Extracellular Matrix Proteoglycans Research Articles

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Assessment of Vitamin D Supplementation on Articular Cartilage Morphology in a Young Healthy Sedentary Rat Model.

Deficiency in vitamin D (Vit D) has been widely associated with several musculoskeletal diseases. However, the effects of the exogenous Vit D supplementation are still unclear in the prevention of the latter, especially in the cartilage developmental period. The aim of this study was to compare the effects of Vit D supplementation and restriction on the articular cartilage development in healthy young sedentary rats. To this aim, twelve nine-week-old healthy Sprague–Dawley male rats were subjected to Vit D-based experimental diets: R, with a content in Vit D of 1400 IU/kg; R-DS, with a Vit D supplementation (4000 IU/kg); R-DR, with a Vit D restriction (0 IU/kg) for 10 weeks. The morphology, thickness and expression of cartilage-associated molecules such as collagen type II/X, lubricin and Vit D receptor (VDR), were assessed. Histological, histomorphometric and immunohistochemical evaluations were made on rat tibial cartilage samples. In the present experimental model, restriction of Vit D intake induced: The lower thickness of cartilage compared both to R (p = < 0.0001) and R-DS (p = < 0.0001); reduction of proteoglycans in the extracellular matrix (ECM) compared both to R (p = 0.0359) and R-DS (p = < 0.0001); decreased collagen II (Col II) with respect both to R (p = 0.0076) and R-DS (p = 0.0016); increased collagen X (Col X) immunoexpression when compared both to R (p = < 0.0001) and R-DS (p = < 0.0001), confirming data from the literature. Instead, supplementation of Vit D intake induced: Higher cartilage thickness with respect both to R (p = 0.0071) and R-DR (p = < 0.0001); increase of ECM proteoglycan deposition compared both to R (p = 0.0175) and R-DR (p = < 0.0001); higher immunoexpression of lubricin with respect both to R (p = 0.001) and R-DR (p = 0.0008). These results suggest that Vit D supplementation with diet, already after 10 weeks, has a favorable impact on the articular cartilage thickness development, joint lubrication and ECM fibers deposition in a young healthy rat model.

Exome-wide search and functional annotation of genes associated in patients with severe tick-borne encephalitis in a Russian population

BackgroundTick-borne encephalitis (TBE) is a viral infectious disease caused by tick-borne encephalitis virus (TBEV). TBEV infection is responsible for a variety of clinical manifestations ranging from mild fever to severe neurological illness. Genetic factors involved in the host response to TBEV that may potentially play a role in the severity of the disease are still poorly understood. In this study, using whole-exome sequencing, we aimed to identify genetic variants and genes associated with severe forms of TBE as well as biological pathways through which the identified variants may influence the severity of the disease.ResultsWhole-exome sequencing data analysis was performed on 22 Russian patients with severe forms of TBE and 17 Russian individuals from the control group. We identified 2407 candidate genes harboring rare, potentially pathogenic variants in exomes of patients with TBE and not containing any rare, potentially pathogenic variants in exomes of individuals from the control group. According to DAVID tool, this set of 2407 genes was enriched with genes involved in extracellular matrix proteoglycans pathway and genes encoding proteins located at the cell periphery. A total of 154 genes/proteins from these functional groups have been shown to be involved in protein-protein interactions (PPIs) with the known candidate genes/proteins extracted from TBEVHostDB database. By ranking these genes according to the number of rare harmful minor alleles, we identified two genes (MSR1 and LMO7), harboring five minor alleles, and three genes (FLNA, PALLD, PKD1) harboring four minor alleles.When considering genes harboring genetic variants associated with severe forms of TBE at the suggestive P-value < 0.01, 46 genes containing harmful variants were identified. Out of these 46 genes, eight (MAP4, WDFY4, ACTRT2, KLHL25, MAP2K3, MBD1, OR10J1, and OR2T34) were additionally found among genes containing rare pathogenic variants identified in patients with TBE; and five genes (WDFY4,ALK, MAP4, BNIPL, EPPK1) were found to encode proteins that are involved in PPIs with proteins encoded by genes from TBEVHostDB. Three genes out of five (MAP4, EPPK1, ALK) were found to encode proteins located at cell periphery.ConclusionsWhole-exome sequencing followed by systems biology approach enabled to identify eight candidate genes (MAP4, WDFY4, ACTRT2, KLHL25, MAP2K3, MBD1, OR10J1, and OR2T34) that can potentially determine predisposition to severe forms of TBE. Analyses of the genetic risk factors for severe forms of TBE revealed a significant enrichment with genes controlling extracellular matrix proteoglycans pathway as well as genes encoding components of cell periphery.

Decorin Expression in Human Vulva Carcinoma: Oncosuppressive Effect of Decorin cDNA Transduction on Carcinoma Cells.

The extracellular matrix proteoglycan decorin is well-known for its oncosuppressive activity. Here, decorin expression was examined in human vulva carcinoma tissue samples and in primary and commercial cell lines representing this malignant disease. Furthermore, the effect of adenovirus-mediated decorin cDNA (Ad-DCN) transduction on the viability, proliferation, and the expression and activity of the epidermal growth factor receptor (ErbB/HER) family members of the cell lines were investigated. Using in situ hybridization and immunohistochemistry for decorin, it was demonstrated that malignant cells in human vulva carcinoma tissues lack decorin expression. This result was true independently on tumor stage, grade or human papillomavirus status. RT-qPCR analyses showed that the human vulva carcinoma cell lines used in this study were also negative for decorin expression. Transduction of the cell lines with Ad-DCN caused a marked reduction in cell viability, while the proliferation of the cells was not affected. Experiments examining potential mechanisms behind the oncosuppressive effect of Ad-DCN transduction revealed that ErbB2/HER2 expression and activity in carcinoma cells were markedly downregulated. In conclusion, the results of this study showed that human vulva carcinoma cells lack decorin expression, and that Ad-DCN transduction of these cells induces oncosuppressive activity in part via downregulation of ErbB2/HER2.

LOXL2-A New Target in Antifibrogenic Therapy?

The concept of liver fibrosis and cirrhosis being static and therefore irreversible is outdated. Indeed, both human and animal studies have shown that fibrogenesis is a dynamic and potentially reversible process that can be modulated either by stopping its progression and/or by promoting its resolution. Therefore, the study of the molecular mechanisms involved in the pathogenesis of liver fibrosis is critical for the development of future antifibrotic therapies. The fibrogenesis process, common to all forms of liver injury, is characterized by the increased deposition of extracellular matrix components (EMCs), including collagen, proteoglycans, and glycoproteins (laminin and fibronectin 2). These changes in the composition of the extracellular matrix components alter their interaction with cell adhesion molecules, influencing the modulation of cell functions (growth, migration, and gene expression). Hepatic stellate cells and Kupffer cells (liver macrophages) are the key fibrogenic effectors. The antifibrogenic mechanism starts with the activation of Ly6Chigh macrophages, which can differentiate into macrophages with antifibrogenic action. The research of biochemical changes affecting fibrosis irreversibility has identified lysyl oxidase-like 2 (LOXL2), an enzyme that promotes the network of collagen fibers of the extracellular matrix. LOXL2 inhibition can decrease cell numbers, proliferation, colony formations, and cell growth, and it can induce cell cycle arrest and increase apoptosis. The development of a new humanized IgG4 monoclonal antibody against LOXL2 could open the window of a new antifibrogenic treatment. The current therapeutic target in patients with liver cirrhosis should focus (after the eradication of the causal agent) on the development of new antifibrogenic drugs. The development of these drugs must meet three premises: Patient safety, in non-cirrhotic phases, down-staging or at least stabilization and slowing the progression to cirrhosis must be achieved; whereas in the cirrhotic stage, the objective should be to reduce fibrosis and portal pressure.

Targeted siRNA Nanoparticles for Mammary Carcinoma Therapy.

Non-viral, polymeric-based, siRNA nanoparticles (NPs) have been proposed as promising gene delivery systems. Encapsulating siRNA in targeted NPs could confer improved biological stability, extended half-life, enhanced permeability, effective tumor accumulation, and therapy. In this work, a peptide derived from apolipoprotein B100 (ApoB-P), the protein moiety of low-density lipoprotein, was used to target siRNA-loaded PEGylated NPs to the extracellular matrix/proteoglycans (ECM/PGs) of a mammary carcinoma tumor. siRNA against osteopontin (siOPN), a protein involved in breast cancer development and progression, was encapsulated into PEGylated poly(d,l-lactic-co-glycolic acid) (PLGA) NPs using the double emulsion solvent diffusion technique. The NPs obtained possessed desired physicochemical properties including ~200 nm size, a neutral surface charge, and high siOPN loading of ~5 µg/mg. ApoB-P-targeted NPs exhibited both enhanced binding to isolated ECM and internalization by MDA-MB-231 human mammary carcinoma cells, in comparison to non-targeted NPs. Increased accumulation of the targeted NPs was achieved in the primary mammary tumor of mice xenografted with MDA-MB-231 mammary carcinoma cells as well as in the lungs, one of the main sites affected by metastases. siOPN NPs treatment resulted in significant inhibition of tumor growth (similar bioactivity of both formulations), accompanied with significant reduction of OPN mRNA levels (~40% knockdown of mRNA levels). We demonstrated that targeted NPs possessed enhanced tumor accumulation with increased therapeutic potential in mice models of mammary carcinoma.

Hypomorphic mutations of TRIP11 cause odontochondrodysplasia.

Odontochondrodysplasia (ODCD) is an unresolved genetic disorder of skeletal and dental development. Here, we show that ODCD is caused by hypomorphic TRIP11 mutations, and we identify ODCD as the nonlethal counterpart to achondrogenesis 1A (ACG1A), the known null phenotype in humans. TRIP11 encodes Golgi-associated microtubule-binding protein 210 (GMAP-210), an essential tether protein of the Golgi apparatus that physically interacts with intraflagellar transport 20 (IFT20), a component of the ciliary intraflagellar transport complex B. This association and extraskeletal disease manifestations in ODCD point to a cilium-dependent pathogenesis. However, our functional studies in patient-derived primary cells clearly support a Golgi-based disease mechanism. In spite of reduced abundance, residual GMAP variants maintain partial Golgi integrity, normal global protein secretion, and subcellular distribution of IFT20 in ODCD. These functions are lost when GMAP-210 is completely abrogated in ACG1A. However, a similar defect in chondrocyte maturation is observed in both disorders, which produces a cellular achondrogenesis phenotype of different severity, ensuing from aberrant glycan processing and impaired extracellular matrix proteoglycan secretion by the Golgi apparatus.

Efficient Synthesis of Heparinoid Bioconjugates for Tailoring FGF2 Activity at the Stem Cell-Matrix Interface.

Heparan sulfate glycosaminoglycans (HS GAGs) attached to proteoglycans harbor high affinity binding sites for various growth factors (GFs) and direct their organization and activity across the cell-matrix interface. Here, we describe a mild and efficient method for generating HS-protein conjugates. The two-step process utilizes a "copper-free click" coupling between differentially sulfated heparinoids primed at their reducing end with an azide handle and a bovine serum albumin protein modified with complementary cyclooctyne functionality. When adsorbed on tissue culture substrates, the glycoconjugates served as extracellular matrix proteoglycan models with the ability to sequester FGF2 and influence mesenchymal stem cell proliferation based on the structure of their HS GAG component.

Novel prognostic markers within the CD44-stromal ligand network in pancreatic cancer.

The dense stroma in pancreatic cancer tumours is rich in secreted extracellular matrix proteins and proteoglycans. Secreted hyaluronan, osteopontin and type IV collagen sustain oncogenic signalling by interactions with CD44s and its variant isoform CD44v6 on cancer cell membranes. Although well established in animal and in vitro models, this oncogenic CD44‐stromal ligand network is less explored in human cancer. Here, we use a pancreatic cancer tissue microarray from 69 primary tumours and 37 metastatic lymph nodes and demonstrate that high tumour cell expression of CD44s and, surprisingly, low stromal deposition of osteopontin correlate with poor survival independent of established prognostic factors for pancreatic cancer. High stromal expression of hyaluronan was a universal trait of both primary tumours and metastatic lymph nodes. However, hyaluronan species of different molecular mass are known to function differently in pancreatic cancer biology and immunohistochemistry cannot distinguish between them. Using gas‐phase electrophoretic molecular mobility analysis, we uncover a shift towards high molecular mass hyaluronan in pancreatic cancer tissue compared to normal pancreas and at a transcriptional level, we find that hyaluronan synthesising HAS2 correlates positively with CD44. The resulting prediction that high molecular mass hyaluronan would then correlate with poor survival in pancreatic cancer was confirmed in serum samples, where we demonstrate that hyaluronan >27 kDa measured before surgery is an independent predictor of postoperative survival. Our findings confirm the prognostic value of CD44 tissue expression and highlight osteopontin tissue expression and serum high molecular mass hyaluronan as novel prognostic markers in pancreatic cancer.

Diet-dependent function of the extracellular matrix proteoglycan Lumican in obesity and glucose homeostasis

ObjectiveExtracellular matrix remodeling is required for adipose expansion under increased caloric intake. In turn, inhibited expandability due to aberrant collagen deposition promotes insulin resistance and progression towards the metabolic syndrome. An emerging role for the small leucine-rich proteoglycan Lumican in metabolically driven nonalcoholic fatty liver disease sparks an interest in further understanding its role in diet-induced obesity and metabolic complications. MethodsWhole body ablation of Lumican (Lum−/−) gene and adeno-associated virus-mediated over-expression were used in combination with control or high fat diet to assess energy balance, glucose homeostasis as well as adipose tissue health and remodeling. ResultsLumican was found to be particularly enriched in the stromal cells isolated from murine gonadal white adipose tissue. Likewise murine and human visceral fat showed a robust increase in Lumican as compared to fat from the subcutaneous depot. Lumican null female mice exhibited moderately increased fat mass, decreased insulin sensitivity and increased liver triglycerides in a diet-dependent manner. These changes coincided with inflammation in adipose tissue and no overt effects in adipose expandability, i.e. adipocyte formation and hypertrophy. Lumican over-expression in visceral fat and liver resulted in improved insulin sensitivity and glucose clearance. ConclusionsThese data indicate that Lumican may represent a functional link between the extracellular matrix, glucose homeostasis, and features of the metabolic syndrome.

Ointment vehicles regulate the wound-healing process by modifying the hyaluronan-rich matrix.

Topical ointment consists of an active ingredient and vehicle, and the vehicle largely comprises the volume of the ointment. During the treatment of chronic wounds, such as pressure ulcers, the vehicle has been considered inactive, only serving as a carrier of the main pharmaceutical. However, recent reports have indicated that the vehicle has distinct clinical effects that depend on its physicochemical properties. Therefore, an understanding of the action mechanism of the ointment vehicle in wound tissue is necessary. In this study, we established a mouse model to analyze tissue reactions induced by the following ointment vehicles, an oil-in-water emulsion (EM) vehicle; a macrogol ointment (MO), which is a water-soluble, hydrophilic vehicle; and a MOs containing sucrose (MS). EM-treated wounds exhibited an inflammatory reaction characterized by tissue edema and thick granulation tissue; however, MO- and MS-treated wounds did not exhibit this reaction. Moreover, EM-treated wounds exhibited infiltration of inflammatory cells unlike MO-treated wounds. In contrast, the formation of collagenous tissue was dominantly observed in MO-treated wounds. Because the vehicle regulates the water environment of the wound, the water-holding extracellular matrix molecules, including hyaluronan (HA) and proteoglycan, were examined using immunohistochemical and biochemical methods. The versican G1 fragment, serum-derived HA-associated protein (SHAP) and HA (the VG1F-SHAP-HA) complex characteristically found in inflammatory conditions of pressure ulcers was found in EM-treated wounds. To histologically analyze the mechanism of action of the vehicle, we evaluated the ointment vehicle-wound tissue interface in an en bloc manner. Formation of the HA-containing complex was observed locally between the vehicle and wound surface. On the basis of these data, ointment vehicles regulate the wound-healing process through the formation of HA-rich extracellular matrices at the ointment-wound interface. This study provides a better understanding of the treatment of deep-pressure ulcers with focus on ointment vehicles.

Extracellular proteoglycan decorin maintains human hair follicle stem cells.

Hair follicle stem cells (HFSC) are localized in the bulge region of the hair follicle and play a role in producing hair. Recently, it has been shown that the number of HFSC decreases with age, which is thought to be a cause of senile alopecia. Therefore, maintaining HFSC may be key for the prevention of age-related hair loss, but the regulatory mechanisms of HFSC and the effects of aging on them are largely unknown. In general, stem cells are known to require regulatory factors in the pericellular microenvironment, termed the stem cell niche, to maintain their cell function. In this study, we focused on the extracellular matrix proteoglycan decorin (DCN) as a candidate factor for maintaining the human HFSC niche. Gene expression analysis showed that DCN was highly expressed in the bulge region. We observed decreases in DCN expression as well as the number of KRT15-positive HFSC with age. In vitro experiments with human plucked hair-derived HFSC revealed that HFSC lost their undifferentiated state with increasing passages, and prior to this change a decrease in DCN expression was observed. Furthermore, knockdown of DCN promoted HFSC differentiation. In contrast, when HFSC were cultured on DCN-coated plates, they showed an even more undifferentiated state. From these results, as a novel mechanism for maintaining HFSC, it was suggested that DCN functions as a stem cell niche component, and that the deficit of HFSC maintenance caused by a reduction in DCN expression could be a cause of age-related hair loss.

Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study

Endogenous vascular endothelial growth factor (VEGF-A) can protect retinal ganglion cells (RGC) from stress-induced cell death in ocular hypertensive glaucoma. To exploit the neuroprotective function of VEGF-A for therapeutic application in ocular disorders such as glaucoma while minimizing unwanted vascular side effects, we engineered two novel VEGF variants, eVEGF-38 and eVEGF-53. These variants of the diffusible VEGF-A isoform VEGF121 are expressed as dimeric concatamers and remain tethered to the cell membrane, thus restricting the effects of the engineered VEGF to the cells expressing the protein. For comparison, we tested a Myc-tagged version of VEGF189, an isoform that binds tightly to the extracellular matrix and heparan sulfate proteoglycans at the cell surface, supporting only autocrine and localized juxtacrine signaling. In human retinal endothelial cells (hREC), expression of eVEGF-38, eVEGF-53, or VEGF189 increased VEGFR2 phosphorylation without increasing expression of pro-inflammatory markers, relative to VEGF165 protein and vector controls. AAV2-mediated transduction of eVEGF-38, eVEGF-53, or VEGF189 into primary mouse RGC promoted synaptogenesis and increased the average total length of neurites and axons per RGC by ~ 12-fold, an increase that was mediated by VEGFR2 and PI3K/AKT signaling. Expression of eVEGF-38 in primary RGC enhanced expression of genes associated with neuritogenesis, axon outgrowth, axon guidance, and cell survival. Transduction of primary RGC with any of the membrane-associated VEGF constructs increased survival both under normal culture conditions and in the presence of the cytotoxic chemicals H2O2 (via VEGFR2/PI3K/AKT signaling) and N-methyl-d-aspartate (via reduced Ca2+ influx). Moreover, RGC number was increased in mouse embryonic stem cell-derived retinal organoid cultures transduced with the eVEGF-53 construct. The novel, engineered VEGF variants eVEGF-38 and eVEGF-53 show promise as potential therapeutics for retinal RGC neuroprotection when delivered using a gene therapy approach.

Metabolic reprogramming of murine cardiomyocytes during autophagy requires the extracellular nutrient sensor decorin

The extracellular matrix is a master regulator of tissue homeostasis in health and disease. Here we examined how the small, leucine-rich, extracellular matrix proteoglycan decorin regulates cardiomyocyte metabolism during fasting in vivo First, we validated in Dcn-/- mice that decorin plays an essential role in autophagy induced by fasting. High-throughput metabolomics analyses of cardiac tissue in Dcn-/- mice subjected to fasting revealed striking differences in the hexosamine biosynthetic pathway resulting in aberrant cardiac O-β-N-acetylglycosylation as compared with WT mice. Functionally, Dcn-/- mice maintained cardiac function at a level comparable with nonfasted animals whereas fasted WT mice showed reduced ejection fraction. Collectively, our results suggest that reduced sensing of nutrient deprivation in the absence of decorin preempts functional adjustments of cardiac output associated with metabolic reprogramming.

Disparate Remodeling of the Extracellular Matrix and Proteoglycans in Failing Pediatric Versus Adult Hearts.

BackgroundDilated cardiomyopathy (DCM) is a common cause of heart failure in adult and pediatric patients, but the underlying mechanism may vary in adults and children, with few studies conducted to date. The objective of the present study was to determine whether differential remodeling of the extracellular matrix contributes to the differences between pediatric and adult DCM hearts.Methods and ResultsExplanted hearts were procured from adult (age, 46–61 years) and pediatric (age, 2–8) patients with DCM‐related heart failure and nonfailing control hearts. Fibrillar and nonfibrillar extracellular matrix (proteoglycans, glycosaminoglycans, glycoprotein), their regulatory enzymes (matrix metalloproteinases, disintegrin and metalloproteinases, and disintegrin and metalloproteinases with a thrombospondin domain), and their inhibitors (tissue inhibitor of metalloproteinases) were assessed. Pediatric DCM hearts exhibited less fibrosis compared with adult DCMs. Total glycosaminoglycans increased similarly in both DCM groups but exhibited a significantly lower affinity for transforming growth factor‐β in adult DCMs versus pediatric DCMs, resulting in increased bioavailability of transforming growth factor‐β1 and a significantly higher activity of the Smad2/3 pathway in adult DCMs. Glycosylated biglycan and versican, and cleaved thrombospondin‐1 increased in both DCMs. Protein expression of disintegrin and metalloproteinases with thrombospondin domains (‐1, ‐2, ‐4, ‐7) and disintegrin and metalloproteinases (‐12, ‐15, ‐17, ‐19) were altered differently in pediatric and adult control and failing hearts. Total matrix metalloproteinase activity increased in both DCMs. Tissue inhibitor of metalloproteinase levels were altered similarly with heart failure in both age groups, and only tissue inhibitor of metalloproteinase 3 decreased in both DCM groups.ConclusionsDifferential remodeling of glycosaminoglycans in pediatric DCMs versus adult DCMs could underlie the enhanced activation of the transforming growth factor‐β pathway, leading to more fibrosis in adult DCM hearts. The distinct remodeling of the fibrillar and nonfibrillar extracellular matrix between pediatric and adult DCM hearts highlights a distinct pathophysiological basis for these cohorts.

The extracellular matrix proteoglycan fibromodulin is upregulated in clinical and experimental heart failure and affects cardiac remodeling.

Pressure overload of the heart leads to cardiac remodeling that may progress into heart failure, a common, morbid and mortal condition. Increased mechanistic insight into remodeling is instrumental for development of novel heart failure treatment. Cardiac remodeling comprises cardiomyocyte hypertrophic growth, extracellular matrix alterations including fibrosis, and inflammation. Fibromodulin is a small leucine-rich proteoglycan that regulates collagen fibrillogenesis. Fibromodulin is expressed in the cardiac extracellular matrix, however its role in the heart remains largely unknown. We investigated fibromodulin levels in myocardial biopsies from heart failure patients and mice, subjected fibromodulin knock-out (FMOD-KO) mice to pressure overload by aortic banding, and overexpressed fibromodulin in cultured cardiomyocytes and cardiac fibroblasts using adenovirus. Fibromodulin was 3-10-fold upregulated in hearts of heart failure patients and mice. Both cardiomyocytes and cardiac fibroblasts expressed fibromodulin, and its expression was increased by pro-inflammatory stimuli. Without stress, FMOD-KO mice showed no cardiac phenotype. Upon aortic banding, left ventricles of FMOD-KO mice developed mildly exacerbated hypertrophic remodeling compared to wild-type mice, with increased cardiomyocyte size and altered infiltration of leukocytes. There were no differences in mortality, left ventricle dilatation, dysfunction or expression of heart failure markers. Although collagen amount and cross-linking were comparable in FMOD-KO and wild-type, overexpression of fibromodulin in cardiac fibroblasts in vitro decreased their migratory capacity and expression of fibrosis-associated molecules, i.e. the collagen-cross linking enzyme lysyl oxidase, transglutaminase 2 and periostin. In conclusion, despite a robust fibromodulin upregulation in clinical and experimental heart failure, FMOD-KO mice showed a relatively mild hypertrophic phenotype. In cultured cardiac fibroblasts, fibromodulin has anti-fibrotic effects.

A complex interplay between the extracellular matrix and the innate immune response to microbial pathogens.

The role of the host extracellular matrix (ECM) in infection tends to be neglected. However, the complex interactions between invading pathogens, host tissues and immune cells occur in the context of the ECM. On the pathogen side, a variety of surface and secreted molecules, including microbial surface components recognizing adhesive matrix molecules and tissue-degrading enzymes, are employed that interact with different ECM proteins to effectively establish an infection at specific sites. Microbial pathogens can also hijack or misuse host proteolytic systems to modify the ECM, evade immune responses or process biologically active molecules such as cell surface receptors and cytokines that direct cell behaviour and immune defence. On the host side, the ECM composition and three-dimensional ultrastructure undergo significant modifications, which have a profound impact on the specific signals that the ECM conveys to immune cells at the forefront of infection. Unexpectedly, activated immune cells participate in the remodelling of the local ECM by synthesizing ECM glycoproteins, proteoglycans and collagen molecules. The close interplay between the ECM and the innate immune response to microbial pathogens ultimately affects the outcome of infection. This review explores and discusses recent data that implicate an active role for the ECM in the immune response to infection, encompassing antimicrobial activities, microbial recognition, macrophage activation, phagocytosis, leucocyte population balance, and transcriptional and post-transcriptional regulation of inflammatory networks, and may foster novel antimicrobial approaches.

Abstract 3146: Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer

Abstract Background: Infiltration of CD8+ T cells has shown important prognostic implications in colorectal cancers (CRC). Our group has recently correlated proteolysis of an extracellular matrix proteoglycan, versican (VCAN), with infiltration of CRCs by CD8+ T cells. Cleavage by ADAMTS proteases generates a bioactive fragment of versican, versikine (VKINE). VKINE stimulates Batf3-driven dendritic cell activation and maturation, which are critical for immunotherapy efficacy. Methods: CT26 murine CRC cells were transfected to constitutively express VKINE. BALB/c mice between 50 and 70 days of age were orthotopically injected with either 105 empty vector (CT26-EV) or VKINE-expressing (CT26-VKINE) cells. Tumor growth was tracked weekly with colonoscopy and growth rate assessed using percent lumen occlusion. Mice were euthanized and dissected once moribund. Tissues were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemical (IHC) staining was performed using standard procedures. Additionally, FFPE samples from human CRC liver metastases (n=9) were obtained under the University of Wisconsin Translational Science Biocore Institutional Review Board-approved protocol and IHC performed for VCAN, VKINE and CD8. The number of CD8+ tumor-infiltrating lymphocytes (TILs) per high-powered field (HPF) within the malignant epithelium was calculated using four separate 400x magnification fields of view. Results: CT26-EV and CT26-VKINE injected mice both had a ~70% tumor initiation rate and these tumors averaged ~50% lumen occlusion by 14 days post injection. Survival data for each group were compared using a log-rank test. A trend towards improved survival was observed in those mice injected with CT26-VKINE (average survival of 27 days for CT26-VKINE compared to 22 days for CT26-EV, p=0.41, n=13). The average number of CD8+ TILs for each tumor were compared. For mice with CT26-VKINE, the average number of CD8+ TILs per HPF was 7.6, compared to 4.7 for mice with CT26-EV (p=0.39). Human CRC liver metastases were analyzed for VCAN, VKINE, and CD8+ T cells. Several lesions had little to no VCAN staining, whereas others had intense pockets or peripheral staining. VKINE staining was more abundant in metastatic CRC lesions than in primary lesions. CD8+ T cells were excluded in the setting of increased VCAN staining and infiltration was noted specifically in areas of VCAN proteolysis (low VCAN and high VKINE staining). These areas of proteolysis were more often present at the tumor margin and correlated with ADAMTS1 expression. Conclusions: Immunotherapies offer a unique treatment option for cancer patients, but their use is limited in CRC. VCAN proteolysis and the generation of bioactive VKINE has potential to influence the ability of CD8+ T cells to infiltrate the tumor in both primary and metastatic CRC, indicating the potential to utilize VCAN and VKINE as immune biomarkers or therapeutic targets. Citation Format: Philip B. Emmerich, Susan N. Payne, Connor J. Maloney, Rosabella T. Pitera, Hanna Rainiero, Gioia Sha, Athanasios T. Papadas, Adam C. Pagenkopf, Michael F. Bassetti, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3146.

Aggrecan-like biomimetic proteoglycans (BPGs) composed of natural chondroitin sulfate bristles grafted onto a poly(acrylic acid) core for molecular engineering of the extracellular matrix

Biomimetic proteoglycans (BPGs) were designed to mimic the three-dimensional (3D) bottlebrush architecture of natural extracellular matrix (ECM) proteoglycans, such as aggrecan. BPGs were synthesized by grafting native chondroitin sulfate bristles onto a synthetic poly(acrylic acid) core to form BPGs at a molecular weight of approximately ∼1.6 MDa. The aggrecan mimics were characterized chemically, physically, and structurally, confirming the 3D bottlebrush architecture as well as a level of water uptake, which is greater than that of the natural proteoglycan, aggrecan. Aggrecan mimics were cytocompatible at physiological concentrations. Fluorescently labeled BPGs were injected into the nucleus pulposus of the intervertebral disc ex vivo and were retained in tissue before and after static loading and equilibrium conditioning. BPGs infiltrated the tissue, distributed and integrated with the ECM on a molecular scale, in the absence of a bolus, thus demonstrating a new molecular approach to tissue repair: molecular matrix engineering. Molecular matrix engineering may compliment or offer an acellular alternative to current regenerative medicine strategies. Statement of SignificanceAggrecan is a natural biomolecule that is essential for connective tissue hydration and mechanics. Aggrecan is composed of negatively charged chondroitin sulfate bristles attached to a protein core in a bottlebrush configuration. With age and degeneration, enzymatic degradation of aggrecan outpaces cellular synthesis resulting in a loss of this important molecule. We demonstrate a novel biomimetic molecule composed of natural chondroitin sulfate bristles grafted onto an enzymatically-resistant synthetic core. Our molecule mimics a 3D architecture and charge density of the natural aggrecan, can be delivered via a simple injection and is retained in tissue after equilibrium conditioning and loading. This novel material can serve as a platform for molecular repair, drug delivery and tissue engineering in regenerative medicine approaches.

IL-1 cytokines promote ADAMTS-mediated remodeling of the lung extracellular matrix during severe influenza A infection in mice

Abstract The lung extracellular matrix (ECM) is extensively remodeled following respiratory viral infection. This remodeling occurs during inflammation when immune cells migrate to the site of infection and during repair as the ECM is modified to regenerate damaged tissue. Matrix proteases, including the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) gene family, play a critical role in these remodeling processes. The immune mechanisms that regulate matrix protease activity and their effect on host responses to lung infection have not been well characterized. Previously, we found that ADAMTS-4 knockout mice exhibited improved survival following lethal influenza A (IAV) challenge and signs of reduced immunopathology, including fewer numbers of inflammatory T cells. Here, by flow cytometric sorting of immune and stromal cell populations, we identified fibroblasts as the main producers of ADAMTS-4 in the lung during severe IAV infection, with ~50-fold higher levels of mRNA compared to other lung cell populations. Stimulation of primary lung fibroblasts in vitro with a wide range of inflammatory cytokines demonstrated that only IL-1β and IL-1α induced expression of ADAMTS-4. Co-stimulation of lung fibroblasts with IL-1 cytokines and TNF-α resulted in a synergistic effect and an increase of ADAMTS-4 expression to greater than 6-fold above baseline levels. IL-1 treatment of lung fibroblasts also induced expression of the lung ECM proteoglycan versican (VCAN), a major enzymatic target of ADAMTS-4. These results suggest a role for IL-1 cytokines in regulating lung ECM remodeling, and highlight the potential of modulating the IL-1/ADAMTS-4 pathway to reduce immunopathology following severe respiratory infection.