Extracellular Matrix Remodeling Enzymes Research Articles

Comparative transcriptome analysis of bone marrow resident versus culture-expanded mouse mesenchymal stem/stromal cells

Background aimsMesenchymal stem/stromal cells (MSCs) are defined as culture-expanded populations, and although these cells recapitulate many properties of bone marrow (BM) resident skeletal stem/progenitor cells, few studies have directly compared these populations to evaluate how culture adaptation and expansion impact critical quality attributes. MethodsWe analyzed by RNA sequencing Lin–SCA1+ MSCs enriched from BM by immunodepletion (ID) and after subsequent culture expansion (Ex) and Lin−LEPR+ MSCs sorted (S) directly from BM. Pairwise comparisons were used to identify differentially expressed genes (DEGs) between populations, and gene set enrichment analysis was employed to identify biological pathways/processes unique to each population. K-means cluster analysis resolved isolation status-dependent changes in transcription in pseudotime. ResultsHierarchical clustering segregated populations by isolation process, and principal component analysis identified transcripts related to vasculature development, ossification and inflammatory/cytokine signaling as key drivers of population variance. Pairwise comparisons identified 3849 DEGs in ID versus S BM-MSCs mapping to Gene Ontology (GO) terms related to immune and metabolic processes and 334 DEGs in Ex versus ID BM-MSCs mapping to GO terms related to tissue development, cell growth and replication and organelle organization. K-means cluster analysis revealed significant differences in transcripts encoding stemness and differentiation markers, extracellular matrix structural constituents and remodeling enzymes and paracrine-acting factors between populations. ConclusionsThese comparative analyses reveal significant differences in gene expression signatures between BM resident and culture-expanded MSCs, thereby providing new insight into how culture adaptation/expansion endows the latter with unique quality attributes.

Cyclic di-AMP Rescues Porphyromonas gingivalis–Aggravated Atherosclerosis

Growing evidence demonstrates the relationship between periodontitis and atherosclerotic cardiovascular diseases. The periodontal pathogen Porphyromonas gingivalis (Pg) has been shown to contribute to the progression of atherosclerosis. Cyclic diadenylate monophosphate (c-di-AMP) has been widely studied as an immune adjuvant for tumor immunotherapy, given its ability to activate the stimulator of interferon genes (STING) and regulate trained immunity. This study sought to elucidate the role of c-di-AMP in Pg-associated atherosclerosis. Periodontitis and atherosclerosis mouse models were established by ligature application around maxillary second molars and feeding ApoE knockout mice with a high-fat diet. We found that periodontitis and atherosclerosis were more severe in mice exposed to Pg than mice that underwent ligature placement only, while prophylactic treatment with c-di-AMP activated trained immunity and elicited significant alleviation of alveolar bone resorption, as well as reduced blood lipid levels and atherosclerotic plaque accumulation. After 3 mo of intervention, c-di-AMP limited the elevation of cytokines interleukin (IL)–6, IL-1β, tumor necrosis factor α, and interferon β; extracellular matrix remodeling enzymes MMP-2 and MMP-9; and adhesion molecules ICAM-1 and VCAM-1 gene expression. The mechanism underlying Pg-aggravated atherosclerosis may be attributed to changes in microbiota composition in oral and aortic plaques and excess inflammatory response, whereas c-di-AMP could prevent the effects of Pg infection due to its potential ability to activate trained immunity and regulate microecological balance. Our findings suggest a positive role of c-di-AMP in alleviating Pg-aggravated atherosclerosis by regulating the immune response and influencing the local microenvironment.

Abstract SY17-03: Age-related changes in the tumor microenvironment drive tumor progression

Abstract Cancer is a disease of the aged. While the accumulation of cell autonomous mutations contributes to tumorigenesis, the central role age-related changes in the tumor microenvironment play in the transformation process is becoming more fully appreciated. Underscoring the importance of an aged microenvironment in cancer development are findings that senescent fibroblasts, which accumulate with age, directly stimulate preneoplastic and neoplastic cell growth and tumor progression. Investigations into how senescent fibroblasts promote tumorigenesis revealed that they express a plethora of growth factors, extracellular matrix remodeling enzymes, chemokines, and cytokines collectively referred to as the senescence associated secretory phenotype (SASP). To identify a role for senescent stromal cells in a spontaneous model of breast cancer, we used single cell RNA-Sequencing (scRNA-Seq) to interrogate the cell types and their functional status in the MMTV-PyMT breast tumor model. This analysis revealed the presence of senescent cancer associated fibroblasts (senCAFs) that we also identified in a human triple negative breast cancer scRNA-Seq database. To establish a role for senCAFs in breast cancer, we mated the PyMT model to the INKATTAC (INK) mouse that allowed us to inducibly eliminate senescent cells. We found that the elimination of senCAFs reduced tumor growth and unleashed the killing power of NK cells. We will discuss the mechanism by which senCAFs impact NK cell killing. Citation Format: Sheila A. Stewart. Age-related changes in the tumor microenvironment drive tumor progression. [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 SY17-03.

Characteristics of circulating small noncoding RNAs in plasma and serum during human aging.

Aging is a complicated process that triggers age-related disease susceptibility through intercellular communication in the microenvironment. While the classic secretome of senescence-associated secretory phenotype (SASP) including soluble factors, growth factors, and extracellular matrix remodeling enzymes are known to impact tissue homeostasis during the aging process, the effects of novel SASP components, extracellular small noncoding RNAs (sncRNAs), on human aging are not well established. Here, by utilizing 446 small RNA-seq samples from plasma and serum of healthy donors found in the Extracellular RNA (exRNA) Atlas data repository, we correlated linear and nonlinear features between circulating sncRNAs expression and age by the maximal information coefficient (MIC) relationship determination. Age predictors were generated by ensemble machine learning methods (Adaptive Boosting, Gradient Boosting, and Random Forest) and core age-related sncRNAs were determined through weighted coefficients in machine learning models. Functional investigation was performed via target prediction of age-related miRNAs. We observed the number of highly expressed transfer RNAs (tRNAs) and microRNAs (miRNAs) showed positive and negative associations with age respectively. Two-variable (sncRNA expression and individual age) relationships were detected by MIC and sncRNAs-based age predictors were established, resulting in a forecast performance where all R 2 values were greater than 0.96 and root-mean-square errors (RMSE) were less than 3.7 years in three ensemble machine learning methods. Furthermore, important age-related sncRNAs were identified based on modeling and the biological pathways of age-related miRNAs were characterized by their predicted targets, including multiple pathways in intercellular communication, cancer and immune regulation. In summary, this study provides valuable insights into circulating sncRNAs expression dynamics during human aging and may lead to advanced understanding of age-related sncRNAs functions with further elucidation.

Methods to Assess the Impact of Hsp90 Chaperone Function on Extracellular Client MMP2 Activity.

Secreted, or extracellular, heat shock protein 90 (eHsp90) is considered a recent discovery in eukaryotes. Over the last two decades, studies have provided significant supporting evidence that implicates eHsp90 both in normal cellular processes such as wound healing and in the development of human pathologies and diseases including fibrosis and cancer. In the early 2000s, Eustace et al. demonstrated that eHsp90 promotes the invasion of breast cancer cells by binding to and regulating the activity of an extracellular matrix (ECM) remodeling enzyme, the matrix metalloproteinase 2 or MMP2. Interestingly, inside mammalian cells, Hsp90 is an essential chaperone that interacts with hundreds of newly synthesized proteins, known as "clients," that require Hsp90's assistance to perform their function. Several methods are routinely used to characterize the role and impact of Hsp90 on a client protein's functionality in vitro and in vivo. However, the mechanistic role of eHsp90 is less well-defined since, so far, only a handful of extracellular client proteins have been identified. Here, we describe methods to characterize the impact of the secreted chaperone on MMP2 activity, the most characterized extracellular client of eHsp90. The procedures described here can be applied and adapted to characterize other extracellular clients, particularly members of the MMP family.

Distinct Expression Patterns of Genes Coding for Biological Response Modifiers Involved in Inflammatory Responses and Development of Fibrosis in Chronic Hepatitis C: Upregulation of SMAD-6 and MMP-8 and Downregulation of CAV-1, CTGF, CEBPB, PLG, TIMP-3, MMP-1, ITGA-1, ITGA-2 and LOX.

Background and Objectives: The aim of this study was to analyze the expression of genes on transcriptomic levels involved in inflammatory immune responses and the development of fibrosis in patients with chronic hepatitis C. Materials and Methods: Expression patterns of 84 selected genes were analyzed with real-time quantitative RT PCR arrays in the peripheral blood of treatment-naive patients with chronic hepatitis C and healthy controls. The panel included pro- and anti-fibrotic genes, genes coding for extracellular matrix (EMC) structural constituents and remodeling enzymes, cell adhesion molecules, inflammatory cytokines, chemokines and growth factors, signal transduction members of the transforming growth factor- beta (TGF-ß) superfamily, transcription factors, and genes involved in epithelial to mesenchymal transition. Results: The expression of SMAD-6 coding for a signal transduction TGF-beta superfamily member as well as MMP-8 coding for an ECM protein were significantly increased in CHC patients compared with controls. Conclusions: Chronic hepatitis C was also characterized by a significant downregulation of a set of genes including CAV-1, CTGF, TIMP-3, MMP-1, ITGA-1, LOX, ITGA-2, PLG and CEBPB encoding various biological response modifiers and transcription factors. Our results suggest that chronic hepatitis C is associated with distinct patterns of gene expression modulation in pathways associated with the regulation of immune responses and development of fibrosis.

A loss-of-function cysteine mutant in fibulin-3 (EFEMP1) forms aberrant extracellular disulfide-linked homodimers and alters extracellular matrix composition.

Fibulin-3 (F3 or EFEMP1) is a disulfide-rich, secreted glycoprotein necessary for maintaining extracellular matrix (ECM) and connective tissue integrity. Three studies have identified distinct autosomal recessive F3 mutations in individuals with Marfan Syndrome-like phenotypes. Herein, we characterize how one of these mutations, c.163T>C; p.Cys55Arg (C55R), disrupts F3 secretion, quaternary structure, and function by forming unique extracellular disulfide-linked homodimers. Dual cysteine mutants suggest that the C55R-induced disulfide species forms because of the new availability of Cys70 on adjacent F3 monomers. Surprisingly, mutation of single cysteines located near Cys55 (i.e., Cys29, Cys42, Cys48, Cys61, Cys70, Cys159, and Cys171) also produced similar extracellular disulfide-linked dimers, suggesting that this is not a phenomenon isolated to the C55R mutant. To assess C55R functionality, F3 knockout (KO) retinal pigmented epithelial (RPE) cells were generated, followed by reintroduction of wild-type (WT) or C55R F3. F3 KO cells produced lower levels of the ECM remodeling enzyme, matrix metalloproteinase 2, and reduced formation of collagen VI ECM filaments, both of which were partially rescued by WT F3 overexpression. However, C55R F3 was unable to compensate for these same ECM-related defects. Our results highlight the unique behavior of particular cysteine mutations in F3 and uncover potential routes to restore C55R F3 loss-of-function.

RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell–cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development

BackgroundSecreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations.Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes.ResultsCultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell–cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient’s keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control.ConclusionsAltogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

Cold exposure and capsaicin promote 1,2-dimethylhyrazine-induced colon carcinogenesis in rats correlates with extracellular matrix remodeling.

BACKGROUNDExtracellular matrix (ECM) remodeling and stiffening, which are correlated with tumor malignancy, drives tumor development. However, the relationship between ECM remodeling and rat experimental model of 1,2-dimethylhyrazine (DMH)-induced colorectal cancer (CRC) imposed by cold and capsaicin exposure remains unclear.AIMTo explore the effects of cold exposure and capsaicin on ECM remodeling and ECM enzymes in DMH-induced CRC.METHODSFor histopathological analysis, the sections of colon tissues were stained with hematoxylin and eosin, Masson’s trichrome, Picrosirius red, and Weigert’s Resorcin-Fuchsin to observe the remodeling of collagen and elastin. Additionally, the protein expression level of type I collagen (COL I), type 3 collagen (COL III0, elastin, matrix metalloproteinase (MMP) 1, MMP2, MMP9, and tissue-specific matrix metalloproteinase 1 (TIMP1) was assessed by immunohistochemistry. The messenger RNA (mRNA) levels of COL I, COL III, elastin, and lysyl oxidase-like-2 (LOXL2) in the colon tissues of rats was measured by reverse-transcriptase quantitative polymerase chain reaction.RESULTSAlthough no differences were observed in the proportion of adenomas, a trend towards the increase of invasive tumors was observed in the cold and capsaicin group. The cold exposure group had a metastasis rate compared with the other groups. Additionally, abnormal accumulation of both collagen and elastin was observed in the cold exposure and capsaicin group. Specifically, collagen quantitative analysis showed increased length, width, angle, and straightness compared with the DMH group. Collagen deposition and straightness were significantly increased in the cold exposure group compared with the capsaicin group. Cold exposure and capsaicin significantly increased the protein levels of COL I, elastin, and LOXL2 along with increases in their mRNA levels in the colon tissues compared with the DMH group, while COL III did not show a significant difference. Furthermore, in immunohistochemical evaluations, MMP1, MMP2, MMP9, and TIMP1 staining increased in the cold exposure and capsaicin group compared with the DMH group.CONCLUSIONThese results suggest that chronic cold and capsaicin exposure further increased the deposition of collagen and elastin in the colonic tissue. Increased COL I and elastin mRNA and protein levels expression may account for the enhanced ECM remodel and stiffness variations of colon tissue. The upregulated expression of the LOXL2 and physiological imbalance between MMP/TIMP activation and deactivation could contribute to the progression of the CRC resulting from cold and capsaicin exposure.

T Cells Promote Metastasis by Regulating Extracellular Matrix Remodeling following Chemotherapy.

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. SIGNIFICANCE: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis.See related commentary by Kolonin and Woodward, p. 197.

Extracellular Matrix: Emerging Roles and Potential Therapeutic Targets for Breast Cancer.

Increasing evidence shows that the extracellular matrix (ECM) is an important regulator of breast cancer (BC). The ECM comprises of highly variable and dynamic components. Compared with normal breast tissue under homeostasis, the ECM undergoes many changes in composition and organization during BC progression. Induced ECM proteins, including fibrinogen, fibronectin, hyaluronic acid, and matricellular proteins, have been identified as important components of BC metastatic cells in recent years. These proteins play major roles in BC progression, invasion, and metastasis. Importantly, several specific ECM molecules, receptors, and remodeling enzymes are involved in promoting resistance to therapeutic intervention. Additional analysis of these ECM proteins and their downstream signaling pathways may reveal promising therapeutic targets against BC. These potential drug targets may be combined with new nanoparticle technologies. This review summarizes recent advances in functional nanoparticles that target the ECM to treat BC. Accurate nanomaterials may offer a new approach to BC treatment.

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease.

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial–mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.

Collagen production and niche engineering: A novel strategy for cancer cells to survive acidosis in DCIS and evolve.

Growing tumors are dynamic and nonlinear ecosystems, wherein cancer cells adapt to their local microenvironment, and these adaptations further modify the environment, inducing more changes. From nascent intraductal neoplasms to disseminated metastatic disease, several levels of evolutionary adaptations and selections occur. Here, we focus on one example of such an adaptation mechanism, namely, “niche construction” promoted by adaptation to acidosis, which is a metabolic adaptation to the early harsh environment in intraductal neoplasms. The avascular characteristics of ductal carcinoma in situ (DCIS) make the periluminal volume profoundly acidic, and cancer cells must adapt to this to survive. Based on discovery proteomics, we hypothesized that a component of acid adaptation involves production of collagen by pre‐cancer cells that remodels the extracellular matrix (ECM) and stabilizes cells under acid stress. The proteomic data were surprising as collagen production and deposition are commonly believed to be the responsibility of mesenchymally derived fibroblasts, and not cells of epithelial origin. Subsequent experiments in 3D culture, spinning disk and second harmonic generation microscopy of DCIS lesions in patients’ samples are concordant. Collagen production assay by acid‐adapted cells in vitro demonstrated that the mechanism of induction involves the RAS and SMAD pathways. Secretome analyses show upregulation of ECM remodeling enzymes such as TGM2 and LOXL2 that are collagen crosslinkers. These data strongly indicate that acidosis in incipient cancers induces collagen production by cancer cells and support the hypothesis that this adaptation initiates a tumor‐permissive microenvironment promoting survival and growth of nascent cancers.

Abstract 1145: Assessing the diagnostic value of the combination of Matrix Metalloproteinase 2 (MMP2) and Progranulin (PGRN) preoperative plasma levels for colorectal cancer (CRC)

Abstract Introduction: MMP 2, also known as Gelatinase A, is an extracellular matrix remodeling enzymes. MMP-2 activity is regulated by various oncogenes, cytokines and growth factors. MMP 2 functions in the breakdown of extracellular matrix (ECM) and participates in tumor neoangiogensis. It is also associated with an angiogenic and metastatic tumor cell phenotype, in vivo. Up-regulation of MMP-2 aggravates the loss of basement membrane type IV collagen, breaks down ECM and promotes tumor progression and invasion. Progranulin (PGRN) is a ubiquitously expressed secreted glycoprotein that activates both the PI3k and ERK pathways, and promotes cyclin D1 and cyclin B expression which supports proliferation and survival of mesenchymal and epithelial origin cells. PGRN stimulates VEGF expression in breast cancer cells, in vitro, and stimulates inflammation, fibroblast accumulation and angiogenesis. Over-expression of MMP2 and PGRN has been reported in many cancers including CRC. The aim of this study was to compare preoperative (preop) plasma MMP2 and PGRN levels in CRC and benign colonic disease (BCD) patients (pts), alone and in combination, as a means of detecting CRC. Method: Preop plasma samples were obtained from consenting CRC and BCD pts undergoing elective resection for whom adequate preop plasma was available from an IRB approved tissue bank. Demographic, clinical, and pathologic data were collected. PreOp plasma levels of MMP2 and PGRN (ng/ml) were determined via ELISA in duplicate and reported as median + 95%CI. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to assess single or multiple plasma protein levels as a diagnostic tool for CRC. The Mann-Whitney test was used for statistical analysis (significance p<0.05). Results: A total of 172 CRC pts (70% colon, 30% rectal) and 102 BCD pts (adenoma 33%, diverticulitis 54%, other 15%) were eligible for the study. The CRC pathologic stage distributions were: Stage-I, 25%; Stage-2, 32%, Stage-3, 31%, Stage- 4, 12%. Median preop plasma protein levels in CRC pts were significantly higher than BCD pts. [MMP2; 205.3,CI 198.2,217.5 vs.165.3,CI 156.8,179.1: PGRN; 54.7,CI 51.8,57.1 vs.43.3,CI 40.1,46.8,P<0.001) The AUC value for ROC curve for MMP2 and PGRN were 0.721 and 0.724 respectively with 73% and 71% specificity. AUC for the combination of these 2 proteins was 0.757 with 97% specificity. Conclusion: Median MMP2 and PGRN levels were higher in CRC pts and the CRC median values were 24% and 26% higher than BCD median levels respectively. ROC curve analysis showed that the two protein combination improved specificity and AUC values. MMP2 and PGRN are potential candidates for a larger diagnostic panel to include other proteins found to be elevated in CRC pts. Further study is warranted. Citation Format: Chandana S K Herath Mudiyanselage, Neil Mitra, Dasuni Niyagama Gamage, Hiromichi Miyagaki, Abhinit Shah, Xiaohong Yan, Vesna Cekic, Richard L. Whelan. Assessing the diagnostic value of the combination of Matrix Metalloproteinase 2 (MMP2) and Progranulin (PGRN) preoperative plasma levels for colorectal cancer (CRC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1145.

Extracellular vesicles influence the pulmonary arterial extracellular matrix in congenital diaphragmatic hernia.

Abnormal pulmonary vasculature directly affects the development and progression of congenital diaphragmatic hernia (CDH)-associated pulmonary hypertension (PH). Though overarching structural and cellular changes in CDH-affected pulmonary arteries have been documented, the precise role of the extracellular matrix (ECM) in the pulmonary artery (PA) pathophysiology remains undefined. Here, we quantify the structural, compositional, and mechanical CDH-induced changes in the main and distal PA ECM and investigate the efficacy of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) as a therapy to ameliorate pathological vascular ECM changes. Pregnant Sprague-Dawley rodents were administered nitrofen to induce CDH-affected pulmonary vasculature in the offspring. A portion of CDH-affected pups was treated with intravenous infusion of MSC-EVs (1 × 1010 /mL) upon birth. A suite of histological, mechanical, and transmission electron microscopic analyses were utilized to characterize the PA ECM. The CDH model main PA presented significantly altered characteristics-including greater vessel thickness, greater lysyl oxidase (LOX) expression, and a relatively lower ultimate tensile strength of 13.6 MPa compared to control tissue (25.1 MPa), suggesting that CDH incurs ECM structural disorganization. MSC-EV treatment demonstrated the potential to reverse CDH-related changes, particularly through rapid inhibition of ECM remodeling enzymes (LOX and MMP-9). Additionally, MSC-EV treatment bolstered structural aspects of the PA ECM and mitigated pathological disorganization as exhibited by increased medial wall thickness and stiffness that, while not significantly altered, trends away from CDH-affected tissue. These data demonstrate notable ECM remodeling in the CDH pulmonary vasculature, along with the capacity of MSC-EVs to attenuate pathological ECM remodeling, identifying MSC-EVs as a potentially efficacious therapeutic for CDH-associated pulmonary hypertension.

Which Biological Properties of Heart Valves Are Relevant to Tissue Engineering?

Over the last 20 years, the designs of tissue engineered heart valves have evolved considerably. An initial focus on replicating the mechanical and structural features of semilunar valves has expanded to endeavors to mimic the biological behavior of heart valve cells as well. Studies on the biology of heart valves have shown that the function and durability of native valves is underpinned by complex interactions between the valve cells, the extracellular matrix, and the mechanical environment in which heart valves function. The ability of valve interstitial cells to synthesize extracellular matrix proteins and remodeling enzymes and the protective mediators released by endothelial cells are key factors in the homeostasis of valve function. The extracellular matrix provides the mechanical strength and flexibility required for the valve to function, as well as communicating with the cells that are bound within. There are a number of regulatory mechanisms that influence valve function, which include neuronal mechanisms and the tight regulation of growth and angiogenic factors. Together, studies into valve biology have provided a blueprint for what a tissue engineered valve would need to be capable of, in order to truly match the function of the native valve. This review addresses the biological functions of heart valve cells, in addition to the influence of the cells' environment on this behavior and examines how well these functions are addressed within the current strategies for tissue engineering heart valves in vitro, in vivo, and in situ.

The ECM path of senescence in aging: components and modifiers.

The extracellular matrix (ECM) is a key noncellular component in all organs and tissues. It is composed of a large number of proteins including collagens, glycoproteins (GP), and ECM-associated proteins, which show diversity of biochemical and biophysical functions. The ECM is dynamic both in normal physiology of tissues and under pathological conditions. One cellular phenomenon associated with changes in both ECM components expression and in ECM remodeling enzymes secretion is cellular senescence. It represents a stable state form of cell cycle arrest induced in proliferating cells by various forms of stress. Short-term induction of senescence is essential for tumor suppression and tissue repair. However, long-term presence of senescent cells in tissues may have a detrimental role in promoting tissue damage and aging. Up to date, there is insufficient knowledge about the interplay between the ECM and senescence cells. Since changes in the ECM occur in many physiological and pathological conditions in which senescent cells are present, a better understanding of ECM-senescence interactions is necessary. Here, we will review the functions of the different ECM components and will discuss the current knowledge about their regulation in senescent cells and their influence on the senescence state.

Effect of Laminin Derived Peptides IKVAV and LRE Tethered to Hyaluronic Acid on hiPSC Derived Neural Stem Cell Morphology, Attachment and Neurite Extension.

Low neural tissue extracellular matrix (ECM) content has led to the understudy of its effects on neural cells and tissue. Hyaluronic acid (HA) and laminin are major neural ECM components, but direct comparisons of their cellular effects could not be located in the literature. The current study uses human-induced pluripotent stem-cell-derived neural stem cells to assess the effects of HA, laminin, and HA with laminin-derived peptides IKVAV and LRE on cellular morphology, attachment, neurite extension and ECM remodeling. Increased attachment was observed on HA with and without IKVAV and LRE compared to laminin. Cellular morphology and neurite extension were similar on all surfaces. Using a direct binding inhibitor of Cav2.2 voltage gated calcium channel activity, a known binding partner of LRE, reduced attachment on HA with and without IKVAV and LRE and altered cellular morphology on surfaces with laminin or IKVAV and LRE. HA with IKVAV and LRE reduced the fluorescent intensity of fibronectin staining, but did not alter the localization of ECM remodeling enzymes matrix metalloprotease 2 and 9 staining compared to HA. Overall, the data indicate HA, IKVAV and LRE have complementary effects on human-induced pluripotent stem-cell-derived neural stem cell behavior.

PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling.

PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6-/- versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6-/- mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.

Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq

Adipose-derived stromal/stem cells (ASCs) are multipotent in nature that can be differentiated into various cells lineages such as adipogenic, osteogenic, and chondrogenic. The commitment of a cell to differentiate into a particular lineage is regulated by the interplay between various intracellular pathways and their resultant secretome. Similarly, the interactions of cells with the extracellular matrix (ECM) and the ECM bound growth factors instigate several signal transducing events that ultimately determine ASC differentiation. In this study, RNA-sequencing (RNA-Seq) was performed to identify the transcriptome profile of osteogenic induced ASCs to understand the associated genotype changes. Gene ontology (GO) functional annotations analysis using Database for Annotation Visualization and Integrated Discovery (DAVID) bioinformatics resources on the differentially expressed genes demonstrated the enrichment of pathways mainly associated with ECM organization and angiogenesis. We, therefore, studied the expression of genes coding for matrisome proteins (glycoproteins, collagens, proteoglycans, ECM-affiliated, regulators, and secreted factors) and ECM remodeling enzymes (MMPs, integrins, ADAMTSs) and the expression of angiogenic markers during the osteogenesis of ASCs. The upregulation of several pro-angiogenic ELR+ chemokines and other angiogenic inducers during osteogenesis indicates the potential role of the secretome from differentiating ASCs in the vascular development and its integration with the bone tissue. Furthermore, the increased expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 involved in the WNT, TGF-β, JNK, HedgeHog and ERK1/2 pathways suggests the regulation of osteogenesis through interplay between these pathways. The RNA-Seq data was also validated by performing QPCR on selected up- and down-regulated genes (COL10A1, COL11A1, FBLN, FERMT1, FN1, FOXF1, LAMA3, LAMA4, LAMB1, IGF1, WNT10B, MMP1, MMP3, MMP16, ADAMTS6, and ADAMTS14).