissue-level architecture. In this review, we highlight advances in three methodological areas: (1) imaging techniques that reveal BM nanostructure and dynamics, (2) manipulation strategies that uncover causal roles of BM components, and (3) omics-based approaches that map BM composition and cellular sources. Integrating these strategies enables the bridging of molecular events and organ-level functions, offering new insights into how the BM is involved in development, homeostasis, and disease progression. The aim of this review is to provide researchers with a comprehensive perspective on evolving tools for dissecting BM structure, dynamics, and function.

Collagens are fundamental components of extracellular matrices, requiring precise intracellular post-translational modifications for proper function. Among the modifications, prolyl 4-hydroxylation is critical to stabilise the collagen triple helix. In humans, this reaction is mediated by collagen prolyl 4-hydroxylases (P4Hs). While humans possess three genes encoding these enzymes (P4H⍺s), Drosophila melanogaster harbour at least 26 candidates for collagen P4H⍺s despite its simple genome, and it is poorly understood which of them are actually working on collagen in the fly. In this study, we addressed this question by carrying out thorough bioinformatic and biochemical analyses. We demonstrate that among the 26 potential collagen P4H⍺s, PH4⍺EFB shares the highest homology with vertebrate collagen P4H⍺s. Furthermore, while collagen P4Hs and their substrates must exist in the same cells, our transcriptomic analyses at the tissue and single cell levels showed a global co-expression of PH4⍺EFB but not the other P4H⍺-related genes with the collagen IV genes. Moreover, expression of PH4⍺EFB during embryogenesis was found to precede that of collagen IV, presumably enabling efficient collagen modification by PH4⍺EFB. Finally, biochemical assays confirm that PH4⍺EFB binds collagen, supporting its direct role in collagen IV modification. Collectively, we identify PH4⍺EFB as the primary and potentially constitutive prolyl 4-hydroxylase responsible for collagen IV biosynthesis in Drosophila. Our findings highlight the remarkably simple nature of Drosophila collagen IV biosynthesis, which may serve as a blueprint for defining the minimal requirements for collagen engineering.

Collagen IV is a major constituent of basement membranes and mutations in the genes COL4A1 and COL4A2 present clinically as a variable, multi-system disorder called COL4A1 (Gould) syndrome. Evidence from case reports supports a cardiac component to this disease, but the phenotypic and functional implications affecting the heart, their progression and underlying mechanisms all remain poorly characterised. Indeed, the role of the basement membrane (BM) in adult cardiac disease remains underexplored. We set out to address these knowledge gaps by combining in-depth phenotypic and molecular analyses of a Col4a1 mutation on cardiac biology in a murine model (Col4a1) of Gould Syndrome. This revealed morphological cardiac defects including cardiomyocyte hypertrophy with myocardial and vascular fibrotic remodelling that impaired cardiac function. The Col4a1 mutation causes systolic and diastolic dysfunction with reduced left ventricular developed pressure. Mechanistically, we show these defects are due to secretion of mutant protein and BM defects rather than collagen misfolding and proteotoxic stress. The BM defects lead to a pro-fibrotic state with increased fibrillar collagen deposition, cardiac stiffness, and ECM compositional defects. These are accompanied by altered regulation of pathways involved in sarcomere formation, sarcolemma stability and cardiomyocyte metabolism, establishing a molecular signature of COL4A1-related cardiac disease. Intriguingly, aspects of this molecular signature including cardiac metabolic pathways, regulation of cardiac muscle contraction and BM component expression, are shared with common cardiomyopathies such as coronary micro-embolism, and dilated, ischemic and hypertrophic obstructive cardiomyopathies. By defining the molecular and phenotypic cardiac components of Gould syndrome these data show that the BM is essential for maintaining systolic and diastolic function and that alterations to the BM leads to a fibrotic response. These data increase insight into the role of the basement membrane and collagen IV in cardiac biology, and highlights mechanisms shared between Gould syndrome and common adult cardiac disease.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer with poor clinical outcomes, due in part to altered fibrotic environments and striking immune dysfunction. Physical properties within tumors, such as aligned extracellular matrix (ECM) fiber architectures, are fundamental to cancer progression and outcome. However, the influence of ECM alignment on immune cell localization and function within tumors, particularly PDA, remains largely unexplored. Here, analysis of mouse and human PDA reveal an inextricable link between collagen architecture and the distribution of immunosuppressive macrophages in both early preinvasive disease and invasive carcinomas. In vitro characterization of primary macrophages demonstrates alignment alone is sufficient to induce elongation, polarization, and immunosuppressive activity, including suppression of CD8+ T cell proliferation and motility. Analysis reveals differential focal adhesion kinase (FAK) activity in aligned macrophages, while FAK inhibition (FAKi) disrupts the immunosuppressive phenotype that emerges from encountering ECM alignment. Furthermore, FAKi in vivo significantly reduces the correlation between elongated immunosuppressive macrophages and aligned collagen, further highlighting the opportunity for FAKi to target stromal immunity. Importantly, the correlation between aligned collagen and immunosuppressive macrophages is also observed in human chronic pancreatitis, a known PDA risk factor that has recently been shown to prime stromal ECM alignments for early dissemination, suggesting that precursor disease is also likely to create stromal memory conducive to early immunosuppression. Taken together, these results support a model in which collagen architecture supports early establishment and maintenance of an immunosuppressive microenvironments and defines a role for targeting stromal matrices to "reprogram" patient immunity.

Collagen VI is a heterotrimeric, ubiquitously expressed microfibrillar collagen with a complex intracellular and extracellular assembly process. In addition to a short collagenous region, it is primarily composed of von Willebrand factor A (VWA) domains. Notably, only the C-terminal end of the α3 chain contains other domain types, including a Kunitz-like C5 domain, which has been reported to be necessary for microfibril formation, to function as a matrikine and exhibit biomarker properties. This region of the α3 chain undergoes proteolytic processing, with cleavage sites identified for proprotein convertases, matrix metalloproteinases (MMPs), and bone morphogenetic protein 1 (BMP1). Cleavage by furin-like convertases results in the generation of a mature collagen VI α3 chain lacking its 70 kDa C2-C5 domains. Here, we provide the first characterization of the functional significance of the furin-like cleavage site, demonstrating that while it is constitutively used, it is not essential for collagen VI assembly, microfibril formation, or skeletal muscle function under physiological conditions, likely due to the presence of redundant cleavage sites. We also present an initial characterization of the biological activity of the released fragments on myoblast cultures showing that they do not affect C2C12 myoblast behaviour or differentiation. These findings deepen our understanding of α3 chain processing and highlight its potential significance for collagen VI assembly and function, including the generation of peptides with potential biomarker and biological activity properties.

The matricellular protein CCN1 is pro-apoptotic, and can be considered as a possible inducer of the senescence-associated secretory phenotype (SASP). Although the SASP facilitates healthy tissue repair when apoptotic cells are properly cleared, pathological fibrosis results when this clearance is defective. Similarly, CCN1 can have both anti-fibrotic and pro-fibrotic properties, depending on the context. Recent data indicate that although CCN1 has anti-fibrotic roles in normal tissue repair and the liver, it also has pro-fibrotic roles in lung, kidney, cardiac, skin and liver fibrosis/scarring. That CCN1 has context-dependent roles in fibrosis deserves consideration when developing anti-fibrotic drugs.

Pathogenic variants in COL4A1 and COL4A2, encoding type IV collagen α1 and α2 chains-core components of all basement membranes-cause a multisystem disorder with variable expressivity. Affected individuals commonly present with cerebral small vessel disease (cSVD), unmanageable intracerebral haemorrhage (ICH), drug-resistant epilepsy, microphthalmia, and congenital cataract. Severe phenotypes are often linked to glycine substitutions that disrupt α1/α2 heterotrimer assembly, though insertions, deletions, and haploinsufficiency seem to also be pathogenic. Limited insight into collagen IV α1 and α2 biology and how specific variants affect their functions-coupled with a lack of rapid in vivo assays for functional variants classification-hampers patient stratification and therapy development. Here, we established and characterized two complementary col4a1 knockdown (KD) models in zebrafish. Taking advantages of their transparency and rapid development we set-up in vivo assays for neurovascular and ocular phenotyping. Both models reproduced key features of human disease, including ventriculomegaly, vascular fragility with spontaneous and trauma-induced ICH, microphthalmia, and cataracts. Notably, expression of human wild-type COL4A1 partially rescued most of the observed defects, while pathogenic glycine-substitution variants failed to do so. These findings validate col4a1 KD in zebrafish as a robust in vivo model of some aspects of COL4A1/A2 syndrome, highlighting a conserved role of collagen IV α1 in neurovascular and ocular development. Our results also support haploinsufficiency as a contributing pathogenic mechanism, alongside dominant-negative effects. This work lays the foundation for the use of zebrafish to support rapid COL4A1 and COL4A2 variants pathogenicity assessment and mechanistic studies, with the potential to accelerate development of targeted therapies.

Laminins are basement membrane components that regulate a plethora of biological processes. Despite decades of research, the exact roles of laminins in different tissues and in organogenesis remain to be elucidated. Here, we investigated the function of laminin γ1 chain in heart, lung and other tissues by generating a mouse that lacks laminin γ1 in cells expressing SM22α (Tagln) (LMγ1 flox/SM22α Cre mouse, referred to as LMγ1KO). Laminin γ1 deletion led to basement membrane disruption around cardiomyocytes, smooth muscle cells, alveolar cells and skeletal muscle. This, in turn, led to perinatal death of conditional LMγ1KO mice. Synchrotron-based imaging revealed developmental heart abnormalities: ventricular and atrioventricular septal defects. Lung tissue from embryos and newborns showed impaired alveolization and this defect was not reversed ex vivo. We also created adult inducible laminin γ1 knockout mice (iLMγ1KO) with targeted knockdown in all tissues, and they exhibited decreased contractility of smooth muscle in colonic and arterial tissue. Finally, both LMγ1KO neonates and iLMγ1KO adults displayed severe dystrophic features in skeletal muscle. In summary, our study reveals novel roles for laminin γ1 chain and basement membranes in heart, lung, skeletal and smooth muscle. Compromising basement membranes around various cell types expressing SM22α during embryonic development did not impair early organogenesis of lung, heart and skeletal muscle, but rather disturbed late developmental events in these tissues. Our results could help to understand clinical implications for patients with laminin α2 chain mutations (muscular dystrophy) and laminin α4 mutations (cardiomyopathy), but also for patients with congenital heart disease and lung diseases.

Basement membranes are key mediators of many biological processes such as epithelial morphogenesis, kidney filtration, and muscle function among others. Basement membranes provide structural support to tissues so understanding their mechanical properties is important for determining how they contribute to tissue form and function. Further, basement membranes are altered in many diseases including cancer, diabetes, and fibrosis, and these changes may contribute to disease pathogenesis and progression. Understanding how basement membrane mechanics integrate with tissue function is the work of both biologists and engineers/material scientists, yet these disciplines have very different foundations. This review discusses basement membrane macromolecular structure with a view to illuminate how this structure confers basement membranes with unique mechanical properties adapted to resisting physiological stresses. The pathological implications of altered basement membrane mechanics are discussed in the context of different diseases. Additionally, we survey methods used to measure basement membrane mechanical properties, including atomic force microscopy, tensile stiffness assays, and non-quantitative assays such as cell bursting, assessing their strengths and limitations and their accessibility for different types of in vivo studies. We focus on explaining and illuminating the complexities of basement membrane material properties for biologists, and explaining the biological aspects for engineers, with the goal of making interdisciplinary science more accessible to experimentalists and readers.

Hyaluronan (HA) metabolism in prostate cancer associates with androgen resistance and metastasis. We showed that binding of low molecular weight HA (≤250kDa) to castration-resistant prostate cancer cells was heterogeneous with most cells binding low amounts of HA (HA) while a minor subset bound higher amounts of this polysaccharide (HA). HA subsets, which were separated by FACS, were stably more metastatic in vivo than HA comparators. Multiplexed flow cytometry analyses indicated that both subsets displayed similar expression of the HA receptor CD44 while an elevated RHAMM cell surface display was unique to HA subsets. Genomic deletion of RHAMM using CRISPR-Cas9 editing reduced the detection of HA subsets by 6mer but not 250kDa HA fluorescent probes, and phenocopied the lower aggressive properties of HA tumor cells. Few differences in the mutation landscape of RHAMM vs. RHAMM tumor cells were detected but pathway analyses of differentially expressed genes predicted RHAMM-loss altered extracellular matrix signaling. Transcriptomic analyses revealed that HA subsets and RHAMM PC3MLN4 cells shared high expression of follistatin (FST), an activin member of the TGF-β family that is clinically linked to metastases in PCA patients. A causal role for FST in RHAMM tumor cell aggression was assessed using motility as a surrogate marker of invasive capability. FST antibodies blocked RHAMM PC3MLN4 cell migration while conversely, recombinant FST protein rescued the migration deficit of RHAMM comparators. These results define a novel form of prostate cancer cell heterogeneity, identify a method for detecting and isolating highly metastatic subsets and highlight a novel RHAMM-regulated pathway that may be targeted to improve patient management by limiting metastasis.

Cerebral small vessel disease (cSVD) is a major cause of vascular dementia and stroke. Our understanding of cSVD pathophysiology is incomplete and our ability to treat patients is limited. Pathogenic variants in type IV collagen alpha 1 (COL4A1) cause a monogenic form of cSVD with variable age of onset, via disturbance of cerebrovascular basement membranes. Zebrafish larvae are a powerful model organism for studying cerebrovascular disease due to their optical clarity and applicability for live imaging. In this study, we characterised a zebrafish crispant model for loss-of-function COL4A1-associated cSVD that successfully recapitulates key disease features, including spontaneous intracerebral haemorrhage and cerebrovascular abnormalities. We also identified evidence for abnormal cerebrovascular basement membranes and elevated matrix metalloproteinase 9 (mmp9) transcription associated with loss of col4a1. Pharmacological inhibition of mmp9 was sufficient to ameliorate some cerebrovascular phenotypes. Finally, we describe the generation of a mutant line carrying a germline-transmissible 20 bp deletion in zebrafish col4a1 (col4a1) which is associated with cerebrovascular abnormalities, swimming defects and increased susceptibility to pharmacologically induced brain haemorrhages during larval stages. In adulthood, mutant col4a1 animals developed spontaneous brain haemorrhages that were observable in free-swimming fish. Overall, this study validates the use of zebrafish disease modelling for preclinical COL4A1-associated cSVD research and highlights its potential for further understanding disease pathophysiology and future drug discovery projects.