Extracellular Matrix Assembly Research Articles

Abstract PO2-25-07: PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer

Abstract Background: Breast cancer (BC) is the most common cancer diagnosed in women. In breast cancers, the extracellular matrix (ECM) is known to play a key role in disease progression by mediating cell signaling processes that drive cancer cell proliferation, migration and invasion. Work by our group identified that aligned collagen fibers perpendicular to the tumor boundary are prognostic of poor patient prognosis. Further, through matrix targeted proteomics we determined that the ECM protein, Fibronectin (FN), organizes with aligned collagen fibers in BC tissues. FN is known as the master regulator of ECM assembly because FN deposition precedes and regulates the deposition of several other ECM proteins. Clinical evidence shows that high expression of FN levels in BC patients correlates with an increased mortality risk. Due to the abundant expression of FN in the tumor microenvironment, FN has been a useful biomarker for cancer imaging and therapy. Despite multiple efforts in developing therapies that target the ECM, currently there are no effective ECM treatments available due to toxicity and lack of specificity. Methods: To target abnormal FN deposition, we used a FN binding peptide, Functional Upstream Domain (FUD), derived from the F1 adhesin from Streptococcus pyogenes. PEGylated-FUD (PEG-FUD) is a potent inhibitor of FN assembly which binds the 70 kDa N-terminal region of FN with high affinity. We hypothesize that PEG-FUD will localize to the tumor site and that targeted disruption of FN assembly with PEG-FUD will reduce ECM deposition, thus block tumor progression in-vivo. In this study, we used an in vivo imaging system to assess the biodistribution of 20-kDa PEG-FUD following subcutaneous injection of Cy5 labeled peptide in 4T1 mammary tumor bearing mice. Additionally, in a second therapeutic experiment, we treated 4T1 mammary tumor bearing mice with 20-kDa PEG-FUD every 48 hr for a total of 10 treatments and measured tumor volume as an indicator of primary tumor burden. Results: As anticipated, we observed PEG-FUD’s accumulation and localization in 4T1 tumors. Additionally, PEG-FUD treatment significantly reduced tumor growth compared to saline treatment. There were no observed changes in standard toxicity assessments such as body weight and spleen weight among treatment groups. After PEG-FUD’s therapeutic treatment, we observed a significant reduction in the deposition of FN matrix and an increase in cleaved caspase-3, a known marker for apoptosis by western blotting providing a potential mechanism of action of PEG-FUD inducing changes in tumor growth. Conclusions: PEG-FUD’s localization in tumors suggests its utility as a solo cancer imaging agent while providing indications that PEG-FUD can be used to deliver other therapeutics to the tumor site as a conjugate in the future. In addition, therapeutic treatment of PEG-FUD inhibited tumor growth providing preliminary evidence for PEG-FUD’s use as a tumor targeting anti-cancer agent. Citation Format: Metti Gari. PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-07.

The Tumor Stroma of Squamous Cell Carcinoma: A Complex Environment That Fuels Cancer Progression.

The tumor microenvironment (TME), a complex assembly of cellular and extracellular matrix (ECM) components, plays a crucial role in driving tumor progression, shaping treatment responses, and influencing metastasis. This narrative review focuses on the cutaneous squamous cell carcinoma (cSCC) tumor stroma, highlighting its key constituents and their dynamic contributions. We examine how significant changes within the cSCC ECM-specifically, alterations in fibronectin, hyaluronic acid, laminins, proteoglycans, and collagens-promote cancer progression, metastasis, and drug resistance. The cellular composition of the cSCC TME is also explored, detailing the intricate interplay of cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), endothelial cells, pericytes, adipocytes, and various immune cell populations. These diverse players modulate tumor development, angiogenesis, and immune responses. Finally, we emphasize the TME's potential as a therapeutic target. Emerging strategies discussed in this review include harnessing the immune system (adoptive cell transfer, checkpoint blockade), hindering tumor angiogenesis, disrupting CAF activity, and manipulating ECM components. These approaches underscore the vital role that deciphering TME interactions plays in advancing cSCC therapy. Further research illuminating these complex relationships will uncover new avenues for developing more effective treatments for cSCC.

The dual role of LOXL4 in the pathogenesis and development of human malignant tumors: a narrative review.

Lysyl oxidase-like protein 4 (LOXL4) is a secreted copper-dependent amine oxidase involved in the assembly and maintenance of extracellular matrix (ECM), playing a critical role in ECM formation and repair. Tumor-stroma interactions and ECM dysregulation are closely associated with the mechanisms underlying tumor initiation and progression. LOXL4 is the latest identified member of the lysyl oxidase (LOX) protein family. Currently, there is limited and controversial research on the role of LOXL4 in human malignancies. Its specific regulatory pathways, mechanisms, and roles in the occurrence, development, and treatment of malignancies remain incompletely understood. This article aims to illustrate the primary protein structure and the function of LOXL4 protein, and the relationship between LOXL4 protein and the occurrence and development of human malignant tumors to provide a reference for further clinical research. We searched the English literature on LOXL4 in the occurrence and development of various malignant tumors in PubMed and Web of Science. The search keywords include "cancer" "LOXL4" "malignant tumor" "tumorigenesis and development", etc. LOXL4 is up-regulated in human gastric cancer, breast cancer, ovarian cancer, head and neck squamous cell carcinoma, esophageal carcinoma and colorectal cancer, but down-regulated in human bladder cancer and lung cancer and inhibits tumor growth. There are two conflicting reports of both upregulation and downregulation in hepatocellular carcinoma, suggesting that LOXL4 has a bidirectional effect of promoting or inhibiting cancer in different types of human malignant tumors. We further explore the application prospect of LOXL4 protein in the study of malignant tumors, laying a theoretical foundation for the clinical diagnosis, treatment and screening of prognostic markers of malignant tumors. LOXL4 exerts a bidirectional regulatory role, either inhibiting or promoting tumors depending on the type of cancer. We still need more research to further confirm the molecular mechanism of LOXL4 in cancer progression.

Dipeptidyl Peptidase-4–Mediated Fibronectin Processing Evokes a Profibrotic Extracellular Matrix

Fibronectin serves as a platform to guide and facilitate deposition of collagen and fibrillin microfibrils. During development of fibrotic diseases, altered fibronectin deposition in the extracellular matrix (ECM) is generally an early event. Following this, dysregulated organization of fibrillins and fibrillar collagens occurs. Because fibronectin is an essential orchestrator of healthy ECM, perturbation of its ECM-organizational capacity may be involved in development of fibrosis. To investigate this, we employed recessive dystrophic epidermolysis bullosa (RDEB) as a disease model with progressive, severe dermal fibrosis. Fibroblasts from RDEB donors in 2D and 3D cultures displayed dysregulated fibronectin deposition. Our analyses revealed that increase of pro-fibrotic DPP4+ fibroblasts coincides with altered fibronectin deposition. DPP4 inhibitors normalized deposition of fibronectin and subsequently of fibrillin microfibrils and collagen I. Intriguingly, proteomics, inhibitor and mutagenesis studies disclosed that DPP4 modulates ECM deposition through proteolysis of the fibronectin N-terminus. Our study provides mechanistic insights to the observed pro-fibrotic activities of DPP4 and extends the understanding of fibronectin-guided ECM assembly in health and disease.

Integration of metabolic flux with hepatic glucagon signaling and gene expression profiles in the conscious dog.

Glucagon rapidly and profoundly stimulates hepatic glucose production (HGP), but for reasons that are unclear, this effect normally wanes after a few hours, despite sustained plasma glucagon levels. This study characterized the time course of glucagon-mediated molecular events and their relevance to metabolic flux in the livers of conscious dogs. Glucagon was either infused into the hepato-portal vein at a sixfold basal rate in the presence of somatostatin and basal insulin, or it was maintained at a basal level in control studies. In one control group, glucose remained at basal, whereas in the other, glucose was infused to match the hyperglycemia that occurred in the hyperglucagonemic group. Elevated glucagon caused a rapid (30 min) and largely sustained increase in hepatic cAMP over 4 h, a continued elevation in glucose-6-phosphate (G6P), and activation and deactivation of glycogen phosphorylase and synthase activities, respectively. Net hepatic glycogenolysis increased rapidly, peaking at 15 min due to activation of the cAMP/PKA pathway, then slowly returned to baseline over the next 3 h in line with allosteric inhibition by glucose and G6P. Glucagon's stimulatory effect on HGP was sustained relative to the hyperglycemic control group due to continued PKA activation. Hepatic gluconeogenic flux did not increase due to the lack of glucagon's effect on substrate supply to the liver. Global gene expression profiling highlighted glucagon-regulated activation of genes involved in cellular respiration, metabolic processes, and signaling, as well as downregulation of genes involved in extracellular matrix assembly and development.NEW & NOTEWORTHY Glucagon rapidly stimulates hepatic glucose production, but these effects are transient. This study links the molecular and metabolic flux changes that occur in the liver over time in response to a rise in glucagon, demonstrating the strength of the dog as a translational model to couple findings in small animals and humans. In addition, this study clarifies why the rapid effects of glucagon on liver glycogen metabolism are not sustained.

The Impact of 3D Nichoids and Matrix Stiffness on Primary Malignant Mesothelioma Cells.

Malignant mesothelioma is a type of cancer that affects the mesothelium. It is an aggressive and deadly form of cancer that is often caused by exposure to asbestos. At the molecular level, it is characterized by a low number of genetic mutations and high heterogeneity among patients. In this work, we analyzed the plasticity of gene expression of primary mesothelial cancer cells by comparing their properties on 2D versus 3D surfaces. First, we derived from primary human samples four independent primary cancer cells. Then, we used Nichoids, which are micro-engineered 3D substrates, as three-dimensional structures. Nichoids limit the dimension of adhering cells during expansion by counteracting cell migration between adjacent units of a substrate with their microarchitecture. Tumor cells grow effectively on Nichoids, where they show enhanced proliferation. We performed RNAseq analyses on all the samples and compared the gene expression pattern of Nichoid-grown tumor cells to that of cells grown in a 2D culture. The PCA analysis showed that 3D samples were more transcriptionally similar compared to the 2D ones. The 3D Nichoids induced a transcriptional remodeling that affected mainly genes involved in extracellular matrix assembly. Among these genes responsible for collagen formation, COL1A1 and COL5A1 exhibited elevated expression, suggesting changes in matrix stiffness. Overall, our data show that primary mesothelioma cells can be effectively expanded in Nichoids and that 3D growth affects the cells' tensegrity or the mechanical stability of their structure.

External Mechanical Stability Regulates Hematoma Vascularization in Bone Healing Rather than Endothelial YAP/TAZ Mechanotransduction.

Bone fracture healing is regulated by mechanobiological cues. Both, extracellular matrix (ECM) deposition and microvascular assembly determine the dynamics of the regenerative processes. Mechanical instability as by inter-fragmentary shear or compression is known to influence early ECM formation and wound healing. However, it remains unclear how these external cues shape subsequent ECM and microvascular network assembly. As transcriptional coactivators, the mechanotransducers yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) translate physical cues into downstream signaling events, yet their role in sprouting angiogenesis into the hematoma after injury is unknown. Using bone healing as model system for scar-free regeneration, the role of endothelial YAP/TAZ in combination with tuning the extrinsic mechanical stability via fracture fixation is investigated. Extrinsically imposed shear across the gap delayed hematoma remodeling and shaped the morphology of early collagen fiber orientations and microvascular networks, suggesting that enhanced shear increased the nutrient exchange in the hematoma. In contrast, endothelial YAP/TAZ deletion haslittle impact on the overall vascularization of the fracture gap, yet slightly increasesthe collagen fiber deposition under semi-rigid fixation. Together, these data provide novel insights into the respective roles of endothelial YAP/TAZ and extrinsic mechanical cues in orchestrating the process of bone regeneration.

Effect of Sparc knockout on the extracellular matrix of mouse periodontal ligament cells

The periodontal ligament (PDL) is a critical component in maintaining tooth stability. It is composed of cells and an extracellular matrix (ECM), each with unique roles in tissue function and homeostasis. Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, plays a crucial role in regulating ECM assembly and turnover, alongside facilitating cellular-ECM interactions. In the present study, mass spectrometry-based proteomics was used to assess the impacts of Sparc-knockout (KO) on PDL-derived cells. Results demonstrated that Sparc-KO significantly reduces ECM production and alters its composition with increased levels of type I collagen. Despite this increase in Sparc-KO, type I collagen was not likely to be effectively integrated into the fibrils due to collagen cross-linking impairment. Furthermore, the pathway and process enrichment analyses suggested that SPARC plays a protective role against ECM degradation by antagonistically interacting with cell-surface collagen receptors. These findings provide detailed insights into the multifaceted role of SPARC in ECM organization, including its impact on ECM production, collagen regulation, and interactions with various cellular compartments. A better understanding of these complex mechanisms is crucial for comprehending the causes of periodontal disease and tissue regeneration, where precise control of ECM organization is necessary.

The Upregulation of the FBLN1 and HS3ST4 Genes May Represent a Potential Mechanism Driving the Fibrotic Progression in Primary Myelofibrosis and Leading to Adverse Prognosis

Myelofibrosis is a group of diseases characterized by increased proliferation of bone marrow fibrous connective tissue and a decrease in hematopoietic cells, leading to impaired bone marrow hematopoietic function. Myelofibrosis includes primary myelofibrosis（PMF）, myelofibrosis secondary to polycythemia vera（post-PV MF）, and myelofibrosis secondary to essential thrombocythemia（post-ET MF）. Studies suggest that certain genes may be responsible for driving the development of myelofibrosis and causing adverse prognosis. By conducting RNA-Seq analysis, we investigated the differentially expressed genes and their impact on prognosis in primary myelofibrosis and secondary myelofibrosis. Furthermore, we analyzed the potential mechanisms driving the progression of fibrosis. Additionally, we performed a comparative analysis of the clinical features among various types of myelofibrosis. In this study, we performed transcriptome sequencing to analyze gene expression differences in 14 overt-PMF samples, 5 post-ET MF samples, and 3 post-PV MF samples.Functional and pathway analyses were conducted on these differentially expressed genes, and cluster heatmaps were used to visualize their expression patterns.Simultaneously, we performed a comparative analysis of clinical characteristics among 370 overt-PMF patients, 96 post-ET MF patients, and 52 post-PV MF patients.The study revealed that compared to post-PV MF, overt-PMF showed 81 upregulated and 2 downregulated differentially expressed genes, while compared to post-ET MF, overt-PMF displayed 19 upregulated and 3 downregulated differentially expressed genes (Figure a) (Figure b). Based on the similarity of gene expression profiles among the samples, a clustering analysis was performed (Figure c).The results demonstrated that in overt-PMF, the FBLN1 gene was significantly upregulated compared to post-PV MF and was enriched in pathways related to extracellular matrix formation and cell adhesion among the differentially expressed genes (Figure d). Additionally, the HS3ST4 gene was significantly upregulated in overt-PMF compared to post-ET MF and was enriched in pathways related to interleukin-8 generation and regulation of cytokine production involved in inflammatory responses.In the analysis of clinical characteristics among different types of myelofibrosis patients, it was observed that overt-PMF was more prone to transform into acute leukemia compared to post-PV MF, had a higher tendency to develop transfusion dependency (Figure e), and exhibited lower hemoglobin levels and hematocrit at the time of diagnosis (Figure f). Moreover, compared to post-ET MF, overt-PMF had a higher likelihood of transforming into acute leukemia and lower platelet counts at the time of diagnosis. The research findings indicate that the expressions of FBLN1 and HS3ST4 genes are significantly upregulated in overt-PMF, and patients with overt-PMF have a worse prognosis compared to those with post-PV MF and post-ET MF. Previous studies have shown that FBLN1 is involved in extracellular matrix assembly, promoting the formation and stabilization of collagen fibers. It can also interact with integrin receptors and other cell adhesion molecules, participating in cell-extracellular matrix signaling processes. On the other hand, HS3ST4 is involved in processes such as cell adhesion, cell migration, and cell signaling. Based on these findings, it is speculated that the upregulation of FBLN1 and HS3ST4 gene expressions may potentially drive the progression of fibrosis and lead to an adverse prognosis in patients with myelofibrosis. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. No. 79 Qingchun Road. Hangzhou, Zhejiang, PR China. househuang@zju.edu.cn

COVID-19 induces more pronounced extracellular matrix deposition than other causes of ARDS

BackgroundLung fibrosis is a major concern in severe COVID-19 patients undergoing mechanical ventilation (MV). Lung fibrosis frequency in post-COVID syndrome is highly variable and even if the risk is proportionally small, many patients could be affected. However, there is still no data on lung extracellular matrix (ECM) composition in severe COVID-19 and whether it is different from other aetiologies of ARDS.MethodsWe have quantified different ECM elements and TGF-β expression in lung tissue of 28 fatal COVID-19 cases and compared to 27 patients that died of other causes of ARDS, divided according to MV duration (up to six days or seven days or more). In COVID-19 cases, ECM elements were correlated with lung transcriptomics and cytokines profile.ResultsWe observed that COVID-19 cases presented significant increased deposition of collagen, fibronectin, versican, and TGF-β, and decreased decorin density when compared to non-COVID-19 cases of similar MV duration. TGF-β was precociously increased in COVID-19 patients with MV duration up to six days. Lung collagen was higher in women with COVID-19, with a transition of upregulated genes related to fibrillogenesis to collagen production and ECM disassembly along the MV course.ConclusionsFatal COVID-19 is associated with an early TGF-β expression lung environment after the MV onset, followed by a disordered ECM assembly. This uncontrolled process resulted in a prominent collagen deposition when compared to other causes of ARDS. Our data provides pathological substrates to better understand the high prevalence of pulmonary abnormalities in patients surviving COVID-19.

Human left ventricular cardiac fibroblasts undergo a dynamic shift in secretome and gene expression toward a cardiac myofibroblast phenotype during early passage in typical culture expansion conditions

Cardiac fibroblasts (CFs) are critical components of the cardiac niche and primarily responsible for assembly and maintenance of the cardiac extracellular matrix (ECM). CFs are increasingly of interest for tissue engineering and drug development applications, as they provide synergistic support to cardiomyocytes through direct cell-to-cell signaling and cell-to-ECM interactions via soluble factors, including cytokines, growth factors and extracellular vesicles. CFs can be activated to a cardiac myofibroblast (CMF) phenotype upon injury or stimulation with transforming growth factor beta 1. Once activated, CMFs assemble collagen-rich ECM, which is vitally important to stabilize scar formation following myocardial infarction, for example. Although there is greater experience with culture expansion of CFs among non-human strains, very little is known about human CF-to-CMF transitions and expression patterns during culture expansion. In this study, we evaluated for shifts in inflammatory and angiogenic expression profiles of human CFs in typical culture expansion conditions. Understanding shifts in cellular expression patterns during CF culture expansion is critically important to establish quality benchmarks and optimize large-scale manufacturing for future clinical applications.

Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we use antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS extracellular matrix, and regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Strontium-driven physiological to pathological transition of bone-like architecture: A dose-dependent investigation.

Whilst strontium (Sr2+) is widely investigated for treating osteoporosis, it is also related to mineralization disorders such as rickets and osteomalacia. In order to clarify the physiological and pathological effects of Sr2+ on bone biomineralization , we performed a dose-dependent investigation in bone components using a 3D scaffold that displays the hallmark features of bone tissue in terms of composition (osteoblast, collagen, carbonated apatite) and architecture (mineralized collagen fibrils hierarchically assembled into a twisted plywood geometry). As the level of Sr2+ is increased from physiological-like to excess, both the mineral and the collagen fibrils assembly are destabilized, leading to a drop in the Young modulus, with strong implications on pre-osteoblastic cell proliferation. Furthermore, the microstructural and mechanical changes reported here correlate with that observed in bone-weakening disorders induced by Sr2+ accumulation, which may clarify the paradoxical effects of Sr2+ in bone mineralization. More generally, our results provide physicochemical insights into the possible effects of inorganic ions on the assembly of bone extracellular matrix and may contribute to the design of safer therapies for treating osteoporosis. STATEMENT OF SIGNIFICANCE: Physiological-like (10% Sr2+) and excess accumulation-like (50% Sr2+) doses of Sr2+ are investigated in 3D biomimetic assemblies possessing the high degree of organization found in the extracellular of bone. Above the physiological dose, the organic and inorganic components of the bone-like scaffold are destabilized, resulting in impaired cellular activity, which correlates with bone-weakening disorders induced by Sr2+.

Unveiling extracellular matrix assembly: Insights and approaches through bioorthogonal chemistry.

Visualizing cells, tissues, and their components specifically without interference with cellular functions, such as biochemical reactions, and cellular viability remains important for biomedical researchers worldwide. For an improved understanding of disease progression, tissue formation during development, and tissue regeneration, labeling extracellular matrix (ECM) components secreted by cells persists is required. Bioorthogonal chemistry approaches offer solutions to visualizing and labeling ECM constituents without interfering with other chemical or biological events. Although biorthogonal chemistry has been studied extensively for several applications, this review summarizes the recent advancements in using biorthogonal chemistry specifically for metabolic labeling and visualization of ECM proteins and glycosaminoglycans that are secreted by cells and living tissues. Challenges, limitations, and future directions surrounding biorthogonal chemistry involved in the labeling of ECM components are discussed. Finally, potential solutions for improvements to biorthogonal chemical approaches are suggested. This would provide theoretical guidance for labeling and visualization of de novo proteins and polysaccharides present in ECM that are cell-secreted for example during tissue remodeling or in vitro differentiation of stem cells.

The proprotein convertase BLI-4 promotes collagen secretion prior to assembly of the Caenorhabditis elegans cuticle.

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

Carrageenan-Based Crowding and Confinement Combination Approach to Increase Collagen Deposition for In Vitro Tissue Development.

Connective tissue models grown from cell monolayers can be instrumental in a variety of biomedical fields such as drug screening, wound healing, and regenerative engineering. However, while connective tissues contain abundant fibrillar collagen, achieving a sufficient assembly and retention of fibrillar collagen in vitro is challenging. Unlike the dilute cell culture environment, the body's environment is characterized by a high density of soluble macromolecules (crowding) and macromolecular networks (confinement), which contribute to extracellular matrix (ECM) assembly in vivo. Consequently, macromolecular crowding (MMC) has been successfully used to enhance the processing of type I procollagen, leading to significant increases in fibrillar collagen assembly and accumulation during in vitro culture of a variety of cell types. In this study, we developed a combination approach using a carrageenan hydrogel, which released soluble macromolecules and served as a confinement barrier. We first evaluated the local carrageenan release and then confirmed the effectiveness of this combination approach on collagen accumulation by the human MG-63 bone cell line. Additionally, computational modeling of oxygen and glucose transport within the culture system showed no negative effects of the hydrogel and its releasates on cell viability.

Corneal Wound Healing in the Presence of Antifibrotic Antibody Targeting Collagen Fibrillogenesis: A Pilot Study.

Highly organized collagen fibrils interlacing with proteoglycans form the crucial architecture of the cornea and facilitate its transparency. Corneal scarring from accidental injury, surgery, or infection alters this highly organized tissue, causing severe consequences, including blindness. There are no pharmacological or surgical methods to effectively and safely treat excessive corneal scarring. Thus, we tested the anticorneal scarring utility of a rationally designed anticollagen antibody (ACA) whose antifibrotic effects have already been demonstrated in nonocular models. Utilizing a rabbit model with an incisional corneal wound, we analyzed ACA's effects on forming collagen and proteoglycan-rich extracellular matrices in scar neotissue. We used microscopic and spectroscopic techniques to quantify these components and measure crucial parameters characterizing the structure and organization of collagen fibrils. Moreover, we analyzed the spatial distribution of collagen and proteoglycans in normal and healing corneas. Our study demonstrated significant changes in the quality and quantity of the analyzed molecules synthesized in scar neotissue. It showed that these changes extend beyond incision margins. It also showed ACA's positive impact on some crucial parameters defining proper cornea structure. This pilot study provides a stepping stone for future tests of therapeutic approaches that target corneal extracellular scar matrix assembly.

Fibronectin-targeted FUD and PEGylated FUD peptides for fibrotic diseases.

Tissue fibrosis is characterized by excessive deposition of extracellular matrix (ECM) molecules. Fibronectin (FN) is a glycoprotein found in the blood and tissues, a key player in the assembly of ECM through interaction with cellular and extracellular components. Functional Upstream Domain (FUD), a peptide derived from an adhesin protein of bacteria, has a high binding affinity for the N-terminal 70-kDa domain of FN that plays a crucial role in FN polymerization. In this regard, FUD peptide has been characterized as a potent inhibitor of FN matrix assembly, reducing excessive ECM accumulation. Furthermore, PEGylated FUD was developed to prevent rapid elimination of FUD and enhance its systemic exposure in vivo. Herein, we summarize the development of FUD peptide as a potential anti-fibrotic agent and its application in experimental fibrotic diseases. In addition, we discuss how modification of the FUD peptide via PEGylation impacts pharmacokinetic profiles of the FUD peptide and can potentially contribute to anti-fibrosis therapy.

SEMA7a primes integrin α5β1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5β1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

Mutations F352A and Y528A in human HSP90α reduce fibronectin association and fibrillogenesis in cell-derived matrices.

HSP90 is a ubiquitously expressed chaperone protein that regulates the maturation of numerous substrate proteins called ‘clients’. The glycoprotein fibronectin (FN) is an important protein of the extracellular matrix (ECM) and a client protein of HSP90. FN and HSP90 interact directly, and the FN ECM is regulated by exogenous HSP90 or HSP90 inhibitors. Here, we extend the analysis of the HSP90 – FN interaction. The importance of the N-terminal 70-kDa fragment of fibronectin (FN70) and FN type I repeat was demonstrated by competition for FN binding between HSP90 and the functional upstream domain (FUD) of the Streptococcus pyogenes F1 adhesin protein. Furthermore, His-HSP90α mutations F352A and Y528A (alone and in combination) reduced the association with full-length FN (FN-FL) and FN70 in vitro. Unlike wild type His-HSP90α, these HSP90 mutants did not enhance FN matrix assembly in the Hs578T cell line model when added exogenously. Interestingly, the HSP90 E353A mutation, which did not significantly reduce the HSP90 – FN interaction in vitro, dramatically blocked FN matrix assembly in Hs578T cell-derived matrices. Taken together, these data extend our understanding of the role of HSP90 in FN fibrillogenesis and suggest that promotion of FN ECM assembly by HSP90 is not solely regulated by the affinity of the direct interaction between HSP90 and FN.