In situ Evidence of Collagen V and Interleukin-6/Interleukin-17 Activation in Vascular Remodeling of Experimental Pulmonary Hypertension.

Several studies have reported the pathophysiologic and molecular mechanisms responsible for pulmonary arterial hypertension (PAH). However, the in situ evidence of collagen V (Col V) and interleukin-17 (IL-17)/interleukin-6 (IL-6) activation in PAH has not been fully elucidated. We analyzed the effects of collagen I (Col I), Col V, IL-6, and IL-17 on vascular remodeling and hemodynamics and its possible mechanisms of action in monocrotaline (MCT)-induced PAH. Twenty male Wistar rats were randomly divided into two groups. In the PAH group, animals received MCT 60 mg/kg intraperitoneally, whereas the control group (CTRL) received saline. On day 21, the pulmonary blood pressure (PAP) and right ventricular systolic pressure (RVSP) were determined. Lung histology (smooth muscle cell proliferation [α-smooth muscle actin; α-SMA] and periadventitial fibrosis), immunofluorescence (Col I, Col V, and α-SMA), immunohistochemistry (IL-6, IL-17, and transforming growth factor-beta [TGF-ß]), and transmission electron microscopy to detect fibronexus were evaluated. The RVSP (40 ± 2 vs. 24 ± 1 mm Hg, respectively; p < 0.0001), right ventricle hypertrophy index (65 ± 9 and 25 ± 5%, respectively; p < 0.0001), vascular periadventitial Col I and Col V, smooth muscle cell α-SMA+, fibronexus, IL-6, IL-17, and TGF-ß were higher in the MCT group than in the CTRL group. In conclusion, our findings indicate in situ evidence of Col V and IL-6/IL-17 activation in vascular remodeling and suggest that increase of Col V may yield potential therapeutic targets for treating patients with PAH.

Physicochemical characterization and self-assembly of human amniotic membrane and umbilical cord collagen: A comparative study.

The diverse application of collagen has created a need to discover renewable and economical sources with prevailing/improved physico-chemical properties. To address this scenario, the present study has extracted collagen from Human Amniotic Membrane (AM) and Umbilical cord, which are treated as medical waste and compared its physico-chemical properties. Collagen was extracted by pepsin solubilization using various salt concentrations (1 M, 2 M and 4 M). Umbilical Cord Collagen (UC) yield was 10% higher than Amniotic Membrane Collagen (AC). UC reported 58% higher sulphated glycosaminoglycan content than AC. Electrophoretic pattern of AC and UC in both disulphide bond reducing and non-reducing conditions showed bands corresponding to collagen type I, III, IV, V and XV. Collagen morphology was examined using SEM and the amino acid content was quantified by HPLC and LC-MS/MS. Triple helicity was confirmed by CD and FTIR spectra. Thermal transition temperature of AC and UC was found equivalent to animal collagen. Self-assembly, fibril morphology and spatial alignment was studied using AFM and DLS. Biocompatibility was analyzed using 3T3 fibroblast cells. In conclusion, UC with higher yield, presented with better physico-chemical, structural and biological properties than AC could serve as an efficient alternative to the existing animal collagen for diverse applications.

Extracellular matrix-derived peptides in tissue remodeling and fibrosis.

Alterations in the composition of the extracellular matrix (ECM) critically regulate the cellular responses in tissue repair, remodeling, and fibrosis. After injury, proteolytic degradation of ECM generates bioactive ECM fragments, named matricryptins, exposing cryptic sites with actions distinct from the parent molecule. Matricryptins contribute to the regulation of inflammatory, reparative, and fibrogenic cascades through effects on several different cell types both in acute and chronic settings. Fibroblasts play a major role in matricryptin generation not only as the main cellular source of ECM proteins, but also as producers of matrix-degrading proteases. Moreover, several matricryptins exert fibrogenic or reparative actions by modulating fibroblast phenotype and function. This review manuscript focuses on the mechanisms of matricyptin generation in injured and remodeling tissues with an emphasis on fibroblast-matricryptin interactions.

Age-related changes to the satellite cell niche are associated with reduced activation following exercise.

Skeletal muscle satellite cell (SC) function and responsiveness is regulated, in part, through interactions within the niche, in which they reside. Evidence suggests that structural changes occur in the SC niche as a function of aging. In the present study, we investigated the impact of aging on SC niche properties. Muscle biopsies were obtained from the vastus lateralis of healthy young (YM; 21 ± 1 yr; n = 10) and older men (OM; 68 ± 1 yr; n = 16) at rest. A separate group of OM performed a single bout of resistance exercise and additional muscle biopsies were taken 24 and 48 hours post-exercise; this was performed before and following 12 wks of combined exercise training (OM-Ex; 73 ± 1; n = 24). Muscle SC niche measurements were assessed using high resolution immunofluorescent confocal microscopy. Type II SC niche laminin thickness was greater in OM (1.86 ± 0.06 µm) as compared to YM (1.55 ± 0.09 µm, P < .05). The percentage of type II-associated SC that were completely surrounded by laminin was greater in OM (13.6%±4.2%) as compared to YM (3.5%±1.5%; P < .05). In non-surrounded SC, the proportion of active MyoD+ /Pax7+ SC were higher compared to surrounded SC (P < .05) following a single bout of exercise. This "incarceration" of the SC niche by laminin appears with aging and may inhibit SC activation in response to exercise.

Detection of collagens by multispectral optoacoustic tomography as an imaging biomarker for Duchenne muscular dystrophy.

Biomarkers for monitoring of disease progression and response to therapy are lacking for muscle diseases such as Duchenne muscular dystrophy. Noninvasive in vivo molecular imaging with multispectral optoacoustic tomography (MSOT) uses pulsed laser light to induce acoustic pressure waves, enabling the visualization of endogenous chromophores. Here we describe an application of MSOT, in which illumination in the near- and extended near-infrared ranges from 680-1,100 nm enables the visualization and quantification of collagen content. We first demonstrated the feasibility of this approach to noninvasive quantification of tissue fibrosis in longitudinal studies in a large-animal Duchenne muscular dystrophy model in pigs, and then applied this approach to pediatric patients. MSOT-derived collagen content measurements in skeletal muscle were highly correlated to the functional status of the patients and provided additional information on molecular features as compared to magnetic resonance imaging. This study highlights the potential of MSOT imaging as a noninvasive, age-independent biomarker for the implementation and monitoring of newly developed therapies in muscular diseases.

Interaction of von Willebrand factor domains with collagen investigated by single molecule force spectroscopy.

von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in primary hemostasis. Sites for collagen binding, an initial event of hemostasis, are located in the VWF-domains A1 and A3. In this study, we investigated single molecule interactions between collagen surfaces and wild type VWF A1A2A3 domain constructs, as well as clinically relevant VWF A3 domain point mutations, such as p.Ser1731Thr, p.Gln1734His, and p.His1786Arg. For this, we utilized atomic force microscopy based single molecular force spectroscopy. The p.Ser1731Thr mutant had no impact on the VWF-collagen type III and VI interactions, while the p.Gln1734His and p.His1786Arg mutants showed a slight increase in bond stability to collagen type III. This effect probably arises from additional hydrogen bonds that come along with the introduction of these mutations. Using the same mutants, but collagen type VI as a binding partner, resulted in a significant increase in bond stability. VWF domain A1 was reported to be essential for the interaction with collagen type VI and thus our findings strengthen the hypothesis that the VWF A1 domain can compensate for mutations in the VWF A3 domain. Additionally, our data suggest that the mutations could even stabilize the interaction between VWF and collagen without shear. VWF-collagen interactions seem to be an important system in which defective interactions between one VWF domain and one type of collagen can be compensated by alternative binding events.

The minor collagens in articular cartilage.

Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.

Collagen cross-linking: insights on the evolution of metazoan extracellular matrix.

Collagens constitute a large family of extracellular matrix (ECM) proteins that play a fundamental role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent cross-links between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Fibrillar collagens are present in a wide variety of animals, therefore often being associated with metazoan evolution, where the emergence of an ancestral collagen chain has been proposed to lead to the formation of different clades. While LOX-generated collagen cross-linking metabolites have been detected in different metazoan families, there is limited information about when and how collagen acquired this particular modification. By analyzing telopeptide and helical sequences, we identified highly conserved, potential cross-linking sites throughout the metazoan tree of life. Based on this analysis, we propose that they have importantly contributed to the formation and further expansion of fibrillar collagens.

Highly biocompatible collagen-Delonix regia seed polysaccharide hybrid scaffolds for antimicrobial wound dressing.

Biomaterials based entirely on biological resources are ideal for tissue engineering applications. Here we report the preparation of hybrid collagen scaffolds comprising gulmohar seed polysaccharide (GSP) and cinnamon bark extract as cross-linking agent. (1)H NMR spectrum of GSP confirms the presence of galactose and mannose in the ratio of 1:1.54, which was further corroborated using FT-IR. The hybrid scaffolds show better enzyme and thermal stability in contrast to pure collagen scaffold probably due to weak interactions from GSP and covalent interaction through cinnamaldehyde. Gas permeability and scanning electron microscopic analysis show that the porosity of the hybrid scaffolds is slightly reduced with the increase in the concentration of GSP. The infrared and circular dichroic spectral studies show that the secondary structure of the collagen did not change after the interaction with GSP and cinnamaldehyde. The hybrid scaffolds stabilized with cinnamaldehyde show good antimicrobial activity against the common multi-drug resistant wound pathogens. These results suggest that the prepared hybrid scaffolds have great potential for antimicrobial wound dressing applications.

Diurnal variation of plasma bone markers in Japanese black calves.

To evaluate diurnal variation of plasma bone markers, blood samples were collected from five calves at 2-hr intervals throughout a 24-hr period. Tartrate-resistant acid phosphatase isoform 5b (TRAP5b), carboxy-terminal collagen crosslinks of type-I collagen (CTX), hydroxyproline, bone specific alkaline phosphatase (BALP) and osteocalcin were measured. Cosinor analysis showed a significant rhythm in all bone markers. The acrophase of each bone marker appeared from the early to late morning. The percentage ratio of the amplitude to mesor and the within-subject variability for CTx and osteocalcin were significantly larger than those for TRAP5b and BALP. This marked diurnal variation in five bone markers suggested that the time of blood sampling should be fixed when studying bone marker concentrations in bovine plasma.

Structure, physiology, and biochemistry of collagens.

Tendons and ligaments are connective tissues that guide motion, share loads, and transmit forces in a manner that is unique to each as well as the anatomical site and biomechanical stresses to which they are subjected. Collagens are the major molecular components of both tendons and ligaments. The hierarchical structure of tendon and its functional properties are determined by the collagens present, as well as their supramolecular organization. There are 28 different types of collagen that assemble into a variety of supramolecular structures. The assembly of specific supramolecular structures is dependent on the interaction with other matrix molecules as well as the cellular elements. Multiple suprastructural assemblies are integrated to form the functional tendon/ligament. This chapter begins with a discussion of collagen molecules. This is followed by a definition of the supramolecular structures assembled by different collagen types. The general principles involved in the assembly of collagen-containing suprastructures are presented focusing on the regulation of tendon collagen fibrillogenesis. Finally, site-specific differences are discussed. While generalizations can be made, differences exist between different tendons as well as between tendons and ligaments. Compositional differences will impact structure that in turn will determine functional differences. Elucidation of the unique physiology and pathophysiology of different tendons and ligaments will require an appreciation of the role compositional differences have on collagen suprastructural assembly, tissue organization, and function.

Analytical methods for measuring collagen XIX in human cell cultures, tissue extracts, and biological fluids.

Type XIX collagen is a minor collagen associated with basement membranes in vascular, neuronal, mesenchymal, and epithelial tissues. We demonstrated that the NC1, C-terminal, domain of collagen XIX inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Other basement membrane collagens or derived fragments were measured in biological fluids such as blood and urine of patients and appeared to be useful for diagnosis, prognosis, or treatment monitoring. The aim of this study was to develop and validate methods to measure collagen XIX and its fragments in human cell cultures, tissue extracts, and human biological fluids. For that purpose, we developed real-time PCR, Western blot, and competitive enzyme-linked immunosorbent assays. We demonstrated that the methods developed in this paper are specific for collagen XIX. We showed that it is expressed in human cell cultures, tissue extracts, and various biological fluids. These methods may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other fluids. Collagen XIX or its fragments could constitute new biomarkers for human diseases as well as for diagnosis and/or prognosis.

The worsening of tibialis anterior muscle atrophy during recovery post-immobilization correlates with enhanced connective tissue area, proteolysis, and apoptosis.

Sustained muscle wasting due to immobilization leads to weakening and severe metabolic consequences. The mechanisms responsible for muscle recovery after immobilization are poorly defined. Muscle atrophy induced by immobilization worsened in the lengthened tibialis anterior (TA) muscle but not in the shortened gastrocnemius muscle. Here, we investigated some mechanisms responsible for this differential response. Adult rats were subjected to unilateral hindlimb casting for 8 days (I8). Casts were removed at I8, and animals were allowed to recover for 10 days (R1 to R10). The worsening of TA atrophy following immobilization occurred immediately after cast removal at R1 and was sustained until R10. This atrophy correlated with a decrease in type IIb myosin heavy chain (MyHC) isoform and an increase in type IIx, IIa, and I isoforms, with muscle connective tissue thickening, and with increased collagen (Col) I mRNA levels. Increased Col XII, Col IV, and Col XVIII mRNA levels during TA immobilization normalized at R6. Sustained enhanced peptidase activities of the proteasome and apoptosome activity contributed to the catabolic response during the studied recovery period. Finally, increased nuclear apoptosis prevailed only in the connective tissue compartment of the TA. Altogether, the worsening of the TA atrophy pending immediate reloading reflects a major remodeling of its fiber type properties and alterations in the structure/composition of the extracellular compartment that may influence its elasticity/stiffness. The data suggest that sustained enhanced ubiquitin-proteasome-dependent proteolysis and apoptosis are important for these adaptations and provide some rationale for explaining the atrophy of reloaded muscles pending immobilization in a lengthened position.

The invasion mode of GH(3) cells is conditioned by collagen subtype, and its efficiency depends on cell-cell adhesion.

The adaptation of GH(3) cells to different microenvironments is a consequence of a partial compromise with the tumor phenotype. A collagen type IV enriched microenvironment favors an invasive phenotype and increases the substrate adhesion capacity, whereas it decreases the phosphorylation of the regulatory myosin light chain and the aggregation capacity. In contrast, the higher internal tension and increased aggregation capacity induced by collagen type I/III are factors that reduce the invasion rate. Our results show, for the first time, the importance of collagen subtypes in determining the migratory strategy: collagen I/III favors mesenchymal-like motility, whereas collagen type IV induces an ameboid-type displacement. The reciprocal modulation of the myosin light chain kinase and the Rho-kinase determines the invasive capacity through changes in tissue cohesion, extracellular matrix affinity, regulatory myosin light chain phosphorylation and spatial distribution. The collagen subtype determines which of the mechano-transduction signaling pathways will regulate the tensional homeostasis and affect the invasion ability as well as the preferred migration strategy of the cells.

The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse.

A chondrocyte progenitor population isolated from the surface zone of articular cartilage presents a promising cell source for cell-based cartilage repair. In this study, equine articular cartilage progenitor cells (ACPCs) and equine bone marrow-derived stromal cells (BMSCs) were compared as potential cell sources for repair. Clonally derived BMSCs and ACPCs demonstrated expression of the cell fate selector gene, Notch-1, and the putative stem cell markers STRO-1, CD90 and CD166. Chondrogenic induction revealed positive labelling for collagen type II and aggrecan. Collagen type X was not detected in ACPC pellets but was observed in all BMSC pellets. In addition, it was observed that BMSCs labelled for Runx2 and matrilin-1 antibodies, whereas ACPC labelling was significantly less or absent. For both cell types, osteogenic induction revealed positive von Kossa staining in addition to positive labelling for osteocalcin. Adipogenic induction revealed a positive result via oil red O staining in both cell types. ACPCs and BMSCs have demonstrated functional equivalence in their multipotent differentiation capacity. Chondrogenic induction of BMSCs resulted in a hypertrophic cartilage (endochondral) phenotype, which can limit cartilage repair as the tissue can undergo mineralisation. ACPCs may therefore be considered superior to BMSCs in producing cartilage capable of functional repair.

Epithelia, an evolutionary novelty of metazoans.

At the point in animal evolution when cells began to adhere to each other they presumably initially functioned as colonies. The formation of an epithelium that enclosed and controlled an internal milieu would have been the first event to distinguish an individual animal from a colony. To better understand when the first epithelium arose and what its characteristics were, we evaluate the morphological, functional, and molecular characters of epithelia in sponges, considered here the extant representatives of the first metazoans. In particular, we show new claudin-like sequences from sponges align most closely with sequences from Drosophila that have a barrier function in septate junctions. We also show that type IV collagen, the main component of the basement membrane (BM), is present in calcareous sponges, and we confirm the presence of type IV-like collagen (spongin short chain collagen) in other sponges. Though in sponges as in other metazoans the epithelium has grades of specialization with varying complexity of junctions and the BM, the main character of a functional epithelium, the ability to seal and control the ionic composition of the internal milieu, is a property of even the simplest sponge epithelium, and therefore the first metazoans likely also had epithelia with these characteristics, which we consider a "true" epithelium.

The collagen family.

Collagens are the most abundant proteins in mammals. The collagen family comprises 28 members that contain at least one triple-helical domain. Collagens are deposited in the extracellular matrix where most of them form supramolecular assemblies. Four collagens are type II membrane proteins that also exist in a soluble form released from the cell surface by shedding. Collagens play structural roles and contribute to mechanical properties, organization, and shape of tissues. They interact with cells via several receptor families and regulate their proliferation, migration, and differentiation. Some collagens have a restricted tissue distribution and hence specific biological functions.