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More than three decades ago, basement membranes (BMs) were described as membrane-like structures capable of isolating a cell from and connecting a cell to its environment. Since this time, it has been revealed that BMs are specialized extracellular matrices (sECMs) with unique components that support important functions including differentiation, proliferation, migration, and chemotaxis of cells during development. The composition of these sECM is as unique as the tissues to which they are localized, opening the possibility that such matrices can fulfill distinct functions. Changes in BM composition play significant roles in facilitating the development of various diseases. Furthermore, tissues have to provide sECM for their stem cells during development and for their adult life. Here, we briefly review the latest research on these unique sECM and their components with a special emphasis on embryonic and adult stem cells and their niches.

The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as one of the earliest-branching extant animal phyla, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here, we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of non-bilaterian animal phyla. We identified basement membrane (BM) and collagen IV in Ctenophora, and show that the structural and genomic features of collagen IV are homologous to those of non-bilaterian animal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Moreover, collagen IV is absent in unicellular sister-groups. Collectively, we conclude that collagen IV and its variant, spongin, are primordial components of the extracellular microenvironment, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis. The emergence of the diversity of multicellular animals involved cells joining together to form tissues and organs. The ‘glue’ that enabled the cells to work together is made of rope-like molecules called collagen, which assemble into scaffolds. These smart scaffolds tether proteins forming basement membranes that connect cells, provide strength to tissues, and transmit information that influences how the cells behave. How did collagen evolve over millions of years to enable the ever-increasing complexity, size and diversity of animals? To investigate, Fidler, Darris, Chetyrkin et al. explored the tissues of the most ancient of currently living animals – the comb jellies and sponges. This revealed that among all the collagens that make up the human body, a type called collagen IV was a key innovation that enabled single celled organisms to evolve into multicellular animals. Collagen IV, as molecular glue, enabled the formation of a fundamental architectural unit of basement membrane and cells that allowed multicellular tissues and organs to evolve. The findings presented by Fidler, Darris, Chetyrkin et al. pose questions about how collagen IV glues cells together, and how information is stored in the rope-like scaffolds to influence cell behavior. Understanding these processes could ultimately lead to the development of new treatments for diseases in which the collagen smart scaffolds play a key role, such as in kidney diseases and cancer.

Background and Objectives: Peripheral nerve adaptation to different pathological conditions is accompanied by the remodelling of the nerve’s extracellular matrix (ECM). Ischemic conditions caused by peripheral vascular disease are known to affect the function of peripheral nerves; however, the morphological changes to their ECM remain insufficiently examined and understood. Bearing in mind that alterations in collagen I, collagen IV, and laminin content may compromise peri- and endoneurial integrity, the aim of our study was to analyse whether peripheral vascular disease (PVD) induces distinct ECM alterations in the human sural nerve compared with the adaptive remodelling observed in ageing. Materials and Methods: The study aimed to determine the amount of type I and IV collagen and laminin in the perineurium and endoneurium of human peripheral nerves from patients with PVD and to compare the results with those of the age-matched controls. Twenty human sural nerves were harvested from cadavers and amputated limbs—10 from each—and were further distributed into two age groups: below and over 75 years of age. The sural nerve tissue samples were stained immunohistochemically for collagen I, collagen IV, and laminin. We measured the percentage content of these ECM components in the perineurium and endoneurium. For morphometric analysis, we used ImageJ software v1.54d. Results: Perineurial collagen type I and laminin were decreased in the older PVD group, relative to both the younger PVD and the older age group. Within the endoneurium, the expression of collagen type IV was higher in older PVD patients, while both collagen type I and laminin were deposited in lower amounts in the same group compared with the younger PVD group. Conclusions: These findings suggest that age-related ECM remodelling in the peripheral nerve may be impaired under ischemic conditions in older adults, with implications for surgical grafting strategies or neural conduit therapies aimed at promoting functional regeneration.

Background Type 2 diabetes mellitus (T2DM) is associated with arterial diseases; particularly, the incidence of atherosclerotic conditions is increased among patients with diabetes. Basement membrane (BM) protein levels are increased in the artery wall of T2DM patients, but the underlying mechanisms and potential consequences remain elusive. This study aimed to identify putative connections between the level of arterial BM components, clinical characteristics, all-cause mortality and major adverse events (MACE) in groups of patients with and without T2DM. Methods Internal thoracic artery samples from 668 patients undergoing coronary artery bypass graft surgery at Odense University Hospital, Denmark, from 2008 to 2018 were included, of which 27% were diagnosed with T2DM. Seventeen vascular proteins were quantified using liquid chromatography tandem mass spectrometry. Results Levels of BM components—collagen IV, laminin, and perlecan—were significantly elevated in T2DM patients compared to non-diabetic controls. In the diabetic group, we found strong correlations between collagen IV and laminin levels and hemoglobin A1c, whereas correlations with plasma lipids, blood pressure, and body mass index (BMI) were weak or absent. Moreover, collagen IV and laminin were associated with both all-cause mortality and major adverse cardiovascular events (MACE) up to 14 years after surgery after adjustment for age, sex, and comorbidities. Conclusions We demonstrate that arterial basement membrane protein accumulation is related to glycemic status in T2DM. Furthermore, the observed associations between collagen IV and laminin levels and clinical outcomes support the notion that generalized alterations in arterial basement membranes may contribute to the development of cardiovascular disease. Graphical abstract

X-linked Alport syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen α5 chain (α5(IV)). Complete absence of α5(IV) in the renal basal membrane is considered a pathological characteristic in male patients; however, positive α5(IV) staining has been found in over 20% of patients. We retrospectively studied 52 genetically diagnosed male X-linked Alport syndrome patients to evaluate differences in clinical characteristics and renal outcomes between 15 α5(IV)-positive and 37 α5(IV)-negative patients. Thirteen patients in the α5(IV)-positive group had non-truncating mutations consisting of nine missense mutations, three in-frame deletions, and one splice-site mutation resulting in small in-frame deletions of transcripts. The remaining two showed somatic mutations with mosaicism. Missense mutations in the α5(IV)-positive group were more likely to be located before exon 25 compared with missense mutations in the α5(IV)-negative group. Furthermore, urinary protein levels were significantly lower and the age at onset of end-stage renal disease was significantly higher in the positive group than in the negative group. These results help to clarify the milder clinical manifestations and molecular characteristics of male X-linked Alport syndrome patients expressing the α5(IV) chain.

Early fibrotic lesions are thought to be the initial findings of fibrogenesis in idiopathic interstitial pneumonias, but little is known about their properties. Type IV collagen comprises six gene products, α1–α6, and although it is known as a major basement membrane component, its abnormal deposition is seen in fibrotic lesions of certain organs. We studied the expression of type I and III collagen and all α chains of type IV collagen in lung specimens from patients with usual interstitial pneumonia (UIP) or organizing pneumonia (OP) via immunohistochemistry. With cultured lung fibroblasts, we analyzed the expression and function of all α chains of type IV collagen via immunohistochemistry, western blotting, real-time quantitative PCR, and a Boyden chamber migration assay after the knockdown of α1 and α2 chains. Although we observed type I and III collagens in early fibrotic lesions of both UIP and OP, we found type IV collagen, especially α1 and α2 chains, in early fibrotic lesions of UIP but not OP. Fibroblasts enhanced the expression of α1 and α2 chains of type IV collagen after transforming growth factor-β1 stimulation. Small interfering RNA against α1 and α2 chains increased fibroblast migration, with upregulated phosphorylation of focal adhesion kinase (FAK), and adding medium containing fibroblast-produced α1 and α2 chains reduced the increased levels of fibroblast migration and phosphorylation of FAK. Fibroblasts in OP were positive for phosphorylated FAK but fibroblasts in UIP were not. These results suggest that fibroblasts in UIP with type IV collagen deposition, especially α1 and α2 chains, have less ability to migrate from early fibrotic lesions than fibroblasts in OP without type IV collagen deposition. Thus, type IV collagen deposition in early fibrotic lesions of UIP may be implicated in refractory pathophysiology including migration of lesion fibroblasts via a FAK pathway.

Many muscular diseases result from abnormal organization of connective tissue and/or collagen network formation. Only a few molecular imaging techniques are able to analyze this collagen network by differentiating collagen types. In this study, FT-IR spectroscopy was used to analyze type I and IV collagens, the most important compounds of which are perimysium and endomysium, respectively. Secondary structure of collagen types was determined by curve-fitting the 1,700–1,480 cm⁻¹ spectral interval. Type I collagen could be differentiated from type IV by its higher amounts of triple helix and α-helix, but lower amounts of β-sheets (P < 0.01). FT-IR imaging was then used to determine structural features of perimysium and endomysium collagen network in bovine Flexor carpi radialis muscle. Secondary structure of proteins contained in perimysium and endomysium was found to be very close to type I and IV collagens, respectively. FT-IR spectroscopy and imaging are thus analytical tools that might be used for investigating biodistribution and assembly of collagen types in connective tissues.

This study aimed to examine whether the diagnostic accuracy of four noninvasive tests (NITs) for detecting advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is maintained or is inferior to with or without the presence of type 2 diabetes. Overall, 874 patients with biopsy‐proven NAFLD were enrolled. After propensity‐score matching by age, sex, and the prevalence of dyslipidemia, 311 patients were enrolled in each group of with or without diabetes. To evaluate the effect of diabetes, we compared the diagnostic accuracy of the fibrosis‐4 (FIB‐4) index, the NAFLD fibrosis score (NFS), the aspartate aminotransferase to platelet ratio index (APRI), and type IV collagen 7S (COL4‐7S) in patients with NAFLD with and without diabetes. The areas under the receiver operating characteristic curve (AUROC) for identifying advanced fibrosis in patients without diabetes were 0.879 for the FIB‐4 index, 0.851 for the NFS, 0.862 for the APRI, and 0.883 for COL4‐7S. The AUROCs in patients with diabetes were 0.790 for the FIB‐4 index, 0.784 for the NFS, 0.771 for the APRI, and 0.872 for COL4‐7S. The AUROC of COL4‐7S was significantly larger than that of the other NITs in patients with NAFLD with diabetes than in those without diabetes. The optimal high and low cutoff points of COL4‐7S were 5.9 ng/mL and 4.8 ng/mL, respectively. At the low cutoff point, the accuracy of COL4‐7S was better than that of the other NITs, especially in patients with diabetes. Conclusion: COL4‐7S measurement might be the best NIT for identifying advanced fibrosis in NAFLD, especially in NAFLD with diabetes.

The ability of tendons to glide smoothly during muscle contraction is impaired after injury by fibrous adhesions that form between the damaged tendon surface and surrounding tissues. To understand how adhesions form we incubated excised tendons in fibrin gels (to mimic the homeostatic environment at the injury site) and assessed cell migration. We noticed cells exiting the tendon from only the cut ends. Furthermore, treatment of the tendon with trypsin resulted in cell extravagation from the shaft of the tendons. Electron microscopy and immunolocalisation studies showed that the tendons are covered by a novel cell layer in which a collagen type IV/laminin basement membrane (BM) overlies a keratinised epithelium. PCR and western blot analyses confirmed the expression of laminin β1 in surface cells, only. To evaluate the cell retentive properties of the BM in vivo we examined the tendons of the Col4a1(+/Svc) mouse that is heterozygous for a G-to-A transition in the Col4a1 gene that produces a G1064D substitution in the α1(IV) chain of collagen IV. The flexor tendons had a discontinuous BM, developed fibrous adhesions with overlying tissues, and were acellular at sites of adhesion formation. In further experiments, tenotomy of wild-type mice resulted in expression of laminin throughout the adhesion. In conclusion, we show the existence of a novel tendon BM-epithelium that is required to prevent adhesion formation. The Col4a1(+/Svc) mouse is an effective animal model for studying adhesion formation because of the presence of a structurally-defective collagen type IV-containing BM.

Small airway fibrosis is the main contributor in airflow obstruction in chronic obstructive pulmonary disease. Epithelial mesenchymal transition (EMT) has been implicated in this process, and in large airways, is associated with angiogenesis, ie, Type-3, which is classically promalignant. In this study we have investigated whether EMT biomarkers are expressed in small airways compared to large airways in subjects with chronic airflow limitation (CAL) and what type of EMT is present on the basis of vascularity. We evaluated epithelial activation, reticular basement membrane fragmentation (core structural EMT marker) and EMT-related mesenchymal biomarkers in small and large airways from resected lung tissue from 18 lung cancer patients with CAL and 9 normal controls. Tissues were immunostained for epidermal growth factor receptor (EGFR; epithelial activation marker), vimentin (mesenchymal marker), and S100A4 (fibroblast epitope). Type-IV collagen was stained to demonstrate vessels. There was increased expression of EMT-related markers in CAL small airways compared to controls: EGFR (P<0.001), vimentin (P<0.001), S100A4 (P<0.001), and fragmentation (P<0.001), but this was less than that in large airways. Notably, there was no hypervascularity in small airway reticular basement membrane as in large airways. Epithelial activation and S100A4 expression were related to airflow obstruction. EMT is active in small airways, but less so than in large airways in CAL, and may be relevant to the key pathologies of chronic obstructive pulmonary disease, small airway fibrosis, and airway cancers.