scholarly journals Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine

2021 ◽  
Vol 22 (24) ◽  
pp. 13584
Author(s):  
Marcin Kolasa ◽  
Grzegorz Galita ◽  
Ireneusz Majsterek ◽  
Ewa Kucharska ◽  
Katarzyna Czerczak ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Porous Materials ◽  
Cell Lines ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Spatial Structures ◽  
Type Iv ◽  
Self Assembling ◽  
Biological Studies ◽  
The Stability

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO− as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1–33 and 2.1–2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1β1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.

scholarly journals Expression of collagen type IV in human kidney during prenatal development

2020 ◽  
pp. 111-111
Author(s):  
Vladimir Petrovic ◽  
Ivan Nikolic ◽  
Marko Jovic ◽  
Vladimir Zivkovic ◽  
Miodrag Jocic ◽  
...  
Keyword(s):  
Type Iv Collagen ◽  
Collagen Type ◽  
Kidney Tissue ◽  
Volume Density ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Collagen Type Iv ◽  
Type Iv ◽  
Metanephric Kidney ◽  
Collecting System

Background / Aim. Type IV collagen belongs to the group of non-fibrillar collagens and is an important component of the basement membranes where it accounts for approximately 50% of its structural elements. The aim of the paper was to describe the expression and distribution of collagen type IV in embryonic and fetal metanephric kidney, and to determine the volume density of collagen type IV in kidney tissue in each trimester of development. Methods. The material consisted of 19 human embryos/fetuses, in the gestational age from 8th to 37th week. Kidney tissue specimens were routinely processed to paraffin molds and stained with hematoxylin and eosin and immunohistochemically using polyclonal anti-collagen IV antibody. Stained slides were examined using light microscope and images of the selected areas, under different lens magnification were captured with digital camera. Volume density of collagen type IV was determined by using ImageJ 1.48v and a plugin of the software which inserted a grid system with 336 points. For the data comparison One-Way Analysis of Variance was used. Results. Strong collagen IV immunopositivity was seen in all specimens, with a distribution in the basement membranes of urinary bud, parietal leaf of Bowman?s capsule, glomerular basement membrane, basement membrane of interstitial blood vessels, and basement membranes of nephron tubules and collecting ducts. No statistically significant difference in the volume density of type IV collagen was found between the different trimesters of development. Conclusion. The synthesis and secretion of collagen type IV simultaneously follows the development of nephron structures, collecting system and blood vessels. The volume density of collagen type IV remains constant throughout all the trimesters of metanephric kidney development, indicating that it plays a crucial role in normal development of nephron and collecting system structures, as well as in maintaining the normal kidney function.

The Effect of Composition and Inter- and Intrafibrillar Interactions on the Structure of Collagen IV Networks in the Computer-Simulated Glomerular Basement Membrane

2009 ◽  
Author(s):  
Michael J. Swickrath ◽  
Kevin Dorfman ◽  
Yoav Segal ◽  
Victor H. Barocas
Keyword(s):  
Basement Membrane ◽  
Blood Plasma ◽  
Glomerular Basement Membrane ◽  
Triple Helix ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Type Iv

The glomerular basement membrane of the kidney, responsible for performing ultrafiltration blood plasma, is largely comprised of type-IV collagen and laminin. Type-IV collagen self-assembles into a heterotrimer composed of three distinct domains (fig. 1A): (1) the globular non-collagenous NCl domain of ∼10 nm in diameter, (2) the non-collagenous 7S domain ∼30 nm in length and ∼3nm in diameter, and (3) the collagenous triple helix of ∼370 nm in length and ∼3 nm in diameter composed of a repeating Gly-X-Y subunit [1]. The heterotrimers associate with remarkable specificity from six genetically distinct α-chains, α1(IV) to α6(IV) forming α1α1α2, α3α4α5, and α5α5α6 heterotrimers [2]. In the healthy glomerulus, α1α1α2 ([α1]2α2) is the predominate collagen while significant α3α4α5 is present; α5α5α6 exists only in negligible quantities [2].

scholarly journals Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody.

1984 ◽  
Vol 99 (4) ◽  
pp. 1405-1409 ◽  
Author(s):  
T F Linsenmayer ◽  
E Gibney ◽  
J M Fitch ◽  
J Gross ◽  
R Mayne
Keyword(s):  
Thermal Stability ◽  
Type Iv Collagen ◽  
Helical Structure ◽  
Basement Membranes ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tissue Sections ◽  
Type Iv ◽  
The Stability ◽  
Thermal Stability Of

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV-specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.

scholarly journals Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

1988 ◽  
Vol 36 (3) ◽  
pp. 271-283 ◽  
Author(s):  
D S Grant ◽  
C P Leblond
Keyword(s):  
Heparan Sulfate ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Enamel Organ ◽  
Sulfate Proteoglycan ◽  
Lamina Densa ◽  
Type Iv ◽  
Reichert's Membrane

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

INVESTIGATION ON THE DIFFERENCE EXPRESSION OF TYPE IV COLLAGEN 1(IV)- 6(IV) CHAIN MRNA IN NORMAL FIBROBLAST AND IN SKIN CELL MALIGNANT MELANOMA

2015 ◽  
Vol 77 (25) ◽  
Author(s):  
Mardhiah Mohammad ◽  
Afzan Mat Yusof ◽  
Syahirah Kaja Mohideen ◽  
Sharifah Norbaizura Syed Bahrom ◽  
Ridhwan Roshdi ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Mrna Expression ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Internal Control ◽  
Collagen Iv ◽  
Normal Fibroblast ◽  
Skin Cell ◽  
Type Iv ◽  
Melanoma A375 Cell

Collagen IV is the major basement membrane protein that influences adhesion, proliferation, and migration of cells. The collagen composed of a network chains a1 to a6. The characterization of this collagen IV will correlates the relationship of collagen gene expression and cancer. This is important in order to provide more detailed understanding of the expression of collagen in tumor cells. . The aim of this study is to determine the a1 to a6 (IV) mRNA expression in the cell lines obtained from skin and melanoma cell. To investigate the mRNA expression, the RNA was extracted from the fibroblast and melanoma (A375) cell lines. The RNA was subjected to reverse transcription and then synthesized. The mRNA expression levels were measured using real time PCR with related to internal control, GAPDH. The study identified that a1, a2, a4, a5 and a6 of the a1-a6 (IV) were expressed in skin fibroblast. This corresponds to the a1a1a2 and a5a5a6 networks. However, in melanoma cell lines the collagen IV a2, a4, a5 and a6 mRNA was observed in low level compared to a1 and this suggested that the tumor has affected the expression of collagen and basement membrane of the cell.

Contribution of the Minor Chain Type IV Collagen Network to the Mechanics of the Ocular Lens Capsule

2012 ◽  
Author(s):  
Lazarina Gyoneva ◽  
Yoav Segal ◽  
Kevin D. Dorfman ◽  
Victor H. Barocas
Keyword(s):  
Alport Syndrome ◽  
Type Iv Collagen ◽  
Dry Weight ◽  
Hereditary Disease ◽  
Collagen Iv ◽  
Lens Capsule ◽  
Basement Membranes ◽  
Ocular Lens ◽  
Ocular Manifestations ◽  
Type Iv

The ocular lens capsule (LC) is a specialized basement membrane which completely surrounds the lens. The LC serves as an attachment point for lens epithelial and fiber cells, controls lens solute and water transport, and makes accommodation possible [1]. It is primarily composed of type IV collagen (65% of dry weight), laminin, nidogen, and proteoglycans, of which type IV collagen is the main-tension resisting element [1,2]. Collagen IV monomers organize into polygonal planar networks resembling chicken wire (Fig.1) [3]. There are six different collagen IV monomers, labeled α1(IV) to α6(IV) each produced by a separate gene – COL4A1 to COL4A6. Monomers form triple helical protomers in a highly selective manner. In nature, only three monomer combinations have been discovered: the [α1(IV)]2α2(IV) protomer, referred to as the major chain, is found in all basement membranes; the α3(IV)α4(IV)α5(IV) protomer (minor chain) is found only in few basement membranes including the LC; the [α5(IV)]2α6(IV) protomer is very rare and will not be discussed further. Protomers of the same type assemble with one another to form separate networks which are known to have some differences [4]. For example, the minor chain network is more cross-linked than the major chain network. In a hereditary disease called Alport syndrome, the minor chain network is completely missing in males due to a mutation in the COL4A5 gene (located on the X chromosome) which prevents production of the α5(IV) monomer. Male Alport syndrome patients have significant ocular manifestations such as anterior lenticonus (protrusion of the lens), cataract, and even lens rupture [5] and they exhibit significant thinning of the LC. Because 1) the minor network is more cross-linked than the major network, 2) its absence affects lens shape, and 3) the LC displays pathological disruptions when it is missing, we theorize that its presence confers additional mechanical strength to the LC. Therefore, the objective of this study is to assess the contribution of the minor chain network to the mechanics of the LC.

Role of Lateral Interactions in Type IV Collagen Network Mechanics

2013 ◽  
Author(s):  
Lazarina Gyoneva ◽  
Mohammad F. Hadi ◽  
Yoav Segal ◽  
Kevin D. Dorfman ◽  
Victor H. Barocas
Keyword(s):  
Mechanical Properties ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Type I Collagen ◽  
Hereditary Disease ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Type I ◽  
Lateral Interactions ◽  
Type Iv

The basement membrane is a specialized part of the extra-cellular matrix. It is usually characterized as a scaffold for epithelial cells but in some tissues it serves other, mechanical, roles [1]. The mechanical properties of the basement membrane are mainly determined by one of its main constituents — type IV collagen. Unlike the well-known fibrous type I collagen, collagen IV assembles into planar networks (Fig. 1) [2]. The α 1(IV) and α 2(IV) collagen IV chains assemble into the so-called major chain network, present in all basement membranes. The α 3(IV), α 4(IV), α 5(IV) collagen IV chains form the minor chain network which is found only in the adult basement membranes of the kidney glomerular capillaries (GBM), ocular lens (LBM), cochlea, and the testes [3]. The minor chains have a higher number of cysteine residues, allowing them to form a higher number of lateral interactions. In the minor chain network, the greater potential to interact laterally manifests in the formation of super-coils, which are rarely observed in the major chain network [4]. Increasing the number of cross-links in a polymeric material is known to increase material stiffness; therefore, it is believed that the minor chain network confers basement membranes with additional strength and stability [5]. In the hereditary disease Alport syndrome, a mutation causes the absence of the minor chain network. The GBM and LBM of Alport patients appear weakened and unable to meet their mechanical demands, further supporting this theory [6]. The objective of this study was to evaluate the importance of cross-linking in the minor chains for the mechanical properties of type IV collagen networks, specifically in the GBM and LBM where the absence of the minor chains has an observed mechanical effect.

Estradiol reverses TGF-β1-stimulated type IV collagen gene transcription in murine mesangial cells

1998 ◽  
Vol 274 (6) ◽  
pp. F1113-F1118 ◽  
Author(s):  
Sharon Silbiger ◽  
Jun Lei ◽  
Fuad N. Ziyadeh ◽  
Joel Neugarten
Keyword(s):  
Reporter Gene ◽  
Gene Transcription ◽  
Collagen Synthesis ◽  
Type Iv Collagen ◽  
Mesangial Cells ◽  
Collagen Iv ◽  
Luciferase Reporter ◽  
Type I ◽  
Type Iv ◽  
Tgf Β1

We have previously shown that estradiol suppresses types I and IV collagen synthesis by mesangial cells grown in the presence of serum. In the present study, we examined the interaction between estradiol and transforming growth factor-β (TGF-β) on collagen IV synthesis. In a luciferase reporter gene construct containing the type IV collagen promoter and α1-chain regulatory sequences, we found that TGF-β1 (2 ng/ml) stimulated α1-collagen IV gene transcription in serum-free media (140.5 ± 6.2 relative luciferase units, expressed as a percent of control untreated cells, P < 0.001). Estradiol reversed the stimulatory effects of TGF-β1 on reporter gene transcription in a dose-dependent manner [for 2.5 × 10−9 M, 114.2 ± 0.2, P < 0.002 vs. TGF-β1; for 10−7 M, 89.5 ± 4.0, P < 0.001 vs. TGF-β1 and P = not significant (NS) vs. control]. Using immunoprecipitation techniques, we found that estradiol (10−7 M) reversed TGF-β1-stimulated type IV collagen synthesis (175.3 ± 14.7 vs. 111.6 ± 7.1, expressed as a percent of control untreated cells, P < 0.001) but did not affect TGF-β1-stimulated type I collagen synthesis (166.9 ± 18.8 vs. 162.2 ± 16.2, P = NS). These results were confirmed with Western blotting. Nuclear extracts from mesangial cells treated with TGF-β1 showed increased binding to a Sp1 consensus binding sequence oligonucleotide and to an Sp1 binding site in the collagen IV promoter. Estradiol reversed this enhanced binding. These data suggest that estradiol antagonizes TGF-β1-stimulated type IV collagen synthesis at a transcriptional level and that this effect may be mediated by interactions with the transcription factor Sp1.

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