Collagen Content Research Articles

426 A Beat Away from Precision Medicine: Characterizing Human Cardiac Fibroblast Responsiveness to Hemodynamic Unloading in Heart Failure with Reduced Ejection Fraction

OBJECTIVES/GOALS: Myocardial interstitial fibrosis leads to high hemodynamic load resulting in heart failure (HFrEF). Previous studies show that treatment with a left ventricular assist device (LVAD) does not reduce fibrosis. We hypothesize that human cardiac fibroblasts are highly activated in HFrEF and remain unresponsive to hemodynamic unloading by LVAD. METHODS/STUDY POPULATION: Forty human subjects with HFrEF undergoing LVAD implantation were enrolled to provide a portion of myocardium routinely removed during LVAD placement. In addition, 7 biopsies previously collected from transplanted hearts with extended LVAD treatment were also evaluated (LVEX). RESULTS/ANTICIPATED RESULTS: Quantification of PSR-stained sections reveals a significant increase in collagen content in the HFrEF tissue (CVF = 2.8) compared to control tissues (CVF = 0.9) that remained elevated in LVEX hearts (CVF = 3.1). HCFs from LV biopsies were isolated and grown to confluence. HCFs from HFrEF patients and control HCFs were plated on substrates with stiffnesses reflective of normal myocardium (2kPa) or HFrEF myocardium (8kPa). Cells were collected at 4- and 7-day time points and levels of collagen I and alpha-smooth muscle actin were quantified by western blot analysis. Control HCFs were responsive to changes in substrate stiffness producing more Col I and a-SMA on 8kPa versus 2kPa, HCFs from HFrEF patients were unresponsive to changes in stiffness exhibiting no significant difference in protein production on 2 vs. 8kPa. DISCUSSION/SIGNIFICANCE: Our data suggests that HCFs isolated from the failing myocardium do not respond to changes in mechanical load and might contribute to persistent increases in fibrosis. These findings bring us one step closer to elucidating mechanisms behind fibrosis in HFrEF which could lead to targeted therapies to improve patient outcomes from LVAD support.

Potential Role of Odanacatib in the Treatment of Postmenopausal Intervertebral Disc Degeneration

Odanacatib (ODN), a cathepsin K (CatK) inhibitor, is widely used for the treatment of postmenopausal osteoporosis. However, whether ODN is efficient in delaying intervertebral disc degeneration (IVDD) remaining unknown. We aimed to explore the effect of ODN in the postmenopausal IVDD using an ovariectomized (OVX) mouse model. We collected the disc tissues from female spinal fracture patients and analyzed the CatK expression in different estrogen levels. Besides, we injected ranged concentration of OND subcutaneously in the OVX mice and tested the disc height index (DHI), aggrecan positive area, collagen I, collagen II, inflammatory factors, and apoptosis-related gene expression comparing to control. The intervertebral disc degraded and the CatK gene expression decreased in the disc lacking of estrogen. OVX method increased the collagen I, MMP-3, MMP-13, caspase-3, and caspase-8 expression, but reduced DHI, and the content of aggrecan and collagen II, indicating an IVDD tendency. However, the ODN treatment could suppress MMP-3, MMP-13, caspase-3, and caspase-8 and protect the stability of extracellular matrix (ECM). ODN is a potential drug to delay the IVDD by suppressing apoptosis and ECM degradation.

The Effects of Slaughter Age and Sex on Carcass Traits, Meat Quality, and Leg Bone Characteristics of Farmed Common Pheasants (Phasianus colchicus L.).

The aim of this study was to determine the effects of slaughter age and sex on the carcass characteristics, meat quality parameters, and leg bone dimensions of common pheasants. The study material consisted of 40 common pheasants, including 10 males and 10 females at 12 weeks of age and 10 males and 10 females at 15 weeks of age. The birds were kept on a farm in a semi-intensive system. The slaughter age had significant effects on the eviscerated carcass weight; the percentage of skin with subcutaneous fat; the wings percentage; electrical conductivity (EC24); the work required to cut the samples (cutting work); redness (a*); the intramuscular fat content in the breast meat; and the protein, intramuscular fat, water, and collagen contents in the leg meat. The sex of the pheasants had significant effects on the eviscerated carcass weight; the contents of leg muscles, skin with subcutaneous fat, and wings in the carcasses; and the electrical conductivity, thermal loss, lightness (L*), and redness (a*) of the breast muscles. It also significantly affected the protein and water contents of the pectoral and leg muscles, the intramuscular fat contents of the leg muscles, the texture traits of the pectoralis major muscle, and all femur and tibia bone dimensions. The results indicate a greater influence of sex compared to slaughter age on the pheasant traits studied. We confirmed the occurrence of a clearly marked sexual dimorphism in birds of this species. Both factors (slaughter age and gender) have significant effects on the nutritional and technological value of pheasant meat. The few studies on meat texture and the dimensions of pheasant leg bones indicate a need for continued research in this area in the future.

Aflatoxin B1 decreased flesh flavor and inhibited muscle development in grass carp (Ctenopharyngodon idella)

In nature, aflatoxins, especially aflatoxin B1 (AFB1), are the common mycotoxins, which cause serious health problems for humans and animals. This paper aimed to study the effects of AFB1 on flesh flavor and muscle development of grass carp (Ctenopharyngodon idella) and its mechanism. There were 1440 individual fish in total, with 6 treatments and each treatment replicated 3 times. The 6 treatments were fed a control diet with different doses of AFB1 (0.04, 29.48, 58.66, 85.94, 110.43 and 146.92 μg/kg diet) for 60 d. AFB1 increased myofiber diameter, as well as decreased myofiber density of grass carp muscle (P < 0.05). The contents of free amino acid decreased gradually (P < 0.05) as dietary AFB1 increased in the muscle of grass carp. The levels of reactive oxygen species, malonaldehyde and protein carbonyl (PC) were increased (P < 0.05) with the dietary AFB1 increased. The levels of antioxidant enzyme (glutathione peroxidase, glutathione, glutathione reductase, total antioxidant capacity, anti-superoxide anion, and anti-hydroxyl radical) were decreased (P < 0.05) with the dietary AFB1 increased. In addition, dietary AFB1 decreased the content of collagen, and downregulated the mRNA and protein levels of transforming growth factor-β (TGF-β)/Smads signaling pathway in grass carp muscle (P < 0.05). The mRNA and protein levels of myogenic regulatory factors were downregulated in grass carp muscle (P < 0.05). Furthermore, the activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were increased (P < 0.05), and the protein levels of phosphorylate-38 mitogen-activated protein kinase (p-p38MAPK), phosphorylate-c-Jun N-terminal kinase, urokinase-type plasminogen activator (uPA), MMP-2 and MMP-9 were upregulated (P < 0.05), but collagen Ⅰ, laminin β1 and fibronectin were downregulated (P < 0.05) with the dietary AFB1 increased in the muscle of grass carp. Based on the results of this study, we can draw the following conclusion: dietary AFB1 might damage flesh flavor and inhibit the muscle development through MAPK/uPA/MMP/extracellular matrix (ECM) signaling pathway in grass carp. Moreover, the recommended safe limit of AFB1 in feed is no more than 26.77 μg/kg diet according to the PC levels in grass carp muscle.

COLLAGEN-CONTAINING CONCENTRATE IN THE PRODUCTION OF DAIRY PRODUCTS SPORTS NUTRITION PRODUCTS

The article substantiates the relevance of the use of collagen-containing concentrate in the production of cottage cheese products for sports nutrition. The inclusion of cottage cheese products with a high content of collagen in the diet of athletes helps to strengthen the cartilage tissue of joints, improve the restoration of connective tissue of the ligamentous apparatus and reduce the pain threshold by increasing collagen production. Based on the study of the chemical composition of secondary products of the poultry processing industry, such chicken parts as skin, bone tissue and paws were selected to obtain a collagen-containing concentrate. A method for producing a dry collagen-containing concentrate has been developed. To obtain a collagen-containing concentrate, a method based on an enzymatic process using the papain enzyme for the hydrolysis of raw materials was chosen. This method allows you to preserve the biological value of collagen due to the gentle processing of raw materials. As a result of experimental studies, it was found that the collagen-containing concentrate contains polyunsaturated omega-3, omega-6 and omega-9 fatty acids with antioxidant properties. The result of the study of nutritional and biological value confirms the validity of the use of collagen-containing concentrate in the production of cottage cheese product for sports nutrition. A curd product enriched with collagen can be intended not only for athletes, but also for people who play sports for recreational purposes.

Two-Photon Microscopy for the Study of Tendons.

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in various biological systems. This technique relies on nonlinear light-matter interactions to detect two distinct signals: the second harmonic generated (SHG) diffusion signal, which facilitates the visualization of collagen fibers and their orientation, and the near-infrared excitation signal for imaging ultraviolet excited autofluorescence. SHG imaging proves especially effective in visualizing collagen fibers due to the non-centrosymmetric crystalline structure of fibrillar collagen I. Given that tendons are matrix-rich tissues with a limited number of cells, their high collagen content makes them ideal candidates for analysis using two-photon microscopy. Consequently, two-photon microscopy offers a valuable means to analyze and characterize collagen abnormalities in tendons. Its application extends to studying tendon development, injuries, healing, and aging, enabling the comprehensive characterization of tendon cells and their interactions with the ECM under various conditions using two-photon microscopy tools. This protocol outlines the use of two-photon microscopy in tendon biology and presents an adapted methodology to achieve effective imaging and characterization of tendon cells during development and after injury. The method allows the utilization of thin microscopic sections to create a comprehensive image of the ECM within tendons and the cells that interact with this matrix. Most notably, the article showcases a technique to generate 3D images using two-photon microscopy in animal models.

Label-free quantification of imaging features in the extracellular matrix of left and right-sided colon cancer tissues

The molecular pathogenesis of colorectal cancer is known to differ between the right and left side of the colon. Several previous studies have focussed on the differences in clinicopathological features, proteomic and genetic biomarkers, the composition of gut microbiota, response to therapy, and the characteristics of the tumour microenvironment. However, the morphology and density of collagen in the extracellular matrix (ECM) have not been studied intensively. In this study, we employed 2-photon laser scanning microscopy (2PLSM) to visualise the intrinsic second-harmonic generation (SHG) signal emitted by collagen fibres in the heterogeneous ECM of human colon tumour tissues. Through texture analysis of the SHG signal, we quantitatively distinguished the imaging features generated by structural differences of collagen fibres in healthy colon and cancers and found marked differences. The fibres inside of tumours exhibited a loss of organisation, particularly pronounced in right-sided colon cancer (RSCC), where the chaotic regions were significantly increased. In addition, a higher collagen content was found in left-sided colon cancer (LSCC). In future, this might aid in subclassification and therapeutic decisions or even in designing new therapy regimens by taking into account the differences between collagen fibres features between colon tumours located at different sides.

Effects of hydroxyproline supplementation in low fish meal diets on collagen synthesis, myofiber development and muscular texture of juvenile Pacific white shrimp (Litopenaeus vannamei)

This experiment aimed to evaluate the impact of dietary hydroxyproline (Hyp) supplementation on the muscle quality of juvenile Pacific white shrimp (Litopenaeus vannamei) fed a low fishmeal diet. Six formulated diets included one high fishmeal (HF; 25% fishmeal content) and five low fishmeal diets (10% fishmeal content) with 0%, 0.2%, 0.4%, 0.6% and 0.8% Hyp (LF0, LF2, LF4, LF6 and LF8, respectively). Each diet was assigned to four replicates, and 40 shrimp (0.32 ± 0.00 g) per replicate were fed four times a day for 8 weeks. Dietary Hyp supplementation had little effects on growth performance, but increased the contents of Hyp, prolyl 4-hydroxylases (P4Hs), and collagen. The meat yield, springiness, hardness, chewiness, and cohesiveness of muscle were the highest in the LF4 group among the low fishmeal groups (P < 0.05). Cooking loss and freezing loss of muscle were the lowest in the LF4 group (P < 0.05). Dietary supplementation with 0.4% Hyp increased the myofiber density and decreased the myofiber diameter of muscle (P < 0.05). Supplementation of Hyp in the diet up-regulated the mRNA expression of smyhc5, smyhc15, col1a1, col1a2, igf-1f, tgf-β and tor and down-regulated the mRNA expression of smyhc1, smyhc2, smyhc6a (P < 0.05). Supplementation of Hyp in the diet up-regulated the protein expression of P-4E-BP1, P-AKT, AKT and P-AKT/AKT (P < 0.05). These results suggested that the addition of 0.4% Hyp to low fishmeal diets improved the muscle quality of L. vannamei.

Biological Activities of Deer Antler-Derived Peptides on Human Chondrocyte and Bone Metabolism.

Orally administered "tortoiseshell and deer antler gelatin" is a common traditional medicine for patients with osteoporosis or osteoarthritis. From the pepsin-digested gelatin, we previously isolated and identified the osteoblast-stimulating pentapeptide, TSKYR. Its trypsin digestion products include the dipeptide YR, enhancing calcium ion uptake, and tripeptide TSK, resulting in remarkable 30- and 50-fold increases in mineralized nodule area and density in human osteoblast cells. These peptides were chemically synthesized in this study. The composition of deer antler preparations comprises not only proteins and peptides but also a significant quantity of metal ion salts. By analyzing osteoblast growth in the presence of peptide YR and various metal ions, we observed a synergistic effect of calcium and strontium on the effects of YR. Those peptides could also stimulate the growth of C2C12 skeletal muscle cells and human chondrocytes, increasing collagen and glycosaminoglycan content in a three-dimensional environment. The maintenance of bone homeostasis relies on a balance between osteoclasts and osteoblasts. Deer antler peptides were observed to inhibit osteoclast differentiation, as evidenced by ROS generation, tartrate-resistant acid phosphatase (TRACP) activity assays, and gene expression in RAW264.7 cells. In summary, our findings provide a deep understanding of the efficacy of this folk medicine.

Anti-skin aging effects and bioavailability of collagen tripeptide and elastin peptide formulations in young and middle-aged women

BackgroundCollagen peptides (CP), including tripeptides and elastin peptides (EP), are known for their in vitro and in vivo anti-skin aging effects. Despite positive results in animal models, the combination effects of CP and EP and the bioavailability of CP in human studies, particularly in young and middle-aged women, remain underexplored. ObjectiveTo evaluate the effects of an orally administered collagen drink combining CP and EP on the skin health of young and middle-aged women. Materials and MethodsA single-center, randomized, double-blind, parallel-controlled trial was conducted, utilizing the WONDERLAB® fish collagen tripeptide beverage. Participants consumed the drink over an 8-week period. ResultsCompared to the placebo group, the collagen drink group showed significant improvements in skin hydration (39.19% increase), transepidermal water loss (33.45% decrease), skin elasticity (25.37% increase), dermal collagen content (21.64% increase), pore size (7.94% decrease), wrinkle length (18.09% decrease), skin smoothness (2.85% improvement), and skin roughness (15.32% decrease). Overall pore volume decreased by 60%, and visual assessments indicated a decrease in skin luminosity by 15.20% and smoothness index by 22.55%. Mass spectrometry demonstrated a significant increase in collagen efficacy components, including blood pH and GPH levels (P < 0.05). ConclusionThe study confirmed the combination nourishing and anti-skin aging effects of EP and CP on the skin of young and middle-aged women, demonstrating significant improvements in various skin parameters and good bioavailability of collagen peptides.

Optimized Decellularization of a Porcine Fasciocutaneaous Flap.

Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps.

Potential of Sea Cucumbers as Fuctional Foods

Sea cucumbers have great potential for managing water resources because they have filter feeder properties that can clean water from harmful substances. Apart from that, sea cucumbers also contain active compounds which have properties as functional foods and can help prevent cancer. Samples of sea cucumbers were collected from Karimunjawa, Central Java, and Surabaya. The research included tests for bacteria, chemicals, and heavy metals on dried sea cucumbers, as well as amino acid analysis. Two species of sea cucumbers were identified: Holothuria atra and Stichopus variegates. H. atra had a higher amino acid content and the highest collagen content among all the amino acids (14,600mg.100g-1). Both types of sea cucumber samples contain amino acids that have anti-inflammatory, antioxidant, and anticancer properties. This is due to their content of saponins, tannins, flavonoids, steroids, and terpenoids, which are beneficial for health. Additionally, S. variegates has been found to have antibacterial activity against Staphylococcus aureus and Escherichia coli bacteria. The amino acid analysis of S. variegates shows the presence of sulfate and alanine compounds, which are not found in H. atra. Research has shown that sea cucumbers can be consumed as supplements or additional food to support health and prevent various diseases, including cancer. The use of sea cucumbers as functional food ingredients also has the potential to manage water resources sustainably. In conclusion, H. atra and S. varigatus sea cucumbers have great potential for managing water resources for functional food and cancer prevention efforts. Their high content of amino acids and other phytochemical compounds makes them a good choice for development as functional food ingredients.

Intracellular Zn2+ promotes extracellular matrix remodeling in dexamethasone-treated trabecular meshwork.

Given the widespread application of glucocorticoids in ophthalmology, the associated elevation of intraocular pressure (IOP) has long been a vexing concern for clinicians, yet the underlying mechanisms remain inconclusive. Much of the discussion focuses on the extracellular matrix (ECM) of trabecular meshwork (TM). It is widely agreed that glucocorticoids impact the expression of matrix metalloproteinases (MMPs), leading to ECM deposition. Since Zn2+ is vital for MMPs, we explored its role in ECM alterations induced by dexamethasone (DEX). Our study revealed that in human TM cells treated with DEX, the level of intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. This correlated with changes in several Zrt-, Irt-related proteins (ZIPs) and metallothionein. ZIP8 knockdown impaired extracellular Zn2+ uptake, but Zn2+ chelation did not affect ZIP8 expression. Resembling DEX's effects, chelation of Zn2+ decreased MMP2 expression, increased the deposition of ECM proteins, and induced structural disarray of ECM. Conversely, supplementation of exogenous Zn2+ in DEX-treated cells ameliorated these outcomes. Notably, dietary zinc supplementation in mice significantly reduced DEX-induced IOP elevation and collagen content in TM, thereby rescuing the visual function of the mice. These findings underscore zinc's pivotal role in ECM regulation, providing a novel perspective on the pathogenesis of glaucoma.NEW & NOTEWORTHY Our study explores zinc's pivotal role in mitigating extracellular matrix dysregulation in the trabecular meshwork and glucocorticoid-induced ocular hypertension. We found that in human trabecular meshwork cells treated with dexamethasone, intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. Zinc supplementation rescues visual function by modulating extracellular matrix proteins and lowering intraocular pressure, offering a direction for further exploration in glaucoma management.

Optimization of Polycaprolactone and Type I Collagen Scaffold for Tendon Tissue Regeneration.

Introduction Collagen synthesis is vital for restoring musculoskeletal tissues, particularly in tendon and ligamentous structures. Tissue engineering utilizes scaffolds for cell adhesion and differentiation. Although synthetic scaffolds offer initial strength, their long-term stability is surpassed by biological scaffolds. Combining polycaprolactone (PCL) toughness with collagen in scaffold design, this study refines fabrication via electrospinning, aiming to deliver enduring biomimetic matrices for widespread applications in musculoskeletal repair. Methods Electrospinning employed four solutions with varied collagen and PCL concentrations, dissolved in chloroform, methanol, and hexafluoro-2-propanol. Solutions were combined to yield 60 mg/mL concentrations with different collagen/PCL ratios. Electrospinning at 12-14kV voltage produced scaffolds, followed by vacuum-drying. Collagen coating was applied to PCL and 15% collagen/PCL scaffolds using a 0.1% collagen solution. SEM characterized fiber morphology, tensile testing was conducted to determine the mechanical properties of the scaffold, and Fourier-transform infrared (FTIR) spectroscopy analyzed scaffold composition. Atomic force microscopy (AFM) analyzed the stiffness properties of individual fibers, and a finite element model was developed to predict the mechanical properties. Cell culture involved seeding human bone marrow mesenchymal stem cells onto scaffolds, which were assessed through Alamar Blue assay and confocal imaging. Results Various scaffolds (100% PCL, PCL-15% collagen, PCL-25% collagen, PCL-35% collagen) were fabricated to emulate the extracellular matrix, revealing collagen's impact on fiber diameter reduction with increasing concentration. Tensile testing highlighted collagen's initial enhancement of mechanical strength, followed by a decline beyond PCL-15% collagen. FTIR spectroscopy detected potential hydrogen bonding between collagen and PCL. A finite element model predicted scaffold response to external forces which was validated by the tensile test data. Cell viability and proliferation assays demonstrated successful plating on all scaffolds, with optimal proliferation observed in PCL-25% collagen. Confocal imaging confirmed stem cell integration into the three-dimensional material. Collagen coating preserved nanofiber morphology, with no significant changes in diameter. Coating of collagen significantly altered the tensile strength of the scaffolds at the macro scale. AFM highlighted stiffness differences between PCL and collagen-coated PCL mats at the single fiber scale. The coating process did not significantly enhance initial cell attachment but promoted increased proliferation on collagen-coated PCL scaffolds. Conclusion The study reveals collagen-induced mechanical and morphological alterations, influencing fiber alignment, diameter, and chemical composition while emphasizing scaffolds' vital role in providing a controlled niche for stem cell proliferation and differentiation. The optimization of each of these scaffold characteristics and subsequent finite element modeling can lead to highly repeatable and ideal scaffold properties for stem cell integration and proliferation.

Comparison of the Effects of Cinnamic Acid and Silver Sulfadiazine on Deep Second-degree Burns in Rabbits

Background: Burns are wounds caused by thermal, chemical, electrical, or radiation injuries. They are complex wounds that are difficult to heal and are associated with thousands of deaths each year. Cinnamic acid (CA) is a natural organic compound found in plants, fruits, vegetables, and honey. CA possesses antimicrobial, anti-inflammatory, antioxidant, and wound healing properties. Objectives: The aim of this study was to compare the healing effects of CA with silver sulfadiazine (SSD), a standard topical agent for burn treatment, on deep second-degree burns. Methods: This experimental study compared the healing effects of CA and SSD on second-degree burns. Fifteen New Zealand white rabbits weighing 2 to 2.3 kg were divided into 5 equal groups. Deep second-degree burn wounds were created on the backs of the rabbits by contacting a heated circular metal plate. The first group was treated with SSD 1% cream, the second, third, and fourth groups were treated with prepared ointments containing 0.1%, 0.5%, and 1% w/w of CA in Eucerin, respectively. The fifth group was treated with Eucerin alone. Wound healing effects were assessed by measuring the rate of wound contraction and the amount of collagen in tissue specimens obtained from different groups. Additionally, histopathological studies were performed on tissue samples. Results: The results showed that CA was significantly more effective than SSD in burn wound healing. Cinnamic acid ointments significantly increased the rate of wound contraction (P &lt; 0.05) and tissue collagen content (P &lt; 0.001) compared to SSD and Eucerin. Conclusions: Our findings suggest that topical CA possesses burn wound healing properties and could be used as an effective topical agent for the treatment of burns.

The inhibition of MARCO by PolyG alleviates pulmonary fibrosis via regulating mitochondrial function in a silicotic rat model.

Silicon dioxide (SiO2)-induced pulmonary fibrosis is potentially associated with the impairment of mitochondrial function. Previous research found that inhibition of macrophage receptor with collagenous structure (MARCO) could alleviate particle-induced lung injury by regulating phagocytosis and mitigating mitochondrial damage. The present study aims to explore the underlying anti-fibrosis mechanism of polyguanylic acid (PolyG, MARCO inhibitor) in a silicotic rat model. Hematoxylin and eosin and Masson staining were performed to visualize lung tissue pathological changes. Confocal microscopy, transmission electron microscope, western blot analysis, quantitative real-time PCR (qPCR), andadenosine triphosphate (ATP) content assay were performed to evaluate collagen content, mitochondrial function, and morphology changes in SiO2-induced rat pulmonary fibrosis. The results suggested that SiO2 exposure contributed to reactive oxygen species aggregation and the reduction of respiratory complexes and ATP synthesis. PolyG treatment could effectively reduce MARCO expression and ameliorate lung injury and fibrosis by rectifying the imbalance of mitochondrial respiration and energy synthesis. Furthermore, PolyG could maintain mitochondrial homeostasis by promoting peroxisome proliferator-activated receptor-coactivator 1 α (PGC1α)-mediated mitochondrial biogenesis and regulating fusion and fission. Together, PolyG could ameliorate SiO2-induced pulmonary fibrosis via inhibiting MARCO to protect mitochondrial function.

Influence of anti-sclerostin monoclonal antibody in the repair of post-extraction sockets of ovariectomized rats

ObjectiveThis study assessed the impact of an anti-sclerostin monoclonal antibody (Scl-Ab)-based osteoporosis drug on the post-extraction alveolar repair of ovariectomized rats. DesignFifteen female rats were randomly distributed into three groups: CTR (healthy animals), OST (osteoporosis induced by ovariectomy), and OST+Scl-Ab (osteoporosis induction followed by Scl-Ab treatment). Ovariectomy or sham surgery was performed 30 days before baseline, and Scl-Ab or a vehicle was administered accordingly in the groups. After seven days, all rats underwent the first lower molar extraction and were euthanized 15 days later. Computed microtomography, histological analysis, and collagen content measurement were performed on post-extraction sockets and intact mandibular and maxillary bone areas. ResultsMicrotomographic analyses of the sockets and mandibles did not reveal significant differences between groups on bone morphometric parameters (p > 0.05), while maxillary bone analyses resulted in better maintenance of bone architecture in OST+Scl-Ab, compared to OST (p < 0.05). Descriptive histological analysis and polarization microscopy indicated better post-extraction socket repair characteristics and collagen content in OST+Scl-Ab compared to OST (p < 0.05). ConclusionsScl-Ab-based medication did not accelerate alveolar bone formation but exhibited better post-extraction repair characteristics, and collagen content compared to ovariectomized animals only.

Abstract 5149: Extracellular matrix remodeling in metastatic mismatch repair deficient endometrial cancer:Implications for immune checkpoint inhibitor response prediction

Abstract Mismatch repair deficient (MMRd) status is a predictive biomarker for Immune checkpoint inhibitors (ICI) in endometrial cancer (EC), however, half of these patients (pts) do not respond. Most studies exploring biomarkers of response to ICI have focused on tumor or immune cell factors. We aimed to describe the extracellular matrix components of the tumor microenvironment (TME) in ICI-Responder (R) versus Non-Responder (NR) MMRd EC pts to identify new predictive biomarkers of response. Clinical data and outcomes of metastatic MMRd EC pts, treated with ICI (2016-2021), were retrospectively collected. Pts were classified as Rs (CR, PR, or SD ≥12 months) or NRs (PD or SD &amp;lt;12 months). Pre-ICI FFPE tumor samples were subjected to Biognosys UltraDeep TrueDiscovery™ Mass Spectrometry (MS) for identification of differentially regulated protein expression between R and NR. Criteria for protein candidate selection were p-value &amp;lt; 0.01 and average absolute log2 fold change (AVG Log ratio) &amp;gt; 0.58. Collagen was visualized on whole slides using Masson’s trichrome and quantified by Qupath. Collagen density was expressed as % positive surface in the intratumoral area. Second-harmonic generation (SHG) microscopy plus polarimetry (p-SHG) was used to estimate collagen content and describe the spatial orientation of fibrillar collagen in the intratumoral area. intraepithelial (ie) and stromal (s) CD3+ and CD8+ cells were quantified by Qupath. Non-parametric t-tests and Spearman’s rank coefficient (r) for linear correlation were used. Twenty-five tumor samples were included in the analysis: 17 from Rs and 8 from NRs. MS identified collagen type V alpha-2 (AVR Log ratio 2.9, p 0.008) and type XXII alpha-1 chains (AVR Log ratio 1.14, p 0.006) as significantly upregulated in NRs. Using spatial imaging, collagen density in the intratumoral area was significantly higher in NRs (median: 14.2% vs 4.6%, p 0.01). Intratumoral collagen density was positively correlated with collagen type V expression (r 0,54, p 0.016), and negatively correlated with ie and s CD3+ cell (r -0.39, p 0.054; r -0.4, p 0.048, respectively) and ie and s CD8+ cell (r -0.39, p 0.053; and r -0.29, p 0.166) infiltration. SHG confirmed that fibrillar collagen content in the tumor area was significantly greater in NRs (p 0.02). p-SHG revealed that NR tumors tended to show a highly disorganized collagen matrix. In contrast, a linear collagen pattern was associated with response to ICI: 89% of tumors displaying this pattern were Rs. Both MS and two different spatial approaches consistently demonstrated that increased collagen expression or density may serve as a potential predictive biomarker of resistance to ICI in MMRd metastatic EC, contributing to create an immunosuppressive TME. The spatial orientation pattern of collagen fibers may also be crucial in shaping immune TME and influencing the response to ICI. Citation Format: Juan Francisco Grau, Carole Aimé, Martin Mehnert, Elisa Yaniz-Galende, Catherine Genestie, Audrey Le Formal, Elodie Edmond, Antoine Amaury Lachaud, Patricia Pautier, Judith Michels, Sébastien Gouy, Amandine Maulard, Emeline Colomba-Blameble, Felix Blanc-Durand, Marie-Claire Schanne-Klein, Alexandra Leary. Extracellular matrix remodeling in metastatic mismatch repair deficient endometrial cancer:Implications for immune checkpoint inhibitor response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5149.

Abstract 720: Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer

Abstract Zelasudil (RXC007) is an orally available, highly selective small molecule inhibitor of Rho-associated coiled-coil containing protein kinase 2 (ROCK2), which has demonstrated pre-clinical anti-fibrotic efficacy and is currently being tested in a Phase 2a clinical trial for the treatment of idiopathic pulmonary fibrosis (IPF) (NCT05570058). Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and therapeutically challenging disease with a dense and highly fibrotic tumor microenvironment which may limit efficacy of conventional therapies. Here, we investigate the benefit of combining an anti-fibrotic, RXC007, with either standard of care chemotherapy or immunotherapy in mouse models of PDAC.In a fibrotic patient-derived metastatic PDAC orthotopic model, RXC007 (10mg/kg, 50mg/kg and 100mg/kg BID 5d on/2d off PO) in combination with SoC chemotherapy Gemcitabine (100mg/kg IP QW)/Abraxane (30 mg/kg IP QW) improved survival in a dose-dependent manner (all P&amp;lt;0.0001) compared to Gemcitabine/Abraxane alone.In the immunocompetent orthotopic LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) model of metastatic PDAC, survival was significantly increased (P&amp;lt;0.0001) upon treatment with a combination of RXC007 (50 mg/kg BID 5d on/2d off PO) and anti-PD1 (200 ug BIW) compared to anti-PD1 alone. ROCK2 inhibition elicited positive immunomodulatory effects in KPC pancreatic tumors, with increase of CD8+ (P=0.0031) and CD4+ (P=0.008) T cell infiltrate into the tumor cortex and reduction in immunosuppressive FOXP3+ regulatory T cells at the tumor border. Furthermore, RXC007 monotherapy exhibited anti-fibrotic effects, via a decrease in collagen content and organization, effects associated with CAF reprogramming and reduced intra-tumoral aSMA+ PDPN+ myofibroblast-like CAFs (myCAFs; P=0.0406).This data highlights the potential for treating highly fibrotic tumours with RXC007 in combination with SoC chemotherapy or immunotherapy in order to improve patient outcomes in this aggressive and treatment-refractory cancer. Citation Format: Marina Pajic, Benjamin McLean, Diego Chacon Fajardo, Sean Porazinski, Dannielle H. Upton, Diana Schuhmacher, Aji Istadi, Thomas Cox, Paul Timpson, Daniel J. Wilcock, Katie J. Anderson, Helen Mason, Nicolas E. Guisot, Clifford D. Jones, Caroline Phillips, Richard Armer. Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 720.

Development of Epigallocatechin 3-gallate-Loaded Hydrogel Nanocomposites for Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) is a chronic progressive disease associated with increased collagen deposition and TGF-β1 release. The current therapy and management have been a limited success due to low efficacy and adverse drug reactions. This study aimed to evaluate epigallocatechin 3-gallate (EGCG) encapsulated nanoparticles loaded mucoadhesive hydrogel nanocomposite (HNC) for OSF. Developed HNC formulations were evaluated for their permeation behaviour using in vitro as well as ex vivo studies, followed by evaluation of efficacy and safety by in vivo studies using areca nut extract-induced OSF in rats. The disease condition in OSF-induced rats was assessed by mouth-opening and biochemical markers. The optimized polymeric nanoparticles exhibited the required particle size (162.93 ± 13.81 nm), positive zeta potential (22.50 ± 2.94 mV) with better mucoadhesive strength (0.40 ± 0.002 N), and faster permeation due to interactions of the positively charged surface with the negatively charged buccal mucosal membrane. HNC significantly improved disease conditions by reducing TGF-β1 and collagen concentration without showing toxicity and reverting the fibroid buccal mucosa to normal. Hence, the optimized formulation can be further tested to develop a clinically alternate therapeutic strategy for OSF.Graphical