Changes In Extracellular Matrix Research Articles

Application of cell laden hydrogels with temporally tunable stiffness in biomedical research.

Extracellular matrix (ECM) is a dynamic and complex environment regulating the cell fate and behavior. It is characterized by biophysical and biochemical properties specific for each tissue. Interestingly, hydrogels can serve as exceptional artificial cellular microenvironments as they can be designed to mimic the key features of the native ECM. They are valuable tools to understand how cells respond to complex microenvironments in normal and pathologic conditions. However, unlike the highly dynamic structure of ECM, nearly all of the conventional hydrogel platforms are primarily static and lack the dynamic properties of native extracellular matrices. Thus, it is necessary to develop dynamic hydrogels to better understand the mechanisms by which dynamic changes of ECM contribute to biological processes. Stiffness is one of the significant dynamic components of ECM which must be appropriately mimicked over time in vitro. In this review, we cover recent advances in engineering strategies to make cell laden hydrogels with temporally tunable stiffness. We also highlight the applications of these hydrogel systems in biomedicine focusing on specific examples in cancer, cardiovascular system, tissue fibrosis and stem cell research. Finally, the challenges regarding the development and application of cell laden hydrogels with temporally tunable stiffness are proposed.

ECM Composition Differentially Regulates Intracellular and Extracellular pH in Normal and Cancer Pancreatic Duct Epithelial Cells

Intracellular pH (pHi) regulation is a challenge for the exocrine pancreas, where the luminal secretion of bicarbonate-rich fluid is accompanied by interstitial flows of acid. This acid–base transport requires a plethora of ion transporters, including bicarbonate transporters and the Na+/H+ exchanger isoform 1 (NHE1), which are dysregulated in Pancreatic Ductal Adenocarcinoma (PDAC). PDAC progression is favored by a Collagen-I rich extracellular matrix (ECM) which exacerbates the physiological interstitial acidosis. In organotypic cultures of normal human pancreatic cells (HPDE), parenchymal cancer cells (CPCs) and cancer stem cells (CSCs) growing on matrices reproducing ECM changes during progression, we studied resting pHi, the pHi response to fluxes of NaHCO3 and acidosis and the role of NHE1 in pHi regulation. Our findings show that: (i) on the physiological ECM, HPDE cells have the most alkaline pHi, followed by CSCs and CPCs, while a Collagen I-rich ECM reverses the acid–base balance in cancer cells compared to normal cells; (ii) both resting pHi and pHi recovery from an acid load are reduced by extracellular NaHCO3, especially in HPDE cells on a normal ECM; (iii) cancer cell NHE1 activity is less affected by NaHCO3. We conclude that ECM composition and the fluctuations of pHe cooperate to predispose pHi homeostasis towards the presence of NaHCO3 gradients similar to that expected in the tumor.

P-628 Prenatally androgenized PCOS-like mice present with dysregulated endometrium during window of implantation (WOI) and decidualization

Abstract Study question How do prenatal androgenization and excessive body weight impact endometrial preparation for implantation? Summary answer Prenatal androgenization rather than excessive body weight causes abnormal uterine function and gene expression during artificially induced window of implantation and decidualization events in vivo. What is known already Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women characterized by reproductive defects including irregular menstruation and pregnancy complications, but also by metabolic derangements such as obesity, insulin resistance and cardiovascular diseases. It is hypothesized that PCOS originates from prenatal androgenization (PNA), hence PNA mice models mimicking PCOS features have been generated. PNA has been shown to disrupt reproductive cycles, ovarian functioning and normal embryonic development; however, endometrial function in PNA mice is currently understudied. Therefore, this project aimed to study endometrial function during endometrial preparation for implantation in a PNA PCOS-like mouse model. Study design, size, duration Female PNA mice received a high-fat high-sugar (HFHS) diet to capture both reproductive and metabolic phenotypes of PCOS. Uteri were collected for histological and transcriptome analysis from 12-week-old ovariectomized PNA mice undergoing hormonal treatments to induce window of implantation (WOI) or decidualization states. Sample collection was done from minimum 6 animals per group from 4 different study groups depending on PNA status (PNA versus control) and diet (HFHS versus normal diet). Participants/materials, setting, methods PNA mice were generated by administrating 250 µg dihydrotestosterone daily to pregnant C57BL/6J dams during E16.5-E18.5. The female pups received a HFHS diet to induce obesity. Estrous cyclicity and anogenital distance were assessed to confirm PCOS phenotype. After ovariectomy, estrogen and progesterone injections were used to artificially induce WOI or decidualization states (intra-uterine injection of 25 µl oil was used for decidual trauma). The collected uteri were analyzed using RNA sequencing (RNAseq) and immunohistochemistry. Main results and the role of chance PNA mice showed increased anogenital distance and disrupted estrous cycles, confirming the PCOS-like phenotype. PNA, but not HFHS diet caused defective uterine luminal closure. PNA mice had altered gene expression during the WOI, including genes important for endometrial receptivity, genes associated with human PCOS, and genes related to reproduction failure. The HFHS diet induced a milder phenotype, i.e., enriched pathways related to mitochondrial functions, cell cycle and DNA damage response. Analysis of artificially decidualized uteri showed a severe morphological decidualization defect in PNA groups, as demonstrated by the reduced weights of the decidualized uterine horns, whereas HFHS diet did not have a significant additive effect. RNA sequencing data showed that PNA decidualized horns clustered together with non-decidualized control and PNA horns, irrespective of diet. Pathways enriched in PNA were associated with changes in extracellular matrix and ion homeostasis. HFHS diet with PNA further aggravated endometrial dysfunction, as shown by reduced prolactin expression, a classical marker for endometrial decidualization. Role of chance: while drastic effects of PNA on endometrial function are detectable, the sample size might be too small to detect more subtle morphological effects caused by the HFHS diet. Limitations, reasons for caution PNA mouse models display selective features of PCOS but cannot mirror the comprehensive PCOS phenotype due to the complexity and heterogeneity of the disorder. Nevertheless, this model provides insights into the impact of PNA and excessive body weight on endometrial physiology. Wider implications of the findings This in-dept investigation of PNA and overweight mice demonstrates the utility of the model for studying PCOS-related endometrial dysfunction and provides cues towards its pathophysiology. This report supports the hypothesis of prenatal androgen exposure as the major factor in the aetiology of PCOS-related subfertility, rather than metabolic status. Trial registration number not applicable

Effect of distinct ECM microenvironments on the genome-wide chromatin accessibility and gene expression responses of hepatic stellate cells

Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Cell microarrays demonstrated the cooperative effects of stiffness and ECM composition on H3K4 and H3K9 methylation/acetylation. ATAC sequencing revealed higher chromatin accessibility in HSCs on 1kPa compared to 25kPa substrates for all ECM conditions. Gene set enrichment analysis using RNA sequencing data of HSCs in defined ECM microenvironments demonstrated higher enrichment of NAFLD and fibrosis-related genes in pre-activated HSCs on 1kPa relative to 25kPa. Overall, these findings are indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs. Statement of SignificanceHepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Overall, these findings were indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs.

Fibrotic Matrix Induces Mesenchymal Transformation of Epithelial Cells in Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) is a potentially malignant disorder of the oral mucosa; however, whether and how the fibrotic matrix of OSF is involved in the malignant transformation of epithelial cells remains unknown. Herein, oral mucosa tissue from patients with OSF, OSF rat models, and their controls were used to observe the extracellular matrix changes and epithelial-mesenchymal transformation (EMT) in fibrotic lesions. Compared with controls, oral mucous tissues from patients with OSF showed an increased number of myofibroblasts, a decreased number of blood vessels, and increased type I and type III collagen levels. In addition, the oral mucous tissues from humans and OSF rats showed increased stiffness, accompanied by increased EMT activities of epithelial cells. The EMT activities of stiff construct-cultured epithelial cells were increased significantly by exogenous piezo-type mechanosensitive ion channel component 1 (Piezo1) activation, and decreased by yes-associated protein (YAP) inhibition. During exvivo implantation, oral mucosal epithelial cells of the stiff group showed increased EMT activities and increased levels of Piezo1 and YAP compared with those in the sham and soft groups. These results indicate that increased stiffness of the fibrotic matrix in OSF led to increased proliferation and EMT of mucosal epithelial cells, in which the Piezo1-YAP signal transduction is important.

Weaving the nest: extracellular matrix roles in pre-metastatic niche formation.

The discovery that primary tumors condition distant organ sites of future metastasis for seeding by disseminating tumor cells through a process described as the pre-metastatic niche (PMN) formation revolutionized our understanding of cancer progression and opened new avenues for therapeutic interventions. Given the inherent inefficiency of metastasis, PMN generation is crucial to ensure the survival of rare tumor cells in the otherwise hostile environments of metastatic organs. Early on, it was recognized that preparing the "soil" of the distal organ to support the outgrowth of metastatic cells is the initiating event in PMN development, achieved through the remodeling of the organ's extracellular matrix (ECM). Remote restructuring of ECM at future sites of metastasis under the influence of primary tumor-secreted factors is an iterative process orchestrated through the crosstalk between resident stromal cells, such as fibroblasts, epithelial and endothelial cells, and recruited innate immune cells. In this review, we will explore the ECM changes, cellular effectors, and the mechanisms of ECM remodeling throughout PMN progression, as well as its impact on shaping the PMN and ultimately promoting metastasis. Moreover, we highlight the clinical and translational implications of PMN ECM changes and opportunities for therapeutically targeting the ECM to hinder PMN formation.

Extracellular matrix mechanobiology in cancer cell migration.

The extracellular matrix (ECM) is pivotal in modulating tumor progression. Besides chemically stimulating tumor cells, it also offers physical support that orchestrates the sequence of events in the metastatic cascade upon dynamically modulating cell mechanosensation. Understanding this translation between matrix biophysical cues and intracellular signaling has led to rapid growth in the interdisciplinary field of cancer mechanobiology in the last decade. Substantial efforts have been made to develop novel in vitro tumor mimicking platforms to visualize and quantify the mechanical forces within the tissue that dictate tumor cell invasion and metastatic growth. This review highlights recent findings on tumor matrix biophysical cues such as fibrillar arrangement, crosslinking density, confinement, rigidity, topography, and non-linear mechanics and their implications on tumor cell behavior. We also emphasize how perturbations in these cues alter cellular mechanisms of mechanotransduction, consequently enhancing malignancy. Finally, we elucidate engineering techniques to individually emulate the mechanical properties of tumors that could help serve as toolkits for developing and testing ECM-targeted therapeutics on novel bioengineered tumor platforms. STATEMENT OF SIGNIFICANCE: Disrupted ECM mechanics is a driving force for transitioning incipient cells to life-threatening malignant variants. Understanding these ECM changes can be crucial as they may aid in developing several efficacious drugs that not only focus on inducing cytotoxic effects but also target specific matrix mechanical cues that support and enhance tumor invasiveness. Designing and implementing an optimal tumor mimic can allow us to predictively map biophysical cue-modulated cell behaviors and facilitate the design of improved lab-grown tumor models with accurately controlled structural features. This review focuses on the abnormal changes within the ECM during tumorigenesis and its implications on tumor cell-matrix mechanoreciprocity. Additionally, it accentuates engineering approaches to produce ECM features of varying levels of complexity which is critical for improving the efficiency of current engineered tumor tissue models.

Matrix disequilibrium in Alzheimer's disease and conditions that increase Alzheimer's disease risk.

Alzheimer's Disease (AD) and related dementias are a leading cause of death globally and are predicted to increase in prevalence. Despite this expected increase in the prevalence of AD, we have yet to elucidate the causality of the neurodegeneration observed in AD and we lack effective therapeutics to combat the progressive neuronal loss. Throughout the past 30 years, several non-mutually exclusive hypotheses have arisen to explain the causative pathologies in AD: amyloid cascade, hyper-phosphorylated tau accumulation, cholinergic loss, chronic neuroinflammation, oxidative stress, and mitochondrial and cerebrovascular dysfunction. Published studies in this field have also focused on changes in neuronal extracellular matrix (ECM), which is critical to synaptic formation, function, and stability. Two of the greatest non-modifiable risk factors for development of AD (aside from autosomal dominant familial AD gene mutations) are aging and APOE status, and two of the greatest modifiable risk factors for AD and related dementias are untreated major depressive disorder (MDD) and obesity. Indeed, the risk of developing AD doubles for every 5 years after ≥ 65, and the APOE4 allele increases AD risk with the greatest risk in homozygous APOE4 carriers. In this review, we will describe mechanisms by which excess ECM accumulation may contribute to AD pathology and discuss pathological ECM alterations that occur in AD as well as conditions that increase the AD risk. We will discuss the relationship of AD risk factors to chronic central nervous system and peripheral inflammation and detail ECM changes that may follow. In addition, we will discuss recent data our lab has obtained on ECM components and effectors in APOE4/4 and APOE3/3 expressing murine brain lysates, as well as human cerebrospinal fluid (CSF) samples from APOE3 and APOE4 expressing AD individuals. We will describe the principal molecules that function in ECM turnover as well as abnormalities in these molecular systems that have been observed in AD. Finally, we will communicate therapeutic interventions that have the potential to modulate ECM deposition and turnover in vivo.

Xeno-Free Biomimetic ECM Model for Investigation of Matrix Composition and Stiffness on Astrocyte Cell Response

Astrocytes, highly specialized glial cells, play a critical role in neuronal function. Variations in brain extracellular matrix (ECM) during development and disease can significantly alter astrocyte cell function. Age-related changes in ECM properties have been linked to neurodegenerative diseases such as Alzheimer's disease. The goal of this study was to develop hydrogel-based biomimetic ECM models with varying stiffness and evaluate the effects of ECM composition and stiffness on astrocyte cell response. Xeno-free ECM models were synthesized by combining varying ratios of human collagen and thiolated hyaluronic acid (HA) crosslinked with polyethylene glycol diacrylate. Results showed that modulating ECM composition yielded hydrogels with varying stiffnesses that match the stiffness of the native brain ECM. Collagen-rich hydrogels swell more and exhibit greater stability. Higher metabolic activity and greater cell spreading was observed in hydrogels with lower HA. Soft hydrogels trigger astrocyte activation indicated by greater cell spreading, high GFAP expression and low ALDH1L1 expression. This work presents a baseline ECM model to investigate the synergistic effects of ECM composition and stiffness on astrocytes, which could be further developed to identify key ECM biomarkers and formulate new therapies to alleviate the impact of ECM changes on the onset and progression of neurodegenerative diseases.

Abstract 575: Impact Of Resident Macrophages On Fibrosis And Lymphatic Growth During Autoimmune Valvular Carditis

Rheumatic diseases are associated with increased cardiovascular disease and the mitral valve (MV) is one of the tissues most commonly affected. Valve disease prevalence continues to rise yet the mechanisms driving autoimmune valvular carditis are incompletely understood, limiting treatment options. Our team has previously demonstrated a role for macrophages in promoting MV disease in the K/B.g7 mouse model of autoantibody-induced arthritis and valvular carditis. To further characterize the MV macrophage populations, we conducted single cell RNA sequencing of healthy (B.g7) and inflamed (K/B.g7) MVs. Analysis revealed multiple macrophage populations including Ccr2 + Bhlhe40 + recruited, pro-inflammatory macrophages and Mrc1 + Cd63 + resident macrophages, which we further validated by flow cytometry. These findings raised new questions regarding which subset(s) of macrophages influence disease progression and how. Within the resident population, we identified a subset of Lyve1 + Folr2 + macrophages. Immunostaining demonstrated that this macrophage subset localizes along the valve endothelium and near lymphatic vessels that we have recently demonstrated emerge in inflamed valves and promote valve disease. Importantly, we also found LYVE1+ macrophages co-localized with lymphatics in human MVs. Prior studies demonstrate that LYVE1+ macrophages are important regulators of vessel growth and extracellular matrix regulation. Thus, we hypothesized that LYVE1+ resident macrophages promote MV disease by augmenting lymphatic growth and contributing to changes in extracellular matrix. To understand the potential functions of these cells, we depleted Lyve1 -expressing macrophages in vivo by crossing Lyve1 -Cre mice with the Csf1r -floxed mice. We found that valves from LYVE1+ macrophage-depleted non-inflamed mice have increased collagen deposition, suggesting these macrophages are important for restraining extracellular matrix deposition. Studies are underway to determine if these resident macrophages promote fibrosis and lymphatic growth in the pro-inflammatory environment of the K/B.g7 model. Outcomes from these studies will have important implications for other cardiovascular and fibrotic diseases.

Histopathological Analysis of Cerebral Microvasculature Pathology with Alzheimer’s Disease Neuropathological Changes: An Autopsy Study (P3-6.007)

<h3>Objective:</h3> This study aimed to investigate the changes in the median tunica and basement membrane-related extracellular matrix (ECM) contents of the microvasculature and correlate this finding between the ADNC-impaired individuals and healthy controls. <h3>Background:</h3> Cerebrovascular lesions are associated with cognitive impairment. However, the implication of Alzheimer’s disease (AD) neuropathological changes (ADNC) on cerebral microvasculature is not completely understood. <h3>Design/Methods:</h3> In this study, 12 decedents with high or intermediate ADNC and 15 matched controls without ADNC were selected from a local brain bank. Tissue blocks were systematically collected from white matter regions of the cortex, putamen, and hippocampus. The proportions of small vessels affected by arteriolosclerosis and venular collagenosis, and the levels of collagen IV, laminin, fibronectin, perlecan, and agrin in the ECM were quantified by immunohistochemistry and compared between the two groups. <h3>Results:</h3> Venular collagenosis was significantly more severe in AD patients than in controls across all selected brain regions (<i>p</i> &lt; 0.001 for all regions). Although arteriolosclerosis was substantially severe in the AD group, only arteriolosclerosis in putamen was significantly more severe (0.63 vs. 0.42, <i>p</i> = 0.040). Similar correlation patterns were observed between these changes in the media tunica and specific AD pathology scores. We found that the levels of collagen IV and fibronectin were decreased and agrin was increased in AD cases, showing that changes in ECM components were significantly correlated with ADNC. <h3>Conclusions:</h3> Our data indicate that venular injuries with severe collagenosis in the media tunica and significant basement membrane-related ECM changes are important contributors to ADNC, providing potential new targets for investigation. <b>Disclosure:</b> Ms. Cao has nothing to disclose. Miss Huang has nothing to disclose. Dr. Mao has nothing to disclose. Dr. Wang has nothing to disclose. Dr. Xu has nothing to disclose. Dr. Qian has nothing to disclose. Dr. Ma has nothing to disclose. Dr. Qiu has nothing to disclose. Yi-Cheng Zhu has nothing to disclose.

P076 Beyond articular cartilage: a systematic review of the extracellular matrix in other components of the osteoarthritic joint

Abstract Background/Aims Traditionally viewed from the perspective of cartilage degeneration, osteoarthritis is increasingly seen as a disease of global joint dysfunction. Connective tissue extracellular matrix (ECM) is a crucial determinant of joint mechanobiology, providing cells with scaffolding, topographical cues, and a reservoir of soluble factors. While ECM dysregulation has been extensively studied in osteoarthritic cartilage, it remains poorly defined in other joint tissues. Here, we systematically review the composition, architecture, and remodelling of non-cartilage soft joint tissue ECM in human osteoarthritis and animal disease models. Methods A systematic search strategy was run through the MEDLINE, EMBASE and Scopus databases on 30 October 2020 and repeated on 1 October 2021. The search criteria included disease nomenclature, relevant tissues, as well as structural ECM components and architectural features. All papers were independently screened by two reviewers on the Covidence platform according to predefined eligibility criteria. Relevant clinical, demographic, and biological data were extracted from included studies, which were assessed for bias using the OHAT Risk of Bias Rating Tool for Human and Animal Studies. Results 148 of 8,156 identified studies met all eligibility criteria. 113 papers evaluated human osteoarthritis; of 35 animal studies, the most frequently used models involved surgical joint destabilisation in small mammals. ECM was best defined in menisci, ligaments, and synovium; fewer papers assessed skeletal muscles, tendons, and fat pads. Compared to the healthy joint, osteoarthritis is associated with qualitative and quantitative alterations in structural ECM components, most notably collagens and proteoglycans. In recent years, whole proteome sequencing has been employed to address these changes systematically. The mechanical properties of ECM change significantly in osteoarthritis in response to post-translational modifications, extensive calcification, and the marked loss of matrix organisation across the joint. Notably, some aspects of ECM remodelling in these tissues appear to precede discernible cartilage dysregulation. Similar ECM dysregulation is also observed in animal models, although inter-model variability in arthritogenic precipitant and the range of reported outcomes make comparisons difficult. Many studies are limited by significant bias, notably in the infrequent reporting of investigator blinding, and in the poor demographic matching of osteoarthritic and control patients. Encouragingly, the quality of methodology reporting and use of age-matched control populations have improved in recent years. Conclusion Current data provide compelling evidence of whole joint ECM changes in osteoarthritis and importantly suggest that these changes occur early in the disease process. How ECM dysfunction affects the behaviour of tissue-resident cells remains less well understood. Our work will support the design of disease-relevant biomaterials used to model osteoarthritis in vitro, helping to address this issue, by more accurately recreating the extracellular environment. Furthermore, the development of imaging modalities sensitive to connective tissue ECM changes warrants investigation from both diagnostic and prognostic perspectives. Disclosure J.Y. Mimpen: None. I.G.A. Raza: None. S.J.B. Snelling: None.

Heterogeneity and versatility of the extracellular matrix during the transition from pleomorphic adenoma to carcinoma ex pleomorphic adenoma: cumulative findings from basic research and new insights.

Pleomorphic adenoma (PA) is the most common salivary gland tumor, accounting for 50%-60% of these neoplasms. If untreated, 6.2% of PA may undergo malignant transformation to carcinoma ex-pleomorphic adenoma (CXPA). CXPA is a rare and aggressive malignant tumor, whose prevalence represents approximately 3%-6% of all salivary gland tumors. Although the pathogenesis of the PA-CXPA transition remains unclear, CXPA development requires the participation of cellular components and the tumor microenvironment for its progression. The extracellular matrix (ECM) comprises a heterogeneous and versatile network of macromolecules synthesized and secreted by embryonic cells. In the PA-CXPA sequence, ECM is formed by a variety of components including collagen, elastin, fibronectin, laminins, glycosaminoglycans, proteoglycans, and other glycoproteins, mainly secreted by epithelial cells, myoepithelial cells, cancer-associated fibroblasts, immune cells, and endothelial cells. Like in other tumors including breast cancer, ECM changes play an important role in the PA-CXPA sequence. This review summarizes what is currently known about the role of ECM during CXPA development.

2023 TERMIS – AMERICAS Conference &amp; Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference &amp; Exhibition Boston Marriott Copley Place April 11–14, 2023

Abstract 2363: Ovarian tumor-intrinsic FAK-dependent regulation of the matrisome

Abstract High-grade serous ovarian cancer (HGSOC) is deadliest of gynecological malignancies. Ovarian tumor cells can directly spread throughout the peritoneal space as single cells that grow into tumorspheres concomitant with the generation of malignant ascites that contains growth factors and extracellular matrix (ECM) proteins. Greater than 70% of advanced stage HGSOC patients exhibit gains or amplifications for the Focal Adhesion Kinase (FAK) gene, a tyrosine kinase that is activated by ECM proteins. In other solid tumors, changes in ECM and associated proteins (termed the Matrisome) are generated in part by tumor associated fibroblasts. Herein we compared transcriptional (RNA sequencing) and proteomic (mass spectrometry) changes using two ascites generating models: MOVCAR, a SV40 T antigen under control of the Mullerian inhibitory substance type II receptor; and KMF, a spontaneously aggressive ID8 derivative containing KRas, Myc, and FAK gene amplifications. FAK inactivation was generated by crossing MOVCAR with FAK floxed mice or CRISPR to disrupt FAK expression in KMF. Comparisons were made with FAK-null or cells re-expressing FAK wildtype or a FAK kinase-inactive point mutant (K454R) by lentiviral transduction with tumor cells grown as spheroids in vitro or harvested after intraperitoneal tumor growth in mice. FAK expression and activity were required for optimal tumor growth in mice and a common set of differentially expressed genes were identified upon FAK loss and re-expression. Gene ontology analyses revealed significant differences in Matrisome, morphogenesis, and epithelial proliferation within spheroids and tumors lacking FAK. Parallel protein mass spectrometry analyses revealed that thirty-four Matrisome proteins were down regulated (&amp;gt; log2 fold change) including collagens -1, -3 and -5, fibulin-1 and -2, elastin, tenascin, and fibronectin. Several of these proteins were regulated by FAK expression and not necessarily intrinsic FAK activity. Bioinformatic analysis of the TCGA ovarian tumor database showed that FAK, collagen-3 and collagen-5 expression negatively correlate with patient survival. As FAK is highly tyrosine phosphorylated in tumor spheroids isolated from patients and from our mouse models, our results support the hypothesis that elevated tumor-intrinsic FAK expression results in increased ECM production that can feed-forward to drive increased FAK activity. These results may impact the clinical testing of small molecule inhibitors of FAK activity. Citation Format: Marjaana Ojalill, Joanna Zhang, Ujjwal Suwal, Pekka Rappu, Jyrki Heino, Denise C. Connolly, Dwayne G. Stupack, David D. Schlaepfer. Ovarian tumor-intrinsic FAK-dependent regulation of the matrisome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2363.

Editorial Highlights

Every organism is a model organism for understanding development, evolution, disease, and regeneration, and we have only begun to scratch the surface of the interdisciplinary genetic, molecular, cellular, and developmental mechanisms that regulate these biological processes. These “Highlights” denote exciting advances recently reported in Developmental Dynamics that illustrate the complex dynamics of developmental biology. Ocular Development and Disease. “Zebrafish model of RERE syndrome recapitulates key ophthalmic defects that are rescued by small molecule inhibitor of shh signaling” by Aman George, Jerry Lee, James Liu, Suzie Kim and Brian Brooks; DevDyn 252:4, pp. 495-509; https://doi.org/10.1002/dvdy.561. Mutations in the human RERE gene (ATROPHIN 2, OMIM *605226) are associated with a neuro-developmental disorder that includes ophthalmic, cardiac, genitourinary and hearing defects. This study demonstrates that rerea mutant (babyface) zebrafish recapitulate the optic fissure closure defects that result from loss of RERE function in humans. Interestingly, the RERE gene acts as a transcriptional corepressor in various nuclear receptor signaling pathways, and the optic fissure phenotype is associated with expansion of proximal retinal optic stalk in concert with deregulated shh signaling. Consistent with this model, inhibiting shh signaling can rescue the coloboma phenotype. Craniofacial Biology. “Frem1 activity is regulated by Sonic Hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis” by Matthew McLaughlin, Miranda Sun, Tyler Beames, Austin Steward, Joshua Theisen, Hannah Chung, Joshua Everson, Ivan Moskowitz, Michael Sheets and Robert Lipinski; DevDyn 252:4, pp. 483-494; https://doi.org/10.1002/dvdy.555. The human face exhibits remarkable variation, and is also highly prone to developmental anomalies. The Frem/Fras family of extracellular matrix proteins has been implicated in regulating face shape variation as well as being mutated in the pathogenesis of disorders such as Manitoba-oculo-tricho-anal bifid nose with or without anorectal and renal anomalies, and Fraser syndromes. This study examined the activity of Frem1 during facial morphogenesis and identified Sonic Hedgehog (Shh) as an upstream regulator that promotes the proliferation of neural crest cells. Shh-Frem1 signaling is disrupted during the pathogenesis of midface hypoplasia, which provides deeper insights into the mechanisms underlying congenital craniofacial anomalies, and variation in midfacial morphogenesis. Organogenesis. “Foxd1 is required for 3D patterning of the kidney interstitial matrix by Sarah Lipp, Kathryn Jacobson, Andrew Schwaderer, David Hains and Sarah Calve; DevDyn 252:4, pp. 463-482; https://doi.org/10.1002/dvdy.545. The extracellular matrix (ECM) comprises a complex network of proteins and glycosaminoglycans that functions as a reservoir of growth factors, while also providing structural integrity and support during kidney development. This study combined 3D imaging and proteomics to investigate the role of Foxd1 in stromal cells, which are responsible for synthesizing the interstitial extracellular matrix, and document how the extracellular matrix dynamically changes during normal murine kidney development and in a model of kidney hypodysplasia. Changes in interstitial extracellular matrix corresponded to disruptions in stromal cell patterning, which helps to shed light on why human patients with connective tissue disorders have an increased incidence of kidney cysts and congenital anomalies of the kidney and urinary tract.

1 H - 13 C cross - polarization kinetics to probe hydration - dependent organic components of bone ECM.

Bone is a living tissue made up of organic proteins, inorganic minerals and water. The organic component of bone (mainly made up of Type-I collagen) provides flexibility and tensile strength. Solid - state nuclear magnetic resonance (ssNMR) is one of the few techniques which can provide atomic - level structural insights of such biomaterials in their native state. In the present article, we employed the variable contact time cross-polarization (1 H - 13 C CP) kinetics experiments to study the hydration - dependent atomic - level structural changes in the bone extracellular matrix (ECM). The natural abundant 13 C CP intensity of the bone ECM is measured by varying CP contact time and best fitted to the non - classical kinetic model. Different relaxation parameters were measured by the best - fit equation corresponding to the different hydration conditions of the bone ECM. The associated changes in the measured parameters due to varying levels of hydration observed at different sites of collagen protein have provided its structural arrangements and interaction with water molecules in bone ECM. Overall, the present study reveals a better understanding of the kinetics of the organic part inside the bone ECM that will help in comprehending the disease - associated pathways.

Extracellular Matrix Changes in Subcellular Brain Fractions and Cerebrospinal Fluid of Alzheimer's Disease Patients.

The brain's extracellular matrix (ECM) is assumed to undergo rearrangements in Alzheimer's disease (AD). Here, we investigated changes of key components of the hyaluronan-based ECM in independent samples of post-mortem brains (N = 19), cerebrospinal fluids (CSF; N = 70), and RNAseq data (N = 107; from The Aging, Dementia and TBI Study) of AD patients and non-demented controls. Group comparisons and correlation analyses of major ECM components in soluble and synaptosomal fractions from frontal, temporal cortex, and hippocampus of control, low-grade, and high-grade AD brains revealed a reduction in brevican in temporal cortex soluble and frontal cortex synaptosomal fractions in AD. In contrast, neurocan, aggrecan and the link protein HAPLN1 were up-regulated in soluble cortical fractions. In comparison, RNAseq data showed no correlation between aggrecan and brevican expression levels and Braak or CERAD stages, but for hippocampal expression of HAPLN1, neurocan and the brevican-interaction partner tenascin-R negative correlations with Braak stages were detected. CSF levels of brevican and neurocan in patients positively correlated with age, total tau, p-Tau, neurofilament-L and Aβ1-40. Negative correlations were detected with the Aβ ratio and the IgG index. Altogether, our study reveals spatially segregated molecular rearrangements of the ECM in AD brains at RNA or protein levels, which may contribute to the pathogenic process.

Reprogramming of VEGF-mediated extracellular matrix changes through autocrine signaling

ABSTRACT Vascular endothelial growth factor (VEGF) plays key roles in angiogenesis, vasculogenesis, and wound healing. In cancers, including triple negative breast cancer (TNBC), VEGF has been associated with increased invasion and metastasis, processes that require cancer cells to traverse through the extracellular matrix (ECM) and establish angiogenesis at distant sites. To further understand the role of VEGF in modifying the ECM, we characterized VEGF-mediated changes in the ECM of tumors derived from TNBC MDA-MB-231 cells engineered to overexpress VEGF. We established that increased VEGF expression by these cells resulted in tumors with reduced collagen 1 (Col1) fibers, fibronectin, and hyaluronan. Molecular characterization of tumors identified an increase of MMP1, uPAR, and LOX, and a decrease of MMP2, and ADAMTS1. α-SMA, a marker of cancer associated fibroblasts (CAFs), increased, and FAP-α, a marker of a subset of CAFs associated with immune suppression, decreased with VEGF overexpression. Analysis of human data from The Cancer Genome Atlas Program confirmed mRNA differences for several molecules when comparing TNBC with high and low VEGF expression. We additionally characterized enzymatic changes induced by VEGF overexpression in three different cancer cell lines that clearly identified autocrine-mediated changes, specifically uPAR, in these enzymes. Unlike the increase of Col1 fibers and fibronectin mediated by VEGF during wound healing, in the TNBC model, VEGF significantly reduced key protein components of the ECM. These results further expand our understanding of the role of VEGF in cancer progression and identify potential ECM-related targets to disrupt this progression.

Proposed Mechanism of Long-Term Intraocular Pressure Lowering With the Bimatoprost Implant.

The purpose of this study was to evaluate the effects of pharmacologically relevant bimatoprost and bimatoprost free acid (BFA) concentrations on matrix metalloproteinase (MMP) gene expression in cells from human aqueous outflow tissues. MMP gene expression by human trabecular meshwork (TM), scleral fibroblast (SF), and ciliary muscle (CM) cells exposed to 10 to 1000 µM bimatoprost or 0.1 to 10 µM BFA (intraocular concentrations after intracameral bimatoprost implant and topical bimatoprost dosing, respectively) was measured by polymerase chain reaction array. Bimatoprost dose-dependently upregulated MMP1 and MMP14 mRNA in all cell types and MMP10 and MMP11 mRNA in TM and CM cells; in TM cells from normal eyes, mean MMP1 mRNA levels were 62.9-fold control levels at 1000 µM bimatoprost. BFA upregulated MMP1 mRNA only in TM and SF cells, to two- to three-fold control levels. The largest changes in extracellular matrix (ECM)-related gene expression by TM cells derived from normal (n = 6) or primary open-angle glaucoma (n = 3) eyes occurred with 1000 µM bimatoprost (statistically significant, ≥50% change for 9-11 of 84 genes on the array, versus 1 gene with 10 µM BFA). Bimatoprost and BFA had differential effects on MMP/ECM gene expression. Dramatic upregulation in MMP1 and downregulation of fibronectin, which occurred only with bimatoprost at high concentrations observed in bimatoprost implant-treated eyes, may promote sustained outflow tissue remodeling and long-term intraocular pressure reduction beyond the duration of intraocular drug bioavailability. Variability in bimatoprost-stimulated MMP upregulation among cell strains from different donors may help explain differential long-term responses of patients to bimatoprost implant.