Collagen Cleavage Research Articles

Degradation of Proteoglycans and Collagen in Equine Meniscal Tissues.

Investigate meniscal extracellular matrix degradation. Equine menisci (n = 34 from 17 horses) were studied. Site-matched sections were cut and scored from three regions (ROIs; n = 102) and stained for histology, proteoglycan (safranin O and fast green), aggrecan, and collagen cleavage (NITEGE, DIPEN, and C1,2C antibodies, respectively). Picrosirius red and second harmonic generation microscopy were performed to investigate collagen ultrastructure. A total of 42 ROIs met the inclusion criteria and were included in the final analysis. The median (range) ROI histological score was 3 (0-9), providing a large spectrum of pathology. The median (range) proteoglycan score was 1 (0-3), representing superficial and central meniscal loss. The median (range) of DIPEN, NITEGE, and C1,2C scores was 1 (0-3), revealing immunostaining of the femoral and tibial surfaces. The proteoglycan scores exhibited significant positive associations with both histologic evaluation (p = 0.03) and DIPEN scores (p = 0.02). Additionally, a robust positive association (p = 0.007) was observed between the two aggrecanolysis indicators, NITEGE and DIPEN scores. A negative association (p = 0.008) was identified between NITEGE and histological scores. The C1,2C scores were not associated with any other scores. Picrosirius red and second harmonic generation microscopy (SHGM) illustrated the loss of the collagen matrix and structure centrally. Proteoglycan and collagen degradation commonly occur superficially in menisci and less frequently centrally. The identification of central meniscal proteoglycan and collagen degradation provides novel insight into central meniscal degeneration. However, further research is needed to elucidate the etiology and sequence of degradative events.

Murine matrix metalloproteinase 13 and human matrix metalloproteinase 1 are involved in remodelling processes after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Centre for Cardiovascular Research (DZHK) Background Cardiovascular diseases such as myocardial infarction (MI) remain a leading cause of death in the world. Matrix metalloproteinases (MMPs) are not only essential for the cleavage of collagen, but are also modifying inflammatory cytokines. Thus, MMPs are playing a substantial role in the context of fibrotic as well as inflammatory processes in tissue remodelling after MI. Purpose Mmp13 is the highest expressed collagenase in the murine left ventricle (LV). After ligation of the left anterior descending artery (LAD) in mice, Mmp13 is highly upregulated in the LV. Therefore, the aim of this study was to characterize the role of MMP13 in MI. In the human LV, the highest expressed collagenase is MMP1. Single nucleotide polymorphisms (SNPs) in the promotor region of MMP1 can lead to alterations in gene expression level. Therefore, we analysed the genotype of 3 SNPs in about 2000 patients admitted to the emergency department with suspected MI to reveal associations with the development of MI and outcome after MI. Methods A MMP13-knockout (KO) mouse model was examined after induction of MI and gene expression analysis, histological staining, in-situ hybridisation and hemodynamic measurements were conducted. Mmp13 expression in different cardiac cell types was investigated at quiescent stage and under ischaemic conditions, to determine the cellular origin of Mmp13 expression. Out of the human cohort, 2 patient groups (non-MI and MI) were restricted. Hazard ratios adjusted to age and male sex were assessed, to evaluate risk for MI and risk for death after MI in dependency of the SNPs. Results After activation with ischaemic secretome of cardiomyocytes, the Mmp13 expression of macrophages (6.6-fold; p=0.0286) and fibroblasts (4.9-fold; p=0.0079) was significantly increased. Under stimulation with ischaemic secretome of fibroblasts, Mmp13 expression of macrophages (4.3-fold; p=0.0286) and leukocytes (2.3-fold; p=0.0260) was significantly elevated. Additionally, in situ hybridisation revealed Mmp13 expression in immune cells in the border zone of the infarct region. While the survival of WT mice was about 50%, only very few KO mice died after MI (p=0.0107). Moreover, KO mice showed benefits in cardiac function compared to WT littermates 28 days post-MI. In the human cohort, risk for death was significantly altered between the examined genotypes in 2 of 3 investigated SNPs, indicating a role of MMP1 in the remodelling process after MI. Conclusion High levels of Mmp13 after MI originate mainly from activated macrophages and from activated fibroblasts in the infarct border zone. MMP13-deficiency is beneficial for mice after MI, leading to an improved cardiac function 28 days post-MI compared to WT controls. Analysis of SNPs of the human functional homologue of Mmp13 – MMP1 – revealed an association of MMP1 with remodelling processes after MI.

Effects of CD8 T-cell transfer on biomechanics of post-myocardial infarction scar formation

CD8+ T-cells are adverse regulators of post-MI remodeling, resulting in attenuated cardiac function and overall survival. Based on previous studies, we hypothesize that CD8+ T-cells impair cardiac function by altering scar composition. MI was induced by ligating the left anterior descending coronary artery on C57BL6/J wildtype (WT; 3-7 months of age, n≥2/sex) and CD8atm1mak (CD8−/−; 3-7 months of age, n≥2/sex/ group) mice. CD8−/− mice were injected with either vehicle or naïve splenic CD8+ T-cells via tail vein, 4-hours post-coronary artery ligation to assess potential effects on the scar. On day 7 post-MI, infarct tissue was collected, undergoing either passive stretch biomechanical analysis or histological and biochemical assays for collagen composition. Effects of granzyme (Gzm) B and K on collagen cleavage were tested using a fluorogenic collagen cleavage assay to examine possible mechanisms of scar alteration. Mice lacking CD8+ T-cells had improved ejection fraction and decreased dilation compared to WT mice at post-MI Day 7. This protective effect was lost in CD8−/− mice that received splenic CD8+ T-cells. Biomechanical analysis demonstrated CD8−/− mice had a 2-fold increase in regional stiffness compared to WT mice. Re-supplementation with splenic CD8+ T-cells decreased scar tissue stiffness mimicking biomechanics of WT mice. Picrosirius red staining surprisingly showed no significant differences in total collagen levels (p=0.51). Immunoblotting for Collagen 1 trended an increase in the 250 kDa band in CD8−/− mice that decreased after supplementation indicating a loss of pro-collagen formation (p=0.051). Ex-vivo cleavage assay demonstrated GzmK cleaved collagen in a concentration (0-50 AU) and temporal-dependent manner (0-24 hrs). Moreover, GzmK demonstrated a 10-fold greater capacity in collagen cleavage compared to GzmB at 25 AU, indicating the role GzmK plays during post-MI remodeling may be distinctly separate from that of GzmB. In conclusion, our data demonstrates that CD8+ T-cells regulate cardiac fibrosis potentially in part through the release of granzymes, leading to alterations in biomechanical capability of the left ventricle. This work was supported by the National Institutes of Health T32GM123055, R25GM113278; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Offce of Research and Development Award IK2BX003922 and BX003922; and South Carolina Translational Research Center UL1TR001450. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparative analysis of the integration of autologous and allogeneic cryopreserved tendons in the femoral canal on a model of laboratory animals

Allogeneic tendon grafts are seriously demand in knee joint plastic surgery. The novel method of tendon cryopreservation, including sterilization with supercritical carbon dioxide, was developed in N.V. Sklifosovsky Research Institute for Emergency Cryopreserved tendons retain their normal fiber structure without significant loss of mechanical properties. At the next stage it was necessary to evaluate cryopreserved tendons′ integration inside bone canal in experimental animals.The aim of study. To evaluate morphologic changes of autologous and allogeneic tendons inside the femur in rats and to determine the effect of tendon transplantation on the physical activity.Material and methods. The study was conducted on white inbreed male rats. Three groups of animals were formed: the control group (animals without tendon transplantation), the 1st experimental group – animals with autologous tendon transplantation, the 2nd experimental group – animals with allogeneic tendon transplantation. In animals of the experimental groups the through channel was formed in the distal metaepiphysis of the femur and a tail tendon graft 0.5 x 0.1 cm was placed there. To assess the physical activity of the animals, we studied maximum distance that the animals could run 3 and 6 weeks after transplantation was determined, using treadmill test. The graft structure was evaluated on histological preparations in transmitted light, stained with hematoxylin-eosin and Van Gieson′s stain. To assess the preservation of collagen fibers we checked the autofluorescence intensity of collagen.Results. According to the treadmill test, the distance run by the animals of both experimental groups did not significantly differ from the values in the control group. Histological analysis after 3 weeks in both experimental groups revealed signs of fibers′ decomposition in the absence of inflammatory infiltration and maintaining close contact with bone trabeculae. The autofluorescence intensity of the collagen fibers in grafts corresponded to normal or was close to normal. After 6 weeks, the animals of both experimental groups revealed areas of graft fusion with their own bone, Sharpe fibers were actively formed. In both groups, numerous small vessels with diameters up to 10 microns were detected in the area of tendon-bone contact. Infiltration of grafts by inflammatory cells was absent or very insignificant, active migration of fibroblasts to the tendon area was also not observed. In both groups, tendon grafts had areas where fiber decompactization was observed. In the area of contact with the bone, the autofluorescence of tendon fibers was sharply increased, which indicates the chemical cleavage of collagen. At 3 and 6 weeks after transplantation the effect of fixation (integration) of the tendon with bone tissue was observed in both experimental groups.Conclusions. Allogeneic tendon grafts did not cause a pronounced inflammatory or immune reaction in experimental animals. 6 weeks after transplantation of autologous and allogeneic tendons, the integration of grafts inside the femoral canal was observed. Cryopreserved allogeneic tendons were able to integrate into the body's own tissues without pronounced structural and functional disorders. According to the treadmill test, the distance covered by the animals of both experimental groups did not differ statistically significantly from the values in the control group (without tendon transplantation) after 3 and 6 weeks

Characterization of Poultry Gelatins Prepared by a Biotechnological Method for Targeted Changes at the Molecular Level.

Chicken collagen is a promising raw material source for the production gelatins and hydrolysates. These can be prepared biotechnologically using proteolytic enzymes. By choosing the appropriate process conditions, such changes can be achieved at the molecular level of collagen, making it possible to prepare gelatins with targeted properties for advanced cosmetic, pharmaceutical, medical, or food applications. The present research aims to investigate model samples of chicken gelatins, focusing on: (i) antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-3-etylbenzotiazolin-6-sulfonic acid (ABTS); (ii) the distribution of molecular weights via gel permeation chromatography with refractometric detection (GPC-RID); (iii) functional groups and the configuration of polypeptide chains related to molecular-level properties using Fourier transform infrared spectroscopy (FTIR); (iv) the microbiological populations on sabouraud dextrose agar (SDA), plate count agar (PCA), tryptic soy agar (TSA), and violet red bile lactose (VRBL) using the matrix-assisted laser desorption ionization (MALDI) method. Antioxidant activity towards ABTS radicals was more than 80%; activity towards DPPH radicals was more than 69%. The molecular weights of all gelatin samples showed typical α-, β-, and γ-chains. FTIR analysis confirmed that chicken gelatins all contain typical vibrational regions for collagen cleavage products, Amides A and B, and Amides I, II, and III, at characteristic wavenumbers. A microbiological analysis of the prepared samples showed no undesirable bacteria that would limit advanced applications of the prepared products. Chicken gelatins represent a promising alternative to products made from standard collagen tissues of terrestrial animals.

11 Extra-label bisphosphonate effects on cartilage turnover in juvenile horses challenged with intra-articular lipopolysaccharide

Perceived chondroprotective benefits of extra-label bisphosphonates, such as clodronate disodium (CD), in juvenile horses led to widespread use despite little scientific understanding of intra-articular impacts. The objective was to determine the effects of CD on articular cartilage hypothesizing that administration of CD would decrease cartilage degradation following an acute inflammatory challenge by lipopolysaccharide (LPS). To test this, 32 yearling Quarter Horses were stratified by age (500 ± 13 d), BW (336 ± 26 kg), sex (n = 16 female; n = 16 male) and initial bone optical density into 4 treatment groups. The 140-d study consisted of 2 phases: phase 1 (d 0–83) emulated sales prep and phase 2 (d 84–140) mimicked early performance training. Horses were housed individually (3.6 m × 7.3 m) and fed to meet exercise requirements. Treatments consisted of control (CON; n = 8), single-dose (1X; n = 8; d 84), 2-doses (2X; n = 8; d 0, 84), and 4-doses (4X; n = 8; d 0, 42, 84, 126). All horses received iso-volumetric intramuscular injections of 1.8mg/kg BW clodronate disodium (OSPHOS®) or saline (placebo) on d 0, 42, 84, and 126. Post treatment administration on d 126, radial carpal joints of each horse were injected with 0.8 mL of either 0.5 ng sterile LPS derived from Escherichia coli O55:B5 or sterile lactated Ringer's solution as a contralateral control. Synovial fluid was collected before LPS injection(h 0) and at 6, 12, 24, and 336 h post induction. Carpal circumference (CC), heart rate (HR), respiration rate (RR), and rectal temperature (RT) were recorded before each collection. Synovial fluid was analyzed for carboxypropeptide of type II collagen (CPII) and collagenase cleavage neopeptide (C2C) by ELISA. Data were analyzed using PROC MIXED of SAS. Treatment had no effect on HR, RR, or RT. As expected, intra-articular LPS increased CC, CPII, C2C, and CPII:C2C (P ≤ 0.01). There was a trend for a treatment effect for CC (P = 0.09) where CC was greater in 2X and 4X compared with 1X with CON being intermediate. There tended to be a treatment by hour interaction in CPII (P = 0.06) where CPII was greater in 4X and 2X compared with 1X and CON at h 24 and 336. Likewise,there was a trend for a treatment effect onCPII:C2C (P = 0.06) with 2X and 4X having greater synthesis:degradation than 1X and CON. In conclusion, LPS administration resulted in an expected increase in carpal circumference. Trends of increasing CPII and CPII:C2C in the 2X and 4X treatment group indicate a net increase in articular cartilage synthesis following 2–4 doses of CD despite larger CC, suggesting a greater inflammatory response.

54 Influence of dietary Saccharomyces cerevisiae fermentation product on cartilage metabolism in young exercising horses challenged with intra-articular lipopolysaccharide

Dietary Saccharomyces cerevisiae fermentation product (SCFP) has the ability to optimize the intra-articular environment of horses, but its efficacy in young, exercising horses is not fully understood. Thirty Quarter Horse yearlings (374 ± 25 kg BW; 562 ± 16 d of age; 15 fillies, 15 geldings) were used in a 60 d study to investigate the effect of dietary SCFP (TruEquine®C, Diamond V Mills Inc.) on cartilage metabolism following an acute inflammatory insult, hypothesizing that SCFP would increase cartilage metabolism. Horses were stratified by BW, age, and sex and randomly assigned to dietary treatments (n = 10/group): 0 mg/kg BW (CON), 46 mg/kg BW SCFP (TRT1), or 92 mg/kg BW SCFP (TRT2) top-dressed to a custom-formulated concentrate void of microbials and offered at 1% BW/d (as-fed) every 12 h. On d 0, 21, 42, and 56 BW was obtained and intake adjusted accordingly. Daily grain and supplement orts were negligible across treatments. Horses were housed individually (3.6 × 7.3 m) and offered ad libitum Coastal bermudagrass hay and water. Horses were exercised on a progressive workload for 30 min/d, 5d/wk using a free-stall exerciser. On d 46, horses underwent an intra-articular lipopolysaccharide (LPS) challenge. Each horse's radial carpal joints were randomly selected to receive either 0.5 ng LPS or sterile lactated Ringer's solution (LRS) as a contralateral control.Synovial fluid obtained pre-injection (h 0) and at 6, 12, 24, and 336 h post-injection was analyzed for c-propeptide of type II collagen (CPII) and collagenase cleavage neopeptide (C2C) by commercial ELISA. Rectal temperature (RT), heart rate (HR), respiration rate (RR), and carpal circumference (CC) were recorded immediately before each arthrocentesis. Data were analyzed using PROC MIXED of SAS v9.4 and mean separation was achieved with orthogonal contrasts. The delta in BW exhibited a quadratic dose response (P ≤ 0.01) where CON horses were heavier than TRT1 and TRT2 on d 56. Diet did not influence HR, RR, or RT (P ≥ 0.25). There was an LPS × time interaction for CPII (P ≤ 0.01), CPII:C2C (P ≤ 0.01), CC (P ≤ 0.01), and a tendency in C2C (P = 0.09) for concentrations to be greater in LPS joints at 6, 12, and 24 h than LRS joints. Regardless of injection, CPII, C2C, and CPII:C2C did not return to baseline through h 336 (P ≤ 0.04). Dietary SCFP did not influence (P ≥ 0.42) biomarkers indicative of cartilage metabolism. Although, intra-articular LPS increased cartilage turnover in young exercising horses, the response was not affected by dietary SCFP.

Tension-suppressed degradation of collagen controls tissue stiffness scaling with fibrillar collagen.

Extremely soft tissues such as developing hearts or adult brain contain far less collagen than highly stiff adult tissues such as tendons, but cell and molecular mechanisms for such homeostatic differences remain unclear. We hypothesized that cell-generated or exogenous forces combine with tension-suppressed collagen degradation in order to sculpt extracellular matrix (ECM) collagen levels in tissues. For various mature mice tissues and beating embryonic chick hearts, we find collagen-sensitive second harmonic generation (SHG) image intensity scales non-linearly versus tissue stiffness, aligning well with the results from cellularized gels of collagen. Chick hearts beating at ∼5% strain maintain collagen levels until their contractile strain is suppressed by myosin-II inhibition and endogenous matrix metalloproteinases (MMPs) then degrade collagens within ∼30-60 minutes - based on SHG and mass spectrometry proteomics. Although tendons composed of oriented collagen fibrils exhibit heterogeneous strain distributions upon deformation, the addition of exogenous MMP or bacterial collagenase suppresses collagen degradation for strains within physiological limits (i.e., up to ∼5-8%). Sequestration of collagen cleavage sites by tissue strain is a likely mechanism because molecular permeation and mobility prove strain-independent whereas artificial collagen cross-links accelerate strain-dependent collagen degradation via collagen molecular unfolding. Tension-suppressed degradation of collagen thus underlies tissue stiffness scaling.

Invadopodia Methods: Detection of Invadopodia Formation and Activity in Cancer Cells Using Reconstituted 2D and 3D Collagen-Based Matrices.

Tumor dissemination involves cancer cell migration through the extracellular matrix (ECM). ECM is mainly composed of collagen fibers that oppose cell invasion. To overcome hindrance in the matrix, cancer cells deploy a protease-dependent program in order to remodel the matrix fibers. Matrix remodeling requires the formation of actin-based matrix/plasma membrane contact sites called invadopodia, responsible for collagen cleavage through the accumulation and activity of the transmembrane type-I matrix metalloproteinase (MT1-MMP). In this article, we describe experimental procedures designed to assay for invadopodia formation and for invadopodia activity using 2D and 3D models based on gelatin (denatured collagen) and fibrillar type-I collagen matrices.

A Highly Sensitive Biomarker of Type II Collagen C-Terminal Pro-Peptide Associated with Cartilage Formation.

The type II collagen C-terminal pro-peptide is one of the most abundant polypeptides in cartilage. The purpose of this study was to develop a competitive chemiluminescence enzyme-linked immunosorbent assay, CALC2, targeting this pro-peptide as a marker of cartilage formation. Technical assay parameters were evaluated. CALC2 level was measured after in vitro cleavage of recombinant type II collagen with bone morphogenetic protein-1 (BMP-1) and treatment of ex vivo human osteoarthritis (OA) cartilage explant model (HEX) with insulin-like growth factor-1 (IGF-1). Serum CALC2 levels were assessed in 18 patients with rheumatoid arthritis (RA), 19 patients with ankylosing spondylitis (AS), and 18 age- and sex-matched controls in cohort 1 and 8 patients with OA and 14 age- and sex-matched controls in cohort 2. Type II collagen cleavage with BMP-1 increased the CALC2 level. IGF-1 treatment increased the CALC2 levels in HEX compared with the untreated explants (p < 0.05). Results were confirmed using Western blot analysis. CALC2 levels were decreased in the patients with RA and AS compared with the healthy controls (p = 0.01 and p = 0.02, respectively). These findings indicate that CALC2 may be a novel biomarker of type II collagen formation. However, further preclinical and clinical studies are required to validate these findings.

Intra-osseous infiltration of adipose mesenchymal stromal cells and plasma rich in growth factors to treat acute full depth cartilage defects in a rabbit model: Serum osteoarthritis biomarkers and macroscopical assessment.

Intra-articular infiltration of plasma rich in growth factors (PRGF) and adipose mesenchymal stromal cells (AMSCs) are known to inhibit osteoarthritis progression. However, in severely affected patients, the treatment cannot reach the deeper layers of the articular cartilage; thus, its potential is limited. To overcome this limitation, intra-osseous infiltrations have been suggested. The purpose of this study is to assess the impact of intra-osseous infiltration therapies on serum biomarkers of osteoarthritis and to assess cartilage regeneration macroscopically. A total of 80 rabbits were divided into four groups based on the intra-osseous treatment administered on the day of surgery: control, PRGF, AMSCs and a combination of PRGF + AMSCs. In addition, all groups received a single intra-articular administration of PRGF on the same day. Serum biomarker levels were measured before infiltration and 28-, 56-, and 84-days post infiltration, and macroscopical assessment was conducted at 56- and 84-days follow-up post infiltration. In the PRGF + AMSCs group, significantly lower concentrations of hyaluronic acid and type II collagen cleavage neoepitope were recorded at all time points during the study, followed by PRGF, AMSCs and control groups. Regarding macroscopical assessment, lower scores were obtained in PRGF + AMSCs group at all study times. The results suggest that the combination of intra-articular PRGF with intra-osseous PRGF or AMSCs achieves better results in rabbits with acute chondral defects and that intra-osseous infiltration is a safe procedure.

Abstract IA014: Collagen cleavage controls PDAC metabolism and malignancy via a DDR1-NRF2 cascade and mitochondrial biogenesis

Abstract PDAC is distinct by being highly desmoplastic, due to the activation and eventual infiltration of cancer associated fibroblasts (CAF) and the extracellular matrix (ECM) they produce, which is mainly composed of collagen type I (Col I). The role of fibroblasts and collagen in PDAC pathology is highly contentious, ranging from tumor stimulatory to tumor inhibitory. Using mouse models and human specimens we found that the way in which Col I affects PDAC progression, metabolism and its response to therapy is highly dependent on its cleavage by matrix metalloproteases (MMP). Whereas noncleaved Col I inhibits PDAC metabolism, inhibits human and mouse PDAC growth in mice and is correlated with improved survival after surgical resection, MMP generated Col I fragments stimulate tumor metabolism and growth and correlate with poor post-resection survival. These differential activities of cleaved and noncleaved Col I depend on its interactions with the receptor tyrosine kinase DDR1. Whereas MMP-generated Col I binds DDR1 and activates it, intact Col I induces DDR1 degradation. By activating DDR1, cleaved Col I triggers an NF-kB-p62-NRF2 signaling cascade, whose major efferent, NRF2, controls many aspects of PDAC metabolism, including macropinocytosis, aerobic glycolysis, respiration and mitochondrial biogenesis. Inhibitors of NRF2 and mitochondrial biogenesis induce regression of established PDAC in-vivo and compromise its growth and survival in-vitro. Citation Format: Michael Karin, Hua Su. Collagen cleavage controls PDAC metabolism and malignancy via a DDR1-NRF2 cascade and mitochondrial biogenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA014.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

Metalloproteinase-1 (MMP-1) catalyzed collagen degradation is essential for a wide variety of normal physiological processes, while at the same time contributing to several diseases in humans. Therefore, a comprehensive understanding of this process is of great importance. Although crystallographic and spectroscopic studies provided fundamental information about the structure and function of MMP-1, the precise mechanism of collagen degradation especially considering the complex and flexible structure of the substrate, remains poorly understood. In addition, how the protein environment dynamically reorganizes at the atomic scale into a catalytically active state capable of collagen hydrolysis remains unknown. In this study, we applied experimentally-guided multiscale molecular modeling methods including classical molecular dynamics (MD), well-tempered (WT) classical metadynamics (MetD), combined quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM MetD simulations to explore and characterize the early catalytic events of MMP-1 collagenolysis. Importantly the study provided a complete atomic and dynamic description of the transition from the open to the closed form of the MMP-1•THP complex. Notably, the formation of catalytically active Michaelis complex competent for collagen cleavage was characterized. The study identified the changes in the coordination state of the catalytic zinc(II) associated with the conformational transformation and the formation of catalytically productive ES complex. Our results confirm the essential role of the MMP-1 catalytic domain's α-helices (hA, hB and hC) and the linker region in the transition to the catalytically competent ES complex. Overall, the results provide unique mechanistic insight into the conformational transformations and associated changes in the coordination state of the catalytic zinc(II) that would be important for the design of effective MMP-1 inhibitors.

Neoepitope fragments as biomarkers for different phenotypes of intervertebral disc degeneration.

BackgroundDuring the intervertebral disc (IVD) degeneration process, initial degenerative events occur at the extracellular matrix level, with the appearance of neoepitope peptides formed by the cleavage of aggrecan and collagen. This study aims to elucidate the spatial and temporal alterations of aggrecan and collagen neoepitope level during IVD degeneration.MethodsBovine caudal IVDs were cultured under four different conditions to mimic different degenerative situations. Samples cultured after 1‐ or 8‐days were collected for analysis. Human IVD samples were obtained from patients diagnosed with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS). After immunohistochemical (IHC) staining of Aggrecanase Cleaved C‐terminus Aggrecan Neoepitope (NB100), MMP Cleaved C‐terminus Aggrecan Neoepitope (MMPCC), Collagen Type 1α1 1/4 fragment (C1α1) and Collagenase Cleaved Type I and II Collagen Neoepitope (C1,2C), staining optical density (OD)/area in extracellular matrix (OECM) and pericellular zone (OPCZ) were analyzed. Conditioned media of the bovine IVD was collected to measure protein level of inflammatory cytokines and C1,2C.ResultsFor the bovine IVD sections, the aggrecan MMPCC neoepitope was accumulated in nucleus pulposus (NP) and cartilage endplate (EP) regions following mechanical overload in the one strike model after long‐term culture; as for the TNF‐α induced degeneration, the OECM and OPCZ of collagen C1,2C neoepitope was significantly increased in the outer AF region after long‐term culture; moreover, the C1,2C was only detected in conditioned medium from TNF‐α injection + Degenerative loading group after 8 days of culture. LDH patients showed higher MMPCC OECM in NP and higher C1,2C OECM in AF region compared with AIS patients.ConclusionsIn summary, aggrecan and collagen neoepitope profiles showed degeneration induction trigger‐ and region‐specific differences in the IVD organ culture models. Different IVD degeneration types are correlated with specific neoepitope expression profiles. These neoepitopes may be helpful as biomarkers of ECM degradation in early IVD degeneration and indicators of different degeneration phenotypes.

Abstract 3996: A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice

Abstract Introduction: Glioblastoma (GBM) is the most frequent treatment resistant and poor prognosis primary brain tumor. Age-adjusted incidence among Caucasians is approx. twice that of Africans. Genetic changes in ethnicity may represent druggable targets for development of new treatments for GBM. Chondroitin sulfate proteoglycan 4 (CSPG4) with sequence homology to neuron-glial-2 (NG2), henceforth CSPG4/NG2, is a transmembrane proteoglycan that is upregulated in GBM, resulting in leaky neovasculature and high cell proliferation, and is independently prognostic for poor survival. Mechanisms regulating CSPG4/NG2 expression remain to be elucidated. Experiments: Tumor-derived DNA whole exome sequences (WES) from mixed American GBM patients (n=300) collected into the TCGA database and blood-derived DNA WES from controls (n = 2504) in the 1000 Genome database were analyzed for mutations in the CSPG4 gene by GATK-3.5. We used prime editing (PE) to introduce a 13-base pair (bp) deletion through design of PE guide RNA (n=27), screening GBM cells (n=14) for CSPG4/NG2 expression, transfecting and expanding PE cells, confirming editing using Sanger sequencing. We functionally characterized the mutation in PE cells and transplanted in orthotopic PDX mouse models compared to wild type (WT) and Cas9 control cells. Western blotting, BrdU proliferation assays, clonogenic- and wound healing assays were used to investigate function. Gelatin zymography assessed CSPG4/NG2 matrix metalloproteinase (MMP) cross-talk during invasion on mixed extracellular matrix- or collagen IV- coated transwells. Results: A novel 13bp frameshift deletion in exon 3 of the CSPG4 gene was identified from WES of GBM samples deposited in TCGA. The mutation was cancer specific (p<0.001). Comparison of frequency in various ethnicities revealed the mutation to be more frequent among African American GBM patients (p=0.019). We generated 3 clones heterozygous for the CSPG4/NG2 deletion in patient derived GBMs. A homozygous deletion was lethal to the cells. The 13bp deletion reduced CSPG4/NG2 expression by approx. 60%, slowed tumor cell proliferation compared to both WT (p<0.001) and Cas9 cells (p<0.0001). PE cells had significantly reduced colony formation (p<0.001), scratch wound healing (p<0.001), had a weaker invasive phenotype when challenged with collagen IV (p<0.001), and reduced cleavage of collagen I by MMP2 that was corroborated by RNAseq gene expression. Reduced growth of PE cells in vivo was verified by multiplex immunohistochemistry on ex vivo brain sections and identified biomarkers for differential tumor development. Tumor growth was reduced in vivo in mice. Conclusions: We identified a novel 13bp deletion in CSPG4/NG2 with phenotypic effect that could be used as a future molecular target in GBM treatment, and present the first successful application of PE in human GBM cells. Citation Format: Victoria Smith Arnesen, Susina Suntharalingam, Żaneta Matuszek, Shahin Sarowar, Stian Knappskog, Stein Atle Lie, David R. Liu, Mohummad Aminur Rahman, Martha Chekenya. A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3996.

Uncovering mediators of collagen degradation in the tumor microenvironment

Increased remodeling of the extracellular matrix in malignant tumors has been shown to correlate with tumor aggressiveness and a poor prognosis. This remodeling involves degradation of the original extracellular matrix (ECM) and deposition of a new tumor-supporting ECM. The main constituent of the ECM is collagen and collagen turnover mainly occurs in a sequential manner, where initial proteolytic cleavage of the insoluble fibers is followed by cellular internalization of large well-defined collagen fragments for lysosomal degradation. However, despite extensive research in the field, a lack of consensus on which cell types within the tumor microenvironment express the involved proteases still exists. Furthermore, the relative contribution of different cell types to collagen internalization is not well-established. Here, we developed quantitative ex vivo collagen degradation assays and show that the proteases responsible for the initial collagen cleavage in two murine syngeneic tumor models are matrix metalloproteinases produced by cancer-associated fibroblasts and that collagen degradation fragments are endocytosed primarily by tumor-associated macrophages and cancer-associated fibroblasts from the tumor stroma. Using tumors from mannose receptor-deficient mice, we show that this receptor is essential for collagen-internalization by tumor-associated macrophages. Together, these findings identify the cell types responsible for the entire collagen degradation pathway, from initial cleavage to endocytosis of fragments for intracellular degradation.

Crosstalk between Irisin Levels, Liver Fibrogenesis and Liver Damage in Non-Obese, Non-Diabetic Individuals with Non-Alcoholic Fatty Liver Disease.

Background: Insulin resistance plays a relevant role in the onset of non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and fibrosis. Irisin is an exercise-induced myokine involved in the regulation of energy homeostasis and glucose metabolism. Additionally, pre-clinical models have shown a potential role of irisin in the pathogenesis of NAFLD. The aim of this study is to explore the association between irisin, histological features and biomarkers of liver fibrogenesis in non-diabetic, non-obese, biopsy-proven NAFLD individuals. Methods: Forty-one patients with histological evidence of NAFLD were included. Circulating irisin and direct markers of fibrogenesis N-terminal type III collagen propeptide (PRO-C3) and type VI collagen cleavage product (PRO-C6) were measured by ELISA. Results: Median age of the cohort was 45 years (41–51) and 80.4% were male. Significant fibrosis (stage ≥ 2) was present in 36.6% of cases. Circulating irisin, PRO-C3 and PRO-C6 levels were significantly higher in subjects with fibrosis stage ≥ 2 when compared to those with fibrosis stage < 2 (5.96 ng/mL (95% CI = 4.42–9.19) vs. 2.42 ng/mL (95% CI = 1.73–5.95), p = 0.033; 9.5 ng/mL (95% CI = 7.7–13.6) vs. 6.2 ng/mL (95% CI = 4.9–8.9), p = 0.016; 6.6 ng/mL (95% CI = 5.6–7.9) vs. 5.1 ng/mL (95% CI = 4.2–5.4), p = 0.013, respectively). Irisin levels were similarly distributed between the features of NASH. Circulating irisin positively correlated with both PRO-C3 and PRO-C6 levels (r = 0.47, p = 0.008 and r = 0.46, p = 0.002). Conclusions: Increased circulating irisin levels may identify a more aggressive phenotype of liver disease with increased fibrogenesis and more severe liver damage.

Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models.

Multicellular tumor spheroids have emerged as well-structured, three-dimensional culture models that resemble and mimic the complexity of the dense and hypoxic cancer microenvironment. However, in brain tumor studies, a variety of glioblastoma multiforme (GBM) cell lines only self-assemble into loose cellular aggregates, lacking the properties of actual glioma tumors in humans. In this study, we used type-I collagen as an extracellular matrix component to promote the compaction of GBM aggregates forming tight spheroids to understand how collagen influences the properties of tumors, such as their growth, proliferation, and invasion, and collagenase to promote collagen degradation. The GBM cell lines U87MG, T98G, and A172, as well as the medulloblastoma cell line UW473, were used as standard cell lines that do not spontaneously self-assemble into spheroids, and GBM U251 was used as a self-assembling cell line. According to the findings, all cell lines formed tight spheroids at collagen concentrations higher than 15.0 μg mL-1. Collagen was distributed along the spheroid, similarly to that observed in invasive GBM tumors, and decreased cell migration with no effect on the cellular uptake of small active molecules, as demonstrated by uptake studies using the photosensitizer verteporfin. The enzymatic cleavage of collagen affected spheroid morphology and increased cell migration while maintaining cell viability. Such behaviors are relevant to the physiological models of GBM tumors and are useful for better understanding cell migration and the in vivo infiltration path, drug screening, and kinetics of progression of GBM tumors.

Evaluation of the influence of chromium and zinc ions on the collagenolysis process in solutions by the method of dynamic light scattering

Using the method of dynamic light scattering, it was found that the rate of collagen cleavage under the influence of bacterial collagenase decreases by almost 4 times when protein molecules are doped with Cr3+ ions, and when the enzyme activator of Zn2+ ions is added, it increases by 1.3 times. Based on the experimental data, the reaction rate constants k1 were calculated. It was found that when collagen molecules are tanned with a chromium salt, it becomes more resistant to degradation in solutions containing zinc ions than in solutions with the addition of calcium ions, as we described in earlier works. Keywords: collagen type 1, bacterial collagenase, chromium chloride, zinc chloride, dynamic light scattering method, translational diffusion coefficient, hydrodynamic radius, reaction rate constant, collagenolysis, Clostridium histolyticum.

Оценка влияния ионов хрома и цинка на процесс коллагенолиза в растворах методом динамического рассеяния света

Using the method of dynamic light scattering, it was found that the rate of collagen cleavage under the influence of bacterial collagenase decreases by almost 4 times when protein molecules are doped with Cr3+ ions, and when the enzyme activator of Zn2+ ions is added, it increases by 1.3 times. Based on the experimental data, the reaction rate constants k1 were calculated. It was found that when collagen molecules are tanned with a chromium salt, it becomes more resistant to degradation in solutions containing zinc ions than in solutions with the addition of calcium ions, as we described in earlier works.