Presence of type III collagen in guinea-pig dermal scar.
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ABSTRACT:
SUBMITTER: Barnes MJ
PROVIDER: S-EPMC1163841 | biostudies-other | 1976 Jul
REPOSITORIES: biostudies-other
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Project description:We investigated whether an uncharacterized protein from guinea pig could be the enzyme behind Kidd's serendipitous discovery, made over 60 years ago, that guinea pig serum has cell killing ability. It has been long known that an enzyme with l-asparaginase activity is responsible for cell killing, although astonishingly, its identity remains unclear. Bacterial asparaginases with similar cell killing properties have since become a mainstay therapy of certain cancers such as acute lymphoblastic leukemia. By hydrolyzing asparagine to aspartate and ammonia, these drugs deplete the asparagine present in the blood, killing cancer cells that rely on extracellular asparagine uptake for survival. However, bacterial asparaginases can elicit an adverse immune response. We propose that replacement of bacterial enzymes with the guinea pig asparaginase responsible for serum activity, by its virtue of being more closely related to human enzymes, will be less immunogenic. To this goal, we investigated whether an uncharacterized protein from guinea pig with putative asparaginase activity, which we call gpASNase3, could be that enzyme. We examined its self-activation process (gpASNase3 requires autocleavage to become active), kinetically characterized it for asparaginase and β-aspartyl dipeptidase activity, and elucidated its crystal structure in both the uncleaved and cleaved states. This work reveals that gpASNase3 is not the enzyme responsible for the antitumor effects of guinea pig serum. It exhibits a low affinity for asparagine as measured by a high Michaelis constant, KM, in the millimolar range, in contrast to the low KM (micromolar range) required for asparaginase to be effective as an anticancer agent.
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Project description:BACKGROUND AND PURPOSE:Adenosine A2A receptor stimulation promotes the synthesis of collagen type I and type III (Col1 and Col3), mediators of fibrosis and scarring. The A2A receptor modulates collagen balance via cAMP/PKA/p38-MAPK/Akt pathways. Wnt signalling is important in fibrosis and the cAMP and Wnt pathways converge. Because the A2A receptor is Gs-linked and increases cAMP, we determined whether A2A receptors and Wnt signalling interact. EXPERIMENTAL APPROACH:Total ?-catenin, de-phosphorylated ?-catenin (canonical activation, de-phospho ?-catenin) and phosphorylated ?-catenin at Ser552 (non-canonical activation, p-Ser552 ?-catenin) levels were determined in primary human dermal fibroblasts, cytosol and nucleus, by western blot analysis and fluorescence microscopy, before and after stimulation by A2A receptor-selective agonist CGS21680, with/without A2A receptor-selective antagonist (SCH56261) pretreatment. ?-Catenin was knocked down by transfection with scrambled-siRNA or specific-siRNA, and Col1 and Col3 levels determined by western blots. KEY RESULTS:CGS21680 stimulation rapidly (15 min) increased cellular ?-catenin levels. Both de-phospho ?-catenin and p-Ser552 ?-catenin levels were also increased. CGS21680 stimulated the translocation of total de-phospho and p-Ser552 ?-catenin to the nucleus. A2A receptor-stimulation increased Col1 synthesis similarly in ?-catenin knockeddown and scrambled cells. However, ?-catenin knockdown abolished the increase in Col3 synthesis induced in A2A receptor-stimulated fibroblasts. CONCLUSIONS AND IMPLICATIONS:A2A receptor stimulation promotes Col3 synthesis via the activation of canonical and non-canonical ?-catenin, consistent with a role for A2A receptors in dermal fibrosis and scarring.
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Project description:Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.
| S-EPMC7415659 | biostudies-literature
Project description:The mammalian heart possesses a poor ability to regenerate after acute ischemic cardiac injury and lost cardiac muscle is replaced by scar tissue. Multiple clinical studies demonstrate that the size of scar tissue following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors that regulate the size of scar after ischemic cardiac injury. In this report, we demonstrate that collagen V, a fibrillar collagen and a minor constituent of heart scars regulates the size of heart scars after ischemic cardiac injury. Depletion of collagen V in heart scars in two independent animal models led to a significant and paradoxical increase in post infarction scar tissue size with worsening of heart function. A systems genetics approach analyzing genes versus traits across 100 in-bred strains of mice independently demonstrated that collagen V is a critical driver of post injury heart function. We show that collagen V deficiency alters the ultra-structure and mechanical properties of scar tissue that make it more vulnerable to expansion. There is altered reciprocal feedback between matrix and cells that induce expression of specific mechanosensitive integrins which drive fibroblast activation and increased ECM gene expression. Scar size increases. Administration of cilengitide, an inhibitor of specific integrins, completely rescues the phenotype of increased post injury scarring, myofibroblast formation and cardiac dysfunction in collagen V deficient mice. These observations demonstrate that collagen V, a structural constituent of heart scar tissue regulates scar size in an integrin dependent manner.
2020-06-05 | GSE151834 | GEO
Project description:The mammalian heart possesses a poor ability to regenerate after acute ischemic cardiac injury and lost cardiac muscle is replaced by scar tissue. Multiple clinical studies demonstrate that the size of scar tissue following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors that regulate the size of scar after ischemic cardiac injury. In this report, we demonstrate that collagen V, a fibrillar collagen and a minor constituent of heart scars regulates the size of heart scars after ischemic cardiac injury. Depletion of collagen V in heart scars in two independent animal models led to a significant and paradoxical increase in post infarction scar tissue size with worsening of heart function. A systems genetics approach analyzing genes versus traits across 100 in-bred strains of mice independently demonstrated that collagen V is a critical driver of post injury heart function. We show that collagen V deficiency alters the ultra-structure and mechanical properties of scar tissue that make it more vulnerable to expansion. There is altered reciprocal feedback between matrix and cells that induce expression of specific mechanosensitive integrins which drive fibroblast activation and increased ECM gene expression. Scar size increases. Administration of cilengitide, an inhibitor of specific integrins, completely rescues the phenotype of increased post injury scarring, myofibroblast formation and cardiac dysfunction in collagen V deficient mice. These observations demonstrate that collagen V, a structural constituent of heart scar tissue regulates scar size in an integrin dependent manner.
2020-06-04 | GSE151695 | GEO
Project description:BACKGROUND:To minimize the inflammatory reaction and improve healing, a new modified dermal substitute composed of an atelocollagen, chondroitin-6-sulfate, and amniotic membrane (AM) was applied to full-thickness skin defects in a pig. Atelocollagen was extracted from bovine skin, and two modified dermal substitutes were generated according to the cross-linking type. METHODS:The AM-collagen dermal substitutes were characterized and compared with currently used dermal substitutes in a pig skin defect model. There were five experimental groups: dehydrothermal (DHT) cross-linking atelocollagen with the AM on the top (AM-DHT), DHT and chemical cross-linking atelocollagen with the AM on the top (AM-DHT/chemical), Terudermis, Integra, and AlloDerm. After 3×3 cm full-thickness skin defects on the back of a pig were created, each dermal substitutes dermal substitutes was randomly grafted on the defects. Two weeks after grafting, autologous partial-thickness skin was over-grafted on the neodermis. The take rate of the dermal substitutes, skin, and histological sections were all assessed at 1, 2, and 4 weeks postoperatively. RESULTS:More rapid healing and a higher take rate were evident in the AM-DHT and Terudermis groups. Histological examination revealed fewer inflammatory cells and more fibroblast hyperplasia in these two groups. Four weeks after surgery, the amount of newly formed collagen was significantly more appropriate in the AM-DHT group. CONCLUSIONS:These observations provide supporting evidence that a newly developed amniotic-collagen dermal substitute may inhibit inflammatory reactions and promote wound healing.
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Project description:The development of commercial collagen inks for extrusion-based bioprinting has increased the amount of research on pure collagen bioprinting, i.e., collagen inks not mixed with gelatin, alginate, or other more common biomaterial inks. New printing techniques have also improved the resolution achievable with pure collagen bioprinting. However, the resultant collagen constructs still appear too weak to replicate dense collagenous tissues, such as the cornea. This work aims to demonstrate the first reported case of bioprinted recombinant collagen films with suitable optical and mechanical properties for corneal tissue engineering. The printing technology used, aerosol jet® printing (AJP), is a high-resolution printing method normally used to deposit conductive inks for electronic printing. In this work, AJP was employed to deposit recombinant human collagen type III (RHCIII) in overlapping continuous lines of 60 µm to form thin layers. Layers were repeated up to 764 times to result in a construct that was considered a few hundred microns thick when swollen. Samples were subsequently neutralised and crosslinked using EDC:NHS crosslinking. Nanoindentation and absorbance measurements were conducted, and the results show that the AJP-deposited RHCIII samples possess suitable mechanical and optical properties for corneal tissue engineering: an average effective elastic modulus of 506 ± 173 kPa and transparency ≥87% at all visible wavelengths. Circular dichroism showed that there was some loss of helicity of the collagen due to aerosolisation. SDS-PAGE and pepsin digestion were used to show that while some collagen is degraded due to aerosolisation, it remains an inaccessible substrate for pepsin cleavage.
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Project description:Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host-pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans.
| S-EPMC8878158 | biostudies-literature
Project description:Eight monoclonal antibodies have been produced against human pepsin-soluble type III collagen. All antibodies were shown to be highly specific for type III collagen and did not cross-react with a range of other collagen types or connective-tissue proteins. Examination of type III collagen from other species showed that these antibodies had a wide range of species specificities, indicating that several distinct epitopes were being recognized. The location of the epitopes was investigated by using reactivity of the antibodies to CNBr fragments and to sequential fragments formed by tryptic digestion of renaturing type III collagen. These data also indicated that several distinct epitopes were recognized and that they were located over the length of the type III collagen.
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