Extracellular Matrix Proteases Research Articles

Inhibiting MMP13 Attenuates Deep Vein Thrombosis in a Mouse Model by Reducing the Expression of Pdpn.

Matrix metalloproteinase 13 (MMP13) is an extracellular matrix protease that affects the progression of atherosclerotic plaques and arterial thrombi by degrading collagens, modifying protein structures and regulating inflammatory responses, but its role in deep vein thrombosis (DVT) has not been determined. The purpose of this study was to investigate the potential effects of MMP13 and MMP13-related genes on the formation of DVT. We altered the expression level of MMP13 in vivo and conducted a transcriptome study to examine the expression and relationship between MMP13 and MMP13-related genes in a mouse model of DVT. After screening genes possibly related to MMP13 in DVT mice, the expression levels of candidate genes in human umbilical vein endothelial cells (HUVECs) and the venous wall were evaluated. The effect of MMP13 on platelet aggregation in HUVECs was investigated in vitro. Among the differentially expressed genes, interleukin 1 beta, podoplanin (Pdpn), and factor VIII von Willebrand factor (F8VWF) were selected for analysis in mice. When MMP13 was inhibited, the expression level of PDPN decreased significantly in vitro. In HUVECs, overexpression of MMP13 led to an increase in the expression level of PDPN and induced platelet aggregation, while transfection of PDPN-siRNA weakened the ability of MMP13 to increase platelet aggregation. Inhibiting the expression of MMP13 could reduce the burden of DVT in mice. The mechanism involves downregulating the expression of Pdpn through MMP13, which could provide a novel gene target for DVT diagnosis and treatment.

Extracellular matrix abnormalities in the hippocampus of subjects with substance use disorder

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.

Molecular profiling of the hippocampus of children with autism spectrum disorder.

Several lines of evidence point to a key role of the hippocampus in Autism Spectrum Disorders (ASD). Altered hippocampal volume and deficits in memory for person and emotion related stimuli have been reported, along with enhanced ability for declarative memories. Mouse models have demonstrated a critical role of the hippocampus in social memory dysfunction, associated with ASD, together with decreased synaptic plasticity. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix molecules, represent a potential key link between neurodevelopment, synaptic plasticity, and immune system signaling. There is a lack of information regarding the molecular pathology of the hippocampus in ASD. We conducted RNAseq profiling on postmortem human brain samples containing the hippocampus from male children with ASD (n = 7) and normal male children (3-14 yrs old), (n = 6) from the NIH NeuroBioBank. Gene expression profiling analysis implicated molecular pathways involved in extracellular matrix organization, neurodevelopment, synaptic regulation, and immune system signaling. qRT-PCR and Western blotting were used to confirm several of the top markers identified. The CSPG protein BCAN was examined with multiplex immunofluorescence to analyze cell-type specific expression of BCAN and astrocyte morphology. We observed decreased expression of synaptic proteins PSD95 (p < 0.02) and SYN1 (p < 0.02), increased expression of the extracellular matrix (ECM) protease MMP9 (p < 0.03), and decreased expression of MEF2C (p < 0.03). We also observed increased BCAN expression with astrocytes in children with ASD, together with altered astrocyte morphology. Our results point to alterations in immune system signaling, glia cell differentiation, and synaptic signaling in the hippocampus of children with ASD, together with alterations in extracellular matrix molecules. Furthermore, our results demonstrate altered expression of genes implicated in genetic studies of ASD including SYN1 and MEF2C.

Abstract C008: Phase I biomarker analysis of farletuzumab ecteribulin (FZEC), formerly MORAb-202, effects on cancer responses in patients with platinum resistant ovarian cancer

Abstract Background: FZEC (formerly MORAb-202) is an antibody-drug conjugate consisting of farletuzumab and eribulin. FZEC is cytotoxic to FRα+ cancer cells (via direct binding) and to adjacent tumor cells (via bystander effects); it also exerts noncytotoxic bystander effects in the tumor microenvironment (Zhang et al, EORTC 2022). Study 101 is a first-in-human dose escalation followed by dose expansion study in Japanese patients (pts) with selected solid tumors to determine safety and preliminary efficacy of FZEC; antitumor activity across several doses was demonstrated in pts with platinum-resistant ovarian cancer (PROC) and FRα+ tumors (Nishio ASCO 2022). This exploratory biomarker study evaluated association of FZEC-induced pharmacological effects and clinical responses in pts with PROC to examine the mechanism of action (MoA) of FZEC. Methods: Serum samples from up to 58 pts with PROC from a FZEC 0.9 mg/kg cohort (equivalent to 33 mg/m2) or from a combined dose cohort (FZEC 0.3, 0.45, 0.68, 0.9, 1.2 mg/kg) were collected predose on day 1 of treatment cycles 1–6 and analyzed using 139 exploratory Luminex, Simoa, or OLINK protein assays. While these doses are no longer being evaluated, as a result of a change to body-surface-area-based dosing, the biomarkers observed to be preliminarily associated with response in this analysis should be further explored in ongoing studies. Markers with &amp;gt;30% unevaluable data points were excluded. The 1-sample Wilcoxon signed-rank test and 2-sample Wilcoxon rank-sum test were used to assess changes from baseline and differences between 2 best overall response groups (responders [CR/PR] and nonresponders [SD/PD]) at each visit. Fisher’s method was used to summarize statistical difference across visits (meta P&amp;lt;0.05) between the 2 groups. Results: Changes from baseline to at least 1 visit postdose (P&amp;lt;0.05) in either the responder or nonresponder groups, and differences between responders and nonresponders (meta P&amp;lt;0.05), were seen in 29 markers in the combined-dose cohort, and in 14 markers in the 0.9 mg/kg cohort. There was an overlap of 11 markers in both the FZEC 0.9 mg/kg and combined-dose cohorts that showed a significant change from baseline and significant differences in changes between responders and nonresponders. Among these 11 markers, SP-D and IGFBP-2 showed persistent changes (≥3 visits) in responders. Decreases in markers with reported protumor functions (eg, ANG-2, KLK-5, KLK-7, MCP-1, FRTN, and MMP-12), and increases in SP-D, were persistent (≥4 of 5 visits) in responders. Some biomarkers (eg, IGFBP-1, FLT3LG, and VEGF-D) showed significant increases in nonresponders. Conclusions: The persistent responder-associated biomarker changes suggest that FZEC may modulate functions associated with these markers (eg, immune regulation, vascular remodeling, extracellular matrix protease activities, iron metabolism) and this may contribute to the antitumor effect of FZEC. In the limited number of pts analyzed, preliminary pharmacodynamic effects trended with the antitumor effects observed with FZEC 0.9 mg/kg. Citation Format: Yan Zhang, Bob Zimmermann, Shuyu Li, Toshimitsu Uenaka, Keiji Furuuchi, Kan Yonemori, Toshio Shimizu, Shin Nishio, Mayu Yunokawa, Koji Matsumoto, Kazuhiro Takehara, Kosei Hasegawa, Yasuyuki Hirashima, Hidenori Kato, Yohei Otake, Takuma Miura, Junji Matsui, Meijuan Li. Phase I biomarker analysis of farletuzumab ecteribulin (FZEC), formerly MORAb-202, effects on cancer responses in patients with platinum resistant ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C008.

A new strategy to identify ADAM12 and PDGFRB as a novel prognostic biomarker for matrine regulates gastric cancer via high throughput chip mining and computational verification

BackgroundGastric cancer is a life-threatening disease that poses a serious risk to human health. Although there are numerous molecular targets for gastric cancer in clinical practice, they often exhibit low specificity and sensitivity. Consequently, this can result in a low early diagnosis rate, delayed treatment, and poor prognosis for patients with gastric cancer. Hence, it remains crucial to identify more precise diagnostic markers for this disease. MethodsThis study utilized ceRNA chips and bioinformatics methods to investigate the key genes and mechanisms involved in matrine intervention in gastric cancer cells. ResultsADAM12 and PDGFRB are the key genes that are down-regulated after matrine intervention in gastric cancer cells. By conducting bioinformatics analysis, two ceRNA regulatory axes were identified, which are associated with the prognosis of gastric cancer. These axes are lncRNA DGCR5/hsa-miR-206/ADAM12 and circRNA ITGA3/hsa-miR-24-3p/PDGFRB. ConclusionThe low expression of ADAM12 may weaken the digestion of extracellular matrix (ECM) molecules, which can result in the invasion and metastasis of tumor cells. This occurs without the catalysis of ECM proteases, thereby impacting the invasion and metastasis of gastric cancer cells. Additionally, the analysis of immune infiltration suggests that ADAM12 and PDGFRB may influence changes in the tumor immune microenvironment, thereby affecting the occurrence and development of gastric cancer. This study contributes to a deeper understanding of the role of the matrine-related ceRNA network in gastric cancer, providing a reference for clinical diagnosis and treatment. It holds significant importance in discovering new drug treatment targets.

Phenotypic Switching of Vascular Smooth Muscle Cells in Atherosclerosis.

The medial layer of the arterial wall is composed mainly of vascular smooth muscle cells (VSMCs). Under physiological conditions, VSMCs assume a contractile phenotype, and their primary function is to regulate vascular tone. In contrast with terminally differentiated cells, VSMCs possess phenotypic plasticity, capable of transitioning into other cellular phenotypes in response to changes in the vascular environment. Recent research has shown that VSMC phenotypic switching participates in the pathogenesis of atherosclerosis, where the various types of dedifferentiated VSMCs accumulate in the atherosclerotic lesion and participate in the associated vascular remodeling by secreting extracellular matrix proteins and proteases. This review article discusses the 9 VSMC phenotypes that have been reported in atherosclerotic lesions and classifies them into differentiated VSMCs, intermediately dedifferentiated VSMCs, and dedifferentiated VSMCs. It also provides an overview of several methodologies that have been developed for studying VSMC phenotypic switching and discusses their respective advantages and limitations.

ADAMTS-7 Modulates Atherosclerotic Plaque Formation by Degradation of TIMP-1.

The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis. Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site. ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events.

Low-density lipoprotein receptor-related protein-1 (LRP1) in the glial lineage modulates neuronal excitability.

The low-density lipoprotein related protein receptor 1 (LRP1), also known as CD91 or α-Macroglobulin-receptor, is a transmembrane receptor that interacts with more than 40 known ligands. It plays an important biological role as receptor of morphogens, extracellular matrix molecules, cytokines, proteases, protease inhibitors and pathogens. In the CNS, it has primarily been studied as a receptor and clearance agent of pathogenic factors such as Aβ-peptide and, lately, Tau protein that is relevant for tissue homeostasis and protection against neurodegenerative processes. Recently, it was found that LRP1 expresses the Lewis-X (Lex) carbohydrate motif and is expressed in the neural stem cell compartment. The removal of Lrp1 from the cortical radial glia compartment generates a strong phenotype with severe motor deficits, seizures and a reduced life span. The present review discusses approaches that have been taken to address the neurodevelopmental significance of LRP1 by creating novel, lineage-specific constitutive or conditional knockout mouse lines. Deficits in the stem cell compartment may be at the root of severe CNS pathologies.

Fibrogenic and fibrolytic potential of differently activated human macrophages

Macrophages are involved in the regulation of fibrogenesis and turnover of the extracellular matrix. One way to perform this function is through the production of profibrotic and fibrolytic factors including fibronectin, laminin, collagen, and extracellular matrix proteases. The production of most of them has been well studied in experimental models; however, much remains unclear regarding human macrophages. Therefore, the aim of this study was to study the content of extracellular matrix proteases (MMP-2 and MMP-9, cathepsin L), their inhibitors (TIMP-1), and collagen (type I) in supernatants of differently activated human macrophages. We compared macrophages differentiated by M-CSF or GM-CSF and further polarized in M1 with lipopolysaccharide, in M2a with IL-4, and in M2c with dexamethasone. Macrophages was obtained from peripheral blood monocytes. The content of MMPs, TIMP, cathepsin, and collagen was determined using appropriate ELISA kits. The results obtained demonstrate that differentiation factors are more important for the production of the above factors compared to polarizing stimuli (lipopolysaccharide, IL-4, dexamethasone). Moreover, macrophages differentiated by M-CSF showed predominantly antifibrotic activity because of pronounced MMPs production, while GM-CSF-induced cultures, on the contrary, were characterized by profibrotic properties due to the high level of TIMP-1 and type I collagen. M1, M2a, and M2c, induced by M-CSF, differed only in MMP-2 production, and M2a produced this metalloproteinase more than other subtypes. In the case of GM-CSF-differentiated cells, a higher level of production of TIMP-1 and, to a lesser extent, type I collagen was characteristic of M1, whereas M2c have minimal concentration of them among GM-CSF-induced macrophage subtypes. Concerning the level of cathepsin L production was relatively constant and did not depend on the generation conditions (differentiation and polarizing signals). Thus, the data obtained help to identify macrophage subtypes with anti- or profibrotic potential and may be useful for the development of cell therapy for diseases associated with fibrogenesis dysregulation.

Missense Mutation in Human CHD4 Causes Ventricular Noncompaction by Repressing ADAMTS1.

Left ventricular noncompaction (LVNC) is a prevalent cardiomyopathy associated with excessive trabeculation and thin compact myocardium. Patients with LVNC are vulnerable to cardiac dysfunction and at high risk of sudden death. Although sporadic and inherited mutations in cardiac genes are implicated in LVNC, understanding of the mechanisms responsible for human LVNC is limited. We screened the complete exome sequence database of the Pediatrics Cardiac Genomics Consortium and identified a cohort with a de novo CHD4 (chromodomain helicase DNA-binding protein 4) proband, CHD4M202I, with congenital heart defects. We engineered a humanized mouse model of CHD4M202I (mouse CHD4M195I). Histological analysis, immunohistochemistry, flow cytometry, transmission electron microscopy, and echocardiography were used to analyze cardiac anatomy and function. Ex vivo culture, immunopurification coupled with mass spectrometry, transcriptional profiling, and chromatin immunoprecipitation were performed to deduce the mechanism of CHD4M195I-mediated ventricular wall defects. CHD4M195I/M195I mice developed biventricular hypertrabeculation and noncompaction and died at birth. Proliferation of cardiomyocytes was significantly increased in CHD4M195I hearts, and the excessive trabeculation was associated with accumulation of ECM (extracellular matrix) proteins and a reduction of ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif 1), an ECM protease. We rescued the hyperproliferation and hypertrabeculation defects in CHD4M195I hearts by administration of ADAMTS1. Mechanistically, the CHD4M195I protein showed augmented affinity to endocardial BRG1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4). This enhanced affinity resulted in the failure of derepression of Adamts1 transcription such that ADAMTS1-mediated trabeculation termination was impaired. Our study reveals how a single mutation in the chromatin remodeler CHD4, in mice or humans, modulates ventricular chamber maturation and that cardiac defects associated with the missense mutation CHD4M195I can be attenuated by the administration of ADAMTS1.

Increased Proteoglycanases in Pulmonary Valves after Birth Correlate with Extracellular Matrix Maturation and Valve Sculpting.

Increased mechanical forces on developing cardiac valves drive formation of the highly organized extracellular matrix (ECM) providing tissue integrity and promoting cell behavior and signaling. However, the ability to investigate the response of cardiac valve cells to increased mechanical forces is challenging and remains poorly understood. The developmental window from birth (P0) to postnatal day 7 (P7) when biomechanical forces on the pulmonary valve (PV) are altered due to the initiation of blood flow to the lungs was evaluated in this study. Grossly enlarged PV, in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from postnatal day 0 (P0) to P7; the Adamts5-/- aortic valves (AV) did not exhibit a phenotypic correction. We hypothesized that blood flow, initiated to the lungs at birth, alters mechanical load on the PV and promotes ECM maturation. In the Adamts5-/- PV, there was an increase in localization of the proteoglycan proteases ADAMTS1, MMP2, and MMP9 that correlated with reduced Versican (VCAN). At birth, Decorin (DCN), a Collagen I binding, small leucine-rich proteoglycan, exhibited complementary stratified localization to VCAN in the wild type at P0 but colocalized with VCAN in Adamts5-/- PV; concomitant with the phenotypic rescue at P7, the PVs in Adamts5-/- mice exhibited stratification of VCAN and DCN similar to wild type. This study indicates that increased mechanical forces on the PV at birth may activate ECM proteases to organize specialized ECM layers during cardiac valve maturation.

Challenges and Opportunities Modeling the Dynamic Tumor Matrisome.

We need novel strategies to target the complexity of cancer and, particularly, of metastatic disease. As an example of this complexity, certain tissues are particularly hospitable environments for metastases, whereas others do not contain fertile microenvironments to support cancer cell growth. Continuing evidence that the extracellular matrix (ECM) of tissues is one of a host of factors necessary to support cancer cell growth at both primary and secondary tissue sites is emerging. Research on cancer metastasis has largely been focused on the molecular adaptations of tumor cells in various cytokine and growth factor environments on 2-dimensional tissue culture polystyrene plates. Intravital imaging, conversely, has transformed our ability to watch, in real time, tumor cell invasion, intravasation, extravasation, and growth. Because the interstitial ECM that supports all cells in the tumor microenvironment changes over time scales outside the possible window of typical intravital imaging, bioengineers are continuously developing both simple and sophisticated in vitro controlled environments to study tumor (and other) cell interactions with this matrix. In this perspective, we focus on the cellular unit responsible for upholding the pathologic homeostasis of tumor-bearing organs, cancer-associated fibroblasts (CAFs), and their self-generated ECM. The latter, together with tumoral and other cell secreted factors, constitute the "tumor matrisome". We share the challenges and opportunities for modeling this dynamic CAF/ECM unit, the tools and techniques available, and how the tumor matrisome is remodeled (e.g., via ECM proteases). We posit that increasing information on tumor matrisome dynamics may lead the field to alternative strategies for personalized medicine outside genomics.

The Role of Hydrogen Sulfide in Plaque Stability.

Atherosclerosis is the greatest contributor to cardiovascular events and is involved in the majority of deaths worldwide. Plaque rapture or erosion precipitates life-threatening thrombi, resulting in the obstruction blood flow to the heart (acute coronary syndrome), brain (ischemic stroke) or low extremities (peripheral vascular diseases). Among these events, major causation dues to the plaque rupture. Although the initiation, procession, and precise time of controlling plaque rupture are unclear, foam cell formation and apoptosis, cell death, extracellular matrix components, protease expression and activity, local inflammation, intraplaque hemorrhage, and calcification contribute to the plaque instability. These alterations tightly associate with the function regulation of intraplaque various cell populations. Hydrogen sulfide (H2S) is gasotransmitter derived from methionine metabolism and exerts a protective role in the genesis of atherosclerosis. Recent progress also showed H2S mediated the plaque stability. In this review, we discuss the progress of endogenous H2S modulation on functions of vascular smooth muscle cells, monocytes/macrophages, and T cells, and the molecular mechanism in plaque stability.

Metalloproteinases in dermal homeostasis.

Maintenance of skin homeostasis is a highly regulated and complex process involving a continuous remodeling by several extracellular matrix proteases, including metalloproteinases. The expression and activity of all metalloproteinases are under strict control, and their deregulation is often associated with diseases or chronic conditions, thereby being considered popular targets for developing new therapeutics. This review will highlight metalloproteinases of the MMP and ADAM families with functions in dermal homeostasis and give some insights into the mechanisms regulating their activity and expression. Furthermore, we discuss how the dysregulation of the most prominent family members affects dermal homeostasis by triggering disease development and influencing progression, focusing on cancer and aging. Here, recent discoveries and new approaches that target or exploit metalloproteinase activity in therapy are emphasized. The potential of naturally derived components in regulating metalloproteinase expression and activity in disease is discussed.

Abstract P1110: Identification Of Timp-1 As A Downstream Target Of The Coronary Artery Disease Risk Factor Adamts-7 In Atherosclerosis

Background: The gene encoding the extracellular matrix (ECM) protease ADAMTS-7 was associated with coronary artery disease (CAD) in genome-wide association studies. ADAMTS-7 is expressed at all stages in human plaques and mice lacking Adamts-7 displayed reduced atherosclerotic plaque formation. While these findings render ADAMTS-7 a promising therapeutic target, the underlying mechanisms remain unknown. Methods and Results: Here, we sought to identify downstream mechanisms of ADAMTS-7 in atherosclerotic plaque formation. Targets of Adamts-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques in Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. The endogenous inhibitor of matrix metalloproteinases (MMP) Timp-1 was identified as a novel target of Adamts-7. Adamts-7 and Timp-1 were found to be co-localized in atherosclerotic plaques and co-immunoprecipitation (Co-IP) studies revealed TIMP-1 as the first putative target to bind to the catalytic domain of ADAMTS-7. In vitro degradation assays demonstrated that ADAMTS-7 degrades TIMP-1. Co-IP furthermore revealed less binding of TIMP-1 to its canonical target MMP-9 when ADAMTS-7 was present. In line, scaffolding and degradation of TIMP-1 by ADAMTS-7 impaired TIMP-1-mediated inhibition of MMP-9 in vitro. As a downstream mechanism, we investigated collagen content in atherosclerotic plaques of Apoe-/- and Apoe-/- Adamts7-/- mice after Western diet. Collagen stainings of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. Since ADAMTS-7 might exert its pro-atherogenic effects through interaction with TIMP-1, we established a TIMP-1-ADAMTS-7-interaction assay based on Förster resonance energy transfer (FRET) to identify inhibitors of this protein-protein interaction. Conclusion and Outlook: TIMP-1 represents a novel downstream target of ADAMTS-7 that can explain the role of this novel risk factor in CAD. Our FRET-based protein-protein interaction assay may be used for high-throughput screening to identifiy inhibitors and prevent the initiation or slow the progression of atherosclerosis.

The adaptive response of the mandibular condyle to increased load is disrupted by ADAMTS5 deficiency

ABSTRACT Objective To determine the impact of increased load on the temporomandibular joint (TMJ) from mice deficient in the extracellular matrix protease ADAMTS5. Materials and Methods Wire springs exerting 0.5 N for 1 h/day for 5 days (Adamts5+/+ -n = 18; Adamts5−/− n = 19) or 0.8 N for 1 h/day for 10 days (Adamts5+/+-n = 18; Adamts5−/− n = 17) were used to increase murine TMJ load. Safranin O-staining was used to determine mandibular condylar cartilage (MCC) morphology. Chondrogenic factors Sox9 and aggrecan were immunolocalized. Microcomputed topography was employed to evaluate mineralized tissues, and Tartrate-Resistant Acid Phosphatase staining was used to quantify osteoclasts. Results Increased load on the mandibular condyle of Adamts5 -/- mice resulted in an increase in the hypertrophic zone of mandibular condylar cartilage (MCC) compared to normal load (NL) (P < 0.01). In the trabecular bone of the mandibular condyle, the total volume (TV), bone volume (BV), trabecular thickness (TbTh), and trabecular separation (TbSp) of the mandibular condyles in Adamts5 -/- mice (n = 27) did not change significantly with increased load, compared to Adamts5 +/+ (n = 38) that exhibited significant responses (TV-P < 0.05; BV-P < 0.001; TbTh-P < 0.01; TbSp-P < 0.01). The bone volume fraction (BV/TV) was significantly reduced in response to increased load in both Adamts5 -/- (P < 0.05) and Adamts5 +/+ mandibular condyles (P < 0.001) compared to NL. Increased load in Adamts5 -/- mandibular condyles also resulted in a dramatic increase in osteoclasts compared to Adamts5 -/- NL (P < 0.001) and to Adamts5 +/+ with increased load (P < 01). Conclusion The trabeculated bone of the Adamts5 -/- mandibular condyle was significantly less responsive to the increased load compared to Adamts5 +/+ . ADAMTS5 may be required for mechanotransduction in the trabeculated bone of the mandibular condyle.

Extracellular Matrix Proteinases

Extracellular Matrix Proteinases

Interleukin-17 modulates uPA and MMP2 expression in human periodontal ligament mesenchymal stem cells: Involvement of the ERK1/2 MAPK pathway

Periodontal disease is a chronic infection of periodontal tissue characterized by extracellular matrix (ECM) degradation due to increased expression of plasminogen activators and matrix metalloproteinases (MMPs) and various proinflammatory cytokines, including interleukin (IL)-17. Successful regeneration of damaged periodontal tissues depends on the proper functionality of periodontal ligament mesenchymal stem cells (PDLMSCs), especially the production of extracellular matrix proteases. We investigated the influence of IL-17 on ECM remodeling through modulation of urokinasetype plasminogen activator (uPA) and MMP2/MMP9 expression in human PDLMSCs at mRNA, protein and activity levels using by RT-PCR, Western blotting and zymography, respectively. Investigation of the involvement of MAPKs in these processes in PDLMSCs was determined by Western blotting, as well as by utilizing specific p38 and MEK1/2 inhibitors. Our results show that IL-17 activates MAPK signaling in PDLMSCs. Moreover, IL-17 had no effect on MMP9 expression, but it stimulated uPA and MMP2 gene and protein expression in PDLMSCs through the activation of the ERK1/2 MAPK signaling pathway. The obtained data suggest that IL-17 contributes to ECM degradation in the periodontal ligament by stimulating uPA and MMP2 expression and activity in PDLMSCs. This information is important for understanding periodontal disease development and defining future directions of its treatment.

Astrocyte tissue plasminogen activator expression during brain development and its role in pyramidal neuron neurite outgrowth

The serine protease tissue plasminogen activator (tPA), encoded by the gene Plat, exerts a wide range of proteolysis-dependent and proteolysis-independent functions. In the developing brain, tPA is involved in neuronal development via the modulation of the proteolytic degradation of the extracellular matrix (ECM). Both lack of and excessive tPA are associated with neurodevelopmental disorders and with brain pathology. Astrocytes play a major role in neurite outgrowth of developing neurons as they are major producers of ECM proteins and ECM proteases. In this study we investigated the expression of Plat in developing and mature hippocampal and cortical astrocytes of Aldh1l1-EGFP-Rpl10a mice in vivo following Translating Ribosome Affinity Purification (TRAP) and the role of tPA in modulating astrocyte-mediated neurite outgrowth in an in vitro astrocyte-neuron co-culture system. We show that Plat is highly enriched in astrocytes in the developing, but not in the mature, hippocampus and cortex. Both the silencing of tPA expression in astrocytes and astrocyte exposure to recombinant tPA reduce neuritogenesis in co-cultured hippocampal neurons. These results suggest that astrocyte tPA is involved in modulating neuronal development and that tight control of astrocyte tPA expression is important for normal neuronal development, with both experimentally elevated and reduced levels of this proteolytic enzyme impairing neurite outgrowth. These results are consistent with the hypothesis that the ECM, by serving as adhesive substrate, enables neurite outgrowth, but that controlled proteolysis of the ECM is needed for growth cone advancement.

Gestational and lactational xenoestrogen exposure disrupts morphology and inflammatory aspects in mammary gland of gerbil mothers during involution

In the mammary gland (MG), the developmental window for gestational/lactational differentiation and growth is highly vulnerable to hormonal disruption. Here we describe that the MG involution process in female gerbil mothers is delayed by bisphenol A (BPA) exposure during gestation and lactation. The process is directly influenced by changes in expression of extracellular matrix proteases MMP-2, MMP-9, and FAP, and the incidence of collagen and elastin is reduced after 7 and 14 days of weaning. A pro-inflammatory environment in the late involution process was confirmed by higher expression of TNF-α, COX-2 and phospho-STAT3 n the MG stroma, allied to increases in the incidence of macrophages and mast cells. These aspects impacted the proliferative pattern of epithelial cells, which decreased on the 14th post-weaning day. These data confirm that the milk production window of susceptibility is vulnerable to the impact of BPA, which promotes a suggestive pro-tumoral microenvironment during mammary involution.