Degradation Of Basement Membrane Research Articles

BIOMEDICAL APPLICATION OF K5 PLASMINOGEN FRAGMENT

Aim. Plasminogen kringle 5 is an endogenous angiogenic inhibitor. The purpose of the present review was to highlight the potential biomedical application of kringle 5 in the regulation of angiogenesis and tumor growth. Methods. Angiogenesis is a complex process that involves endothelial cell proliferation, migration, basement membrane degradation, and neovessel organization. Since the uncontrolled growth of new blood vessels causes the progression of many common diseases, first of all, oncological diseases, autoimmune disorders, neovascular damage of the eye, the use of angiostatins can be a promising pharmacotherapeutic approach to the prevention and adjuvant therapy of these pathological conditions. The advantages of angiostatins application are their non-toxicity even at high doses, non-immunogenicity, lack of tolerance of target cells to their action. Angiostatins comprise a group of kringle-containing proteolytically-derived fragments of plasminogen/plasmin, which act as potent inhibitory mediators of endothelial proliferation and migration. Among all known angiostatin species, isolated K5 plasminogen fragment was shown to display the most potent inhibitory activity against proliferation of endothelial cells via triggering multiple signaling pathways, which lead to cell death and resulting angiogenesis suppression. Results. Current literature data suggest that in addition to expressed and highly specific cytotoxicity in relation to endotheliocytes and some types of tumor cells, the kringle domain 5 of human plasminogen has other advantages as an antiangiogenic and antitumor regulator, including its specific inhibitory activity, which affects only activated, proliferating endothelial cells, and therefore is non-toxic to other types of normal cells. As an endogenous protein, which is formed in the human organism, K5 does not provoke an immune response. K5 as a small polypeptide molecule with a stable structure can be obtained as a recombinant protein in E. coli cells, and can also be used in pharmacokinetic systems of targeted delivery and sustained release. Conclusions. The prospect of successful use of K5 as a therapeutic agent to manage pathological processes associated with dysregulation of angiogenesis makes it necessary to develop and improve methods of its production and to further test its plausible pleiotropic biological activities.

Fgf signalling is required for gill slit formation in the skate, Leucoraja erinacea

The gill slits of fishes develop from an iterative series of pharyngeal endodermal pouches that contact and fuse with surface ectoderm on either side of the embryonic head. We find in the skate (Leucoraja erinacea) that all gill slits form via a stereotypical sequence of epithelial interactions: 1) endodermal pouches approach overlying surface ectoderm, with 2) focal degradation of ectodermal basement membranes preceding endoderm–ectoderm contact; 3) endodermal pouches contact and intercalate with overlying surface ectoderm, and finally 4) perforation of a gill slit occurs by epithelial remodelling, without programmed cell death, at the site of endoderm–ectoderm intercalation. Skate embryos express Fgf8 and Fgf3 within developing pharyngeal epithelia during gill slit formation. When we inhibit Fgf signalling by treating skate embryos with the Fgf receptor inhibitor SU5402 we find that endodermal pouch formation, basement membrane degradation and endodermal–ectodermal intercalation are unaffected, but that epithelial remodelling and gill slit perforation fail to occur. These findings point to a role for Fgf signalling in epithelial remodelling during gill slit formation in the skate and, more broadly, to an ancestral role for Fgf signalling during pharyngeal pouch epithelial morphogenesis in vertebrate embryos.

β-elemene Suppresses Migration of Esophageal Squamous Cell Carcinoma by Modulating Expression of MMP9 through the PI3K/Akt/NF-κB Pathway.

β-elemene is a plant-derived drug with broad-spectrum anticancer activity. Studies have found that β-elemene can inhibit tumor cell proliferation, induce tumor cell apoptosis, and resist tumor cell migration and invasion. Esophageal cancer is a common digestive tract malignant tumor. Progress has been made in the treatment of esophageal cancer, including the use of β-elemene, but the mechanism of anti-migration is unclear. PI3K/Akt/NF- κB/MMP9 signaling pathway is involved in the regulation of tumor cell proliferation, migration, extracellular matrix(ECM), and basement membrane(BM) degradation. This study aims to investigate the effect of β-elemene on the migration of esophageal squamous cell carcinoma (ESCC) and its related mechanisms by bioinformatics, network pharmacology, and molecular docking methods. In this study, the differentially expressed genes (DEGs) of ESCC were screened through GeneCards and BATMAN-TCM databases combined with the Gene Expression Omnibus (GEO) database (GSE17351). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify the functions and related pathways of the genes. The protein-protein interaction (PPI) network of these DEGs was constructed with the STRING database. Five hub genes were screened by CytoHubba plug-in Cytoscape according to the principle of degree value and the expressions of which were validated by the UALCAN database from the Cancer Genome Atlas (TCGA). The hub gene with the strongest binding energy was identified by molecular docking. A wound healing assay was subjected to assess the migration ability. RT-PCR was used to detect the content of migration-related mRNA. Western blotting was performed to examine the expression rates of Akt, NF-κB, and MMP9 in ESCC tissues by β-elemene and SC79. 71 target genes were obtained which were mainly involved in biological processes such as epidermal development and extracellular matrix decomposition. In addition, critical pathways, including PI3K/AKT signaling pathway and focal adhesion, were verified to be subject to β-elemene regulation. It exhibited marked binding affinity between β-elemene and MMP9 with an excellent docking score of -6.56 kcal/mol. The expression levels of Akt, NF-κB, and MMP9 in ESCC tissues were significantly upregulated compared to normal tissues. Western blot detection demonstrated that β-elemene specifically reduced the phosphorylation level of Akt, and its downstream target molecule NF-κB, thus resulting in reduced levels of their target proteins, including MMP9 in ESCC. A wound healing assay showed β-elemene inhibited the migration of ESCC cells. RT-PCR results found that the mRNA expression of Akt, NF-κB, and MMP9 in the β-elemene group was significantly lower than that in the control group. However, the application of SC79 partially reversed the effect of β-elemene. In summary, our study suggests that the anti-tumor migration effect of β-elemene on ESCC is associated with the inhibition of PI3K/Akt/NF-κB/MMP9 signalling pathway, and it provides a theoretical reference for further rational clinical application.

Matrix Metalloproteinases and the Pathogenesis of Recurrent Corneal Erosions and Epithelial Basement Membrane Dystrophy.

Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes which are members of the zinc endopeptidase family. They have the ability to degrade extracellular matrix elements, allowing for the release of binding molecules and cell migration. Although metalloproteinases regulate numerous physiological processes within the cornea, overexpression of metalloproteinase genes and an imbalance between the levels of metalloproteinases and their inhibitors can contribute to the inhibition of repair processes, the development of inflammation and excessive cellular proliferation. The involvement of MMPs in the pathogenesis of dystrophic corneal diseases needs clarification. Our analyses focus on the involvement of individual metalloproteinases in the pathogenesis of recurrent corneal erosions and highlight their impact on the development of corneal epithelial basement membrane dystrophy (EBMD). We hypothesize that abnormalities observed in patients with EBMD may result from the accumulation and activation of metalloproteinases in the basal layers of the corneal epithelium, leading to basement membrane degradation. A barrier formed from degradation materials inhibits the normal migration of epithelial cells to the superficial layers, which contributes to the development of the aforementioned lesions. This hypothesis seems to be lent support by the elevated concentrations of metalloproteinases in the corneal epithelium of these patients found in our previous studies on the relationships between MMPs and recurrent corneal erosions.

The Role of MMP-9 in Regulating Degradation of the Splenic Microvascular Basement Membrane Induced by High Altitude Hypoxia

The Role of MMP-9 in Regulating Degradation of the Splenic Microvascular Basement Membrane Induced by High Altitude Hypoxia

Cardiac pericytes mediate the remodeling response to myocardial infarction.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-β signaling. Deleting TGF-β receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Verrucous Carcinoma, A Very Well Differentiated Form of OSCC? - An Immunochemical Exploratory Study

Introduction: Verrucous carcinoma shows an analogous biological behaviour to well-differentiated Oral Squamous Cell Carcinoma (OSCC). So, it is a difficult task to differentiate the above two entities based on clinical and pathological behaviour. Immunohistochemical studies can help in investigating the differences between the biological behaviours of these carcinomas. Laminin may be used as a status indicator of intact or degraded basement membrane during tumorigenesis. Thus, the present study aimed to assess the immunohistochemical expression of laminin in different grades histological grades of OSCC and VC. Methods: Laminin expression was evaluated in 31 biopsy-proven cases of Oral Squamous Cell Carcinoma (OSCC) and 10 cases of Verrucous Carcinoma (VC). Results: We observed that most cases of VC (90%) were negative or mildly stained for cytoplasmic laminin while the majority of cases of OSCC (74.2%) stained moderately for the same. Further, VC cases showed a total absence of linear laminin staining around the tumour-host interface in contrast to 13% of OSCC cases that were correspondingly stained (p<0.001). Conclusion: The present study, therefore concludes that the interface expression in Verrucous Carcinoma distinctly differed from that in well-differentiated Oral Squamous Cell Carcinoma. Cytoplasmic laminin expression increased and interface staining decreased with unfavourable clinicopathological outcomes thereby rendering laminin an expedient marker in evaluating the histological differentiation and aggressiveness of oral carcinoma

Changes in the Extracellular Matrix in Endometrial and Cervical Cancer: A Systematic Review

Endometrial and cervical cancers are the two most common gynaecological malignancies and among the leading causes of death worldwide. The extracellular matrix (ECM) is an important component of the cellular microenvironment and plays an important role in developing and regulating normal tissues and homeostasis. The pathological dynamics of the ECM contribute to several different processes such as endometriosis, infertility, cancer, and metastasis. Identifying changes in components of ECM is crucial for understanding the mechanisms of cancer development and its progression. We performed a systematic analysis of publications on the topic of changes in the extracellular matrix in cervical and endometrial cancer. The findings of this systematic review show that matrix metalloproteinases (MMP) play an important role impacting tumour growth in both types of cancer. MMPs degrade various specific substrates (collagen, elastin, fibronectin, aggrecan, fibulin, laminin, tenascin, vitronectin, versican, nidogen) and play a crucial role in the basal membrane degradation and ECM components. Similar types of MMPs were found to be increased in both cancers, namely, MMP-1, MMP-2, MMP-9, and MMP-11. Elevated concentrations of MMP-2 and MMP-9 were correlated with the FIGO stage and are associated with poor prognosis in endometrial cancer, whereas in cervical cancer, elevated concentrations of MMP-9 have been associated with a better outcome. Elevated ADAMTS levels were found in cervical cancer tissues. Elevated disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) levels were also found in endometrial cancer, but their role is still unclear. Following these findings, this review reports on tissue inhibitors of ECM enzymes, MMPs, and ADAMTS. The present review demonstrates changes in the extracellular matrix in cervical and endometrial cancers and compared their effect on cancer development, progression, and patient prognosis.

Pterostilbene alleviated cerebral ischemia/reperfusion-induced blood-brain barrier dysfunction via inhibiting early endothelial cytoskeleton reorganization and late basement membrane degradation.

Pterostilbene, an important analogue of the star molecule resveratrol and a novel compound naturally occurring in blueberries and grapes, exerts a significant neuroprotective effect on cerebral ischemia/reperfusion (I/R), but its mechanism is still unclear. This study aimed to follow the molecular mechanisms behind the potential protective effect of pterostilbene against I/R induced injury. For fulfilment of our aim, we investigated the protective effects of pterostilbene on I/R injury caused by middle cerebral artery occlusion (MCAO) in vivo and oxygen-glucose deprivation (OGD) in vitro. Machine learning models and molecular docking were used for target exploration and validated by western blotting. Pterostilbene significantly reduced the cerebral infarction volume, improved neurological deficits, increased cerebral microcirculation and improved blood-brain barrier (BBB) leakage. Machine learning models confirmed that the stroke target MMP-9 bound to pterostilbene, and molecular docking demonstrated the strong binding activity. We further found that pterostilbene could depolymerize stress fibers and maintain the cytoskeleton by effectively increasing the expression of the non-phosphorylated actin depolymerizing factor (ADF) in the early stage of I/R. In the late stage of I/R, pterostilbene could activate the Wnt pathway and inhibit the expression of MMP-9 to decrease the degradation of the extracellular basement membrane (BM) and increase the expression of junction proteins. In this study, we explored the protective mechanisms of pterostilbene in terms of both endothelial cytoskeleton and extracellular matrix. The early and late protective effects jointly maintain BBB stability and attenuate I/R injury, showing its potential to be a promising drug candidate for the treatment of ischemic stroke.

Lycium Barbarum polysaccharides ameliorates hyperglycemia-exacerbated cerebral ischemia/reperfusion injury via protecting blood-brain barrier

BackgroundHyperglycemia exacerbates brain damage in cerebral ischemia/reperfusion injury. Previous study found that Lycium barbarum polysaccharides (LBP) has a neuroprotective effect on hyperglycemia-aggravated ischemic brain injury, which raising the possibility for treatment of neurodegenerative diseases. However, the underlying mechanism of LBP-induced protection by ameliorating hyperglycemia-aggravated ischemia/reperfusion injury needs to be tested. This study aimed to investigate the effects of LBP on blood–brain barrier (BBB) integrity with a hyperglycemia-aggravated cerebral ischemia/reperfusion injury model. MethodsSprague-Dawley male rats were randomly divided into three groups: normoglycemic (NG), hyperglycemic (HG), and LBP-pretreated hyperglycemic (HG + LBP). Animals underwent middle cerebral artery occlusion (MCAO) for 30 min, followed by 1-, 3-, and 7-day of reperfusion. ResultsOur results showed that the neurological deficit, infarct volume, cell apoptosis, and IgG leakage in the HG group significantly increased separately, compared with that of the NG group, (p < 0.05). Pre-treatment with LBP reversed these injury indicators (p < 0.05). And much more severe degree of swelling endothelium, swollen astrocyte, and decreased tight junctions in the micro-vessel were detected in the HG group comparing to that of the NG group. In addition, increased degree of basement membrane degradation, dissociation between the astrocyte endfeet and basement membrane, and tight junction's protein degradation was found in the HG group compared with the NG group (p < 0.05). However, when exposure to LBP therapy could reverse the above alterations (p < 0.05). ConclusionsThese results demonstrated that LBP could ameliorate hyperglycemia-exacerbated cerebral ischemia/reperfusion injury via protecting the blood-brain barrier.

Effects of stretching on the basement membrane structure in the soleus muscle of Wistar rats.

The basement membrane (BM), mainly composed of collagen IV, plays an important role in the maintenance, protection, and recovery of muscle fibers. Collagen IV expression is maintained by the balance between synthetic and degradative factors, which changes depending on the level of muscle activity. For example, exercise increases collagen IV synthesis, whereas inactivity decreases collagen IV synthesis. However, the effects of stretching on the BM structure remain unclear. Therefore, to investigate the effects of stretching on the BM of the skeletal muscle, we continuously applied stretching to the rat soleus muscle and examined the altered expression of BM-related factors and structure using quantitative polymerase chain reaction (qPCR), western blotting, zymography, immunohistochemistry, and electron microscopy. The results show that stretching increased the matrix metalloproteinase 14 (MMP14) expression and MMP2 activity, and decreased the collagen IV expression and width of the lamina densa in the soleus muscle. These results suggest that stretching promotes BM degradation in the rat soleus muscle. The findings of this study indicate a new influence of stretching on skeletal muscles, and may contribute to the new use of stretching in rehabilitation and sports fields.

Novel TCF21high pericyte subpopulation promotes colorectal cancer metastasis by remodelling perivascular matrix

ObjectiveHaematogenous dissemination is a prevalent route of colorectal cancer (CRC) metastasis. However, as the gatekeeper of vessels, the role of tumour pericytes (TPCs) in haematogenous metastasis remains largely unknown. Here,...

Tissue growth: Basement membrane degradation triggers cell proliferation

Building of the Drosophila abdomen relies on the removal of larval cells and expansion, through proliferation, of a population of progenitor epithelial cells. A new study shows that matrix metalloproteinases produced by larval cells drive basement membrane degradation and proliferative growth of the progenitor epithelial population.

Modulation of the expression and activity of cathepsin S in reconstructed human skin by neohesperidin dihydrochalcone

Dysregulation of cathepsin S (Cat S), a cysteine protease involved in extracellular-matrix and basement membrane (BM) degradation, is a concomitant feature of several inflammatory skin diseases. Therefore, Cat S has been suggested as a potential therapeutic target. Flavonoids, which were identified as regulatory molecules of various proteolytic enzymes, exert beneficial effects on skin epidermis. Herein, thirteen flavonoid compounds were screened in vitro and in silico and neohesperidin dihydrochalcone (NHDC) was identified as a potent, competitive, and selective inhibitor (Ki=8±1 µM) of Cat S. Furthermore, Cat S-dependent hydrolysis of nidogen-1, a keystone protein of BM architecture, as well elastin, collagens I and IV was impaired by NHDC, while both expression and activity of Cat S were significantly reduced in NHDC-treated human keratinocytes. Moreover, a reconstructed human skin model showed a significant decrease of both mRNA and protein levels of Cat S after NHDC treatment. Conversely, the expression of nidogen-1 was significantly increased. NHDC raised IL-10 expression, an anti-inflammatory cytokine, and mediated STAT3 signaling pathway, which in turn dampened Cat S expression. Our findings support that NHDC may represent a valuable scaffold for structural improvement and development of Cat S inhibitors to preserve the matrix integrity and favor skin homeostasis during inflammatory events.

Bleeding and Thrombosis: Insights into Pathophysiology of Bothrops Venom-Related Hemostasis Disorders.

Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A2 and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood.

RasV12; scrib-/- Tumors: A Cooperative Oncogenesis Model Fueled by Tumor/Host Interactions.

The phenomenon of how oncogenes and tumor-suppressor mutations can synergize to promote tumor fitness and cancer progression can be studied in relatively simple animal model systems such as Drosophila melanogaster. Almost two decades after the landmark discovery of cooperative oncogenesis between oncogenic RasV12 and the loss of the tumor suppressor scribble in flies, this and other tumor models have provided new concepts and findings in cancer biology that has remarkable parallels and relevance to human cancer. Here we review findings using the RasV12; scrib−/− tumor model and how it has contributed to our understanding of how these initial simple genetic insults cooperate within the tumor cell to set in motion the malignant transformation program leading to tumor growth through cell growth, cell survival and proliferation, dismantling of cell–cell interactions, degradation of basement membrane and spreading to other organs. Recent findings have demonstrated that cooperativity goes beyond cell intrinsic mechanisms as the tumor interacts with the immediate cells of the microenvironment, the immune system and systemic organs to eventually facilitate malignant progression.

P394 Serological biomarkers of type III and IV collagen remodeling predict and monitor infliximab treatment response in patients with Inflammatory Bowel Disease

Abstract Background Immunotherapeutic drugs such as anti-TNFα antibodies have greatly improved the treatment of inflammatory bowel disease (IBD). However, up to 20-40% of patients still fail to respond to therapy as the pattern of disease is variable. Better patient profiling could increase treatment response and improve quality of life. The aim of this study was to assess whether serological biomarkers reflecting tissue formation and different forms of basement membrane (BM) degradation could monitor or predict treatment response to anti-TNFα in patients with Crohn’s disease (CD) and Ulcerative Colitis (UC). Methods Using competitive ELISA, serum biomarkers of matrix metalloproteinase (MMP)-mediated type IV collagen degradation (C4M), T-cell related collagen degradation (C4G) and type III and IV collagen formation (PRO-C3, PRO-C4) were measured in 89 patients (CD=63, UC=26) receiving infliximab (IFX) induction therapy. Disease activity was defined by composite assessment of the Harvey-Bradshaw Index (HBI) for CD and Simple Clinical Colitis Activity Index (SCCAI) for UC together with physician’s global assessments (PGA). Clinical remission (HBI &amp;lt;5, SCCAI &amp;lt;2.5) was assessed at week 14. Logistic regression models adjusted for confounding factors (CD: age of diagnosis and ileoceacal surgery, UC: steroid use at week 14) were used to examine prediction value for treatment response. Results In patients with CD, baseline ratios of C4M/C4G (P&amp;lt;0.001, AUC=0.95) and PRO-C4/C4G (P&amp;lt; 0.05, AUC=0.92) were significantly lower at baseline in responders (n=57) compared to non-responders (n=6). PRO-C3 levels were increased at week 14 in CD patients responding to therapy compared to those not responding (P&amp;lt;0.01, AUC=0.93). Finally, UC patients responding to treatment (n=19) showed decreased levels of C4M/C4G (P&amp;lt;0.01, AUC=0.96) at week 14 compared to non-responders (n=7). Figure 1: A) Serum levels at baseline or week 14 between non-responders and responders for CD and UC. B) ROC curves showing discriminative capacity of the biomarkers (ratios) between responders and non-responders. Conclusion Increased baseline ratios of type IV collagen remodeling (C4M/C4G, PRO-C4/C4G) in serum predict treatment response to IFX in patients with CD, whereas PRO-C3 may serve as a monitoring tool reflecting tissue restoration. The ratio between MMP- and granzyme B-mediated degradation of type IV collagen (C4M/C4G) in serum was elevated in non-responders compared to responders and may serve as non-invasive tool to monitor treatment response in patients with UC. Together, these biomarkers may help reveal different profiles of ECM turnover and inflammation and thus aid in better prediction and monitoring of response to anti-TNFα treatment.

P278 Type IV collagen formation/degradation ratio predicts response to infliximab induction therapy in patients with Ulcerative Colitis

Abstract Background Up to 50% of patients with ulcerative colitis (UC) do not respond to biological therapies, including TNF-α-antagonists (e.g., infliximab, IFX) and migration inhibitors (e.g., the α4β7-integrin inhibitor vedolizumab, VEDO). Therefore, serological biomarkers that predict therapy response are urgently needed. In this respect, biomarkers of extracellular matrix (ECM) remodeling, including basement membrane (BM) remodeling, and intestinal inflammation may hold promise as these processes play an important role in the pathophysiology of UC. In this study, we aimed to assess the predictive ability of serological biomarkers of ECM turnover and intestinal inflammation with regard to clinical response to IFX and VEDO therapy in patients with UC. Methods Serological biomarkers of collagen formation (PRO-C3, PRO-C4), matrix metalloproteinase (MMP)-mediated collagen degradation (reC1M, C3M, C4M, C4G, C6Ma3) and intestinal inflammation (macrophage activity marker [VICM] and calprotectin degradation fragments [CPa9-HNE]) were measured using neo-epitope solid-phase competitive enzyme-linked immunosorbent assay (ELISA) technology in 80 patients with UC who received either infliximab (IFX, n=26) or vedolizumab (VEDO, n=54) induction therapy. Clinical response to therapy was defined by composite assessment of available Simple Clinical Colitis Activity Index (SCCAI) scores and physician’s global assessments (PGA) derived from clinical records. Logistic regression modelling and receiver operating characteristics (ROC) statistics were used to assess discriminative capacity regarding response to IFX and VEDO. Results Only baseline ratios of type IV collagen formation and degradation (PRO-C4/C4M) were significantly elevated in patients with UC who clinically responded to IFX therapy (P&amp;lt;0.05). In addition, PRO-C4/C4M ratios accurately discriminated responders and non-responders to IFX therapy (area under the curve [AUC]=0.77, P&amp;lt;0.05), also after adjustment for confounding factors (AUC=0.88, P&amp;lt;0.01). Serum VICM levels were elevated in patients not responding to IFX therapy, but did not reach statistical significance (AUC=0.76, P=0.06). None of the biomarkers predicted response to VEDO therapy. Conclusion The baseline ratio of type IV collagen formation/degradation demonstrated high predictive accuracy with regard to response to IFX therapy in patients with UC, which may imply that patients with increased BM formation vs. degradation are more likely to respond. Furthermore, VICM may hold promise as a predictor for non-response to IFX therapy. Serological biomarkers of ECM turnover and intestinal inflammation may aid in predicting response to biological therapy and deserve validation in larger, prospective cohort studies.

Ozanimod Preserves Endothelial Health by Attenuating Cerebrovascular MMP‐9 Activity Following Acute Ischemic Injury

The loss of vascular integrity at the level of the blood brain barrier leads to a vicious cascade of secondary injuries following acute ischemic stroke (AIS). Elevated MMP-9 activity within the cerebral vasculature has been implicated with severe degradation of the vascular basement membrane leading to abnormal cerebrovascular permeability and detrimental stroke outcome. Sphingosine-1-phosphate receptor (S1PR) modulation improves stroke outcome in AIS patients, however the influence of selective S1PR1ligands, such as ozanimod, on brain endothelial health and MMP-9 activity following AIS has not been investigated. Thus, the aim of this study was to determine the impact of acute ischemic injury on MMP-9 activity in both the rat and the human cerebrovasculature as well as the vascular specific role of ozanimod on human endothelial health and MMP-9 activity. Using an in vivo thromboembolic stroke model, cerebral vessels were isolated from male Wistar rats that underwent a right middle cerebral artery (MCA) thrombin injection. Sham-operated animals underwent the same surgical procedure; however, nothing was injected. Vascular MMP-9 enzymatic activity of was evaluated at 3, 6 and 24h post injury using zymography. Using an in vitro ischemic injury model (HGD; hypoxia plus glucose deprivation), male human brain microvascular endothelial cells (HBMECs; P7) were treated with ozanimod (0.5nM) or vehicle (DMSO) and then exposed to normoxia (21% O2) or HGD (1% O2). In some experiments, W146 (a selective antagonist), verified S1PR1 dependence. Morphology and vacuole formations were assessed using crystal violet staining. Enzymatic activity of MMP-9 was evaluated via zymography and the extracellular H2O2 concentration, inducer of MMP-9 activity, was measured using a colorimetric assay. Following in vivo thromboembolic occlusion, ipsilateral MMP-9 activity increased at 6h post injury and returned to baseline when compared to sham. In comparison to the contralateral side, thromboembolic occlusion induced a time dependent modulation of ipsilateral MMP-9 activity compared to the contralateral side with the highest activity peaking at 6h. In our in vitro HBMEC stroke model, MMP-9 enzymatic activity was increased following 3h HGD exposure, and this response was attenuated by ozanimod. Concomitantly, during HGD, H2O2 production, a partial inducer of MMP-9 activity, was increased in a time dependent manner when compared to normoxic controls. Furthermore, HGD induced an increase in HBMEC vacuole formations and a decrease in cell viability at 3h. Loss of cell viability was rescued with ozanimod. In conclusion, increased MMP-9 activity, in part due to H2O2, is an acute response to ischemic injury in the cerebrovasculature. Specifically, ozanimod's ability to attenuate endothelial MMP-9 activity may play an important beneficial role in mitigating blood brain barrier integrity loss following an acute ischemic injury.

Active MT1-MMP is tethered to collagen fibers in DDR2-containing remnants.

Type I collagen is a major extracellular matrix (ECM) component in the interstitial stroma of solid tumors, and it represents the first barrier against tumor cell invasion after basement-membrane degradation. The collagen receptors that convey molecular signals into the cells are collagen-binding discoidin domain receptors (DDRs) and integrins. Collagen-activated DDR2 clusters form DDR2-containing remnants in an integrin-dependent manner in three-dimensional (3D) collagen matrix. Although DDR2-containing remnants in the collagen matrix may generate sustained perturbation to ECM remodeling, the molecular components and function of the remnants are largely unknown. Here we determined the interaction and co-localization between DDR2 and membrane type I-matrix metalloproteinase (MT1-MMP) in the cells and the DDR2-containing remnants on collagen fibers, and we found that MT1-MMP was co-tethered to collagen fibers in the remnants. These collagen fiber-associated MT1-MMP remained active. Furthermore, DDR2 enhanced MT1-MMP proteolytic activity. These results demonstrate that DDR2 ensures the remnant-associated MT1-MMP to continue the degradation of ECM in addition to pericellular ECM degradation mediated by cell surface tethered MT1-MMP. Thus, our findings reveal a new alternative ECM degradation mechanism mediated by MT1-MMP in the DDR2-containing remnants.