Kinetics of LYVE-1-Positive M2-Like Macrophages in Developing and Repairing Dental Pulp in Vivo and Their Pro-Angiogenic Activity in Vitro

Tissue-resident macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) are found in multiple tissues and organs. We aimed to evaluate the dynamics and biological function of LYVE-1+ macrophages in dental pulp during post-injury tissue remodeling. Immunofluorescence staining of mouse embryos revealed that LYVE-1+ macrophages colonized dental pulp before birth. In mature rat molar dental pulp, LYVE-1+ macrophages were the main subset of macrophages expressing CD163, an M2 marker, and were distributed throughout the tissue. In response to dental pulp injury induced by cavity preparation, LYVE-1+ macrophages quickly disappeared from the affected area of the pulp and gradually repopulated during the wound healing process. RAW264.7 mouse macrophages cultured with a mixture of macrophage colony-stimulating factor, interleukin-4, and dexamethasone increased LYVE-1 expression, whereas lipopolysaccharide-stimulation decreased LYVE-1 expression. Enforced expression of Lyve1 in RAW264.7 cells resulted in increased mRNA expression of matrix metalloproteinase 2 (Mmp2), Mmp9, and vascular endothelial growth factor A (Vegfa). Lyve1-expressing macrophages promoted the migration and tube formation of human umbilical vein endothelial cells. In conclusion, LYVE-1+ tissue-resident M2-like macrophages in dental pulp showed dynamism in response to pulp injury, and possibly play an important role in angiogenesis during wound healing and tissue remodeling.

Plasmatic MMP9 released from tumor-infiltrating neutrophils is predictive for bevacizumab efficacy in glioblastoma patients: an AVAglio ancillary study

We previously identified matrix metalloproteinase 2 (MMP2) and MMP9 plasma levels as candidate biomarkers of bevacizumab activity in patients with recurrent glioblastoma. The aim of this study was to assess the predictive value of MMP2 and MMP9 in a randomized phase III trial in patients with newly diagnosed glioblastoma and to explore their tumor source. In this post hoc analysis of the AVAglio trial (AVAGlio/NCT00943826), plasma samples from 577 patients (bevacizumab, n = 283; placebo, n = 294) were analyzed for plasma MMP9 and MMP2 levels by enzyme-linked immunosorbent assay. A prospective local cohort of 38 patients with newly diagnosed glioblastoma was developed for analysis of tumor characteristics by magnetic resonance imaging and measurement of plasma and tumor levels of MMP9 and MMP2. In this AVAglio study, MMP9, but not MMP2, was correlated with bevacizumab efficacy. Patients with low MMP9 derived a significant 5.2-month overall survival (OS) benefit with bevacizumab (HR 0.51, 95% CI 0.34–0.76, p = 0.0009; median 13.6 vs. 18.8 months). In multivariate analysis, a significant interaction was seen between treatment and MMP9 (p = 0.03) for OS. In the local cohort, we showed that preoperative MMP9 plasma levels decreased after tumor resection and were correlated with tumor levels of MMP9 mRNA (p = 0.03). However, plasma MMP9 was not correlated with tumor size, invasive pattern, or angiogenesis. Using immunohistochemistry, we showed that MMP9 was expressed by inflammatory cells but not by tumor cells. After cell sorting, we showed that MMP9 was expressed by CD45+ immune cells. Finally, using flow cytometry, we showed that MMP9 was expressed by tumor-infiltrating neutrophils. In conclusion, circulating MMP9 is predictive of bevacizumab efficacy and is released by tumor-infiltrating neutrophils.

Features of the level of matrix metalloproteinase-2, -3, -9 and tissue inhibitors of metalloproteinases-1, -2, -3, -4 in the aqueous humor of patients with primary open-angle glaucoma

Aim. To study the content of matrix metalloproteinase (MMP)-2, -3, -9 and tissue inhibitors of metalloproteinases (TIMPs) -1, -2, -3, -4 in the aqueous humor of patients with moderate primary open-angle glaucoma (POAG).Materials and methods. The experimental group included 47 patients with verified moderate primary open-angle glaucoma. The control group consisted of 26 patients with uncomplicated cataract. The levels of MMP-2, -3, -9 were determined with Luminex Performance Human MMP Magnetic Panel 3-plex kit (R&D Systems, USA), the concentration of TIMPs-1, -2, -3, - 4 was determined with the Human TIMP Magnetic Luminex Performance Assay 4-plex kit (R&D Systems, USA). The study was carried out using flow-through field fluorometry on a Bio-Plex 200 double-beam laser analyzer (Bio-Rad, USA).Results. The study showed a statistically significant increase in the levels of matrix metalloproteinase-2 and tissue inhibitors of matrix metalloproteinases-1, -2, -3, -4 in the aqueous humor of patients with moderate POAG compared with patients with uncomplicated cataract.Conclusion. The obtained data on high concentrations and imbalance in the levels of matrix metalloproteinases and their tissue inhibitors in the aqueous humor of patients with moderate POAG confirm the role of local inflammation, as well as impairments in the structure of the extracellular matrix and its remodeling in the mechanisms of development of this pathology.

Sesquiterpene from Polygonum Barbatum Disrupt Mitochondrial Membrane Potential to Induce Apoptosis and Inhibits Metastasis by Downregulating Matrix Metalloproteinase and Osteopontin in NCI-H460 Cells

Background: Globally, lung cancer accounts for 18% of cancer-associated mortalities. Among the subtypes, non-small-cell lung cancer (NSCLC) is the most prevalent one. The increased resistance and poor survival rates, signifies disease aggressiveness and thus require a search for an alternative anticancer molecule. Hypothesis/Purpose: Earlier, the isolated sesquiterpene i.e. compound 1 ((E)-Methyl 6-acetoxy-7-methoxy-1-(2-methylpropylidene)-1H-indene-3-carboxylate) from P. barbatum was isolated, characterized by us and reported for preliminary anticancer activity. Considering its potent activity, this study was designed to explore the underlying molecular mechanism of apoptosis and metastasis against NCI-H460 cells. Method: The molecular mechanism of compound 1 inducing apoptosis and inhibiting metastasis was elucidated by analyzing mitochondrial membrane potential, DNA fragmentation, clonogenic assay, invasion assay and expression of apoptotic (Caspases 3, 6, 8, 9 and BAK) and metastatic markers (MMP 2, 9 and osteopontin). Results: Compound 1 significantly inhibited cell proliferation and induced apoptosis via intrinsic route i.e. the mitochondrial pathway by disrupting mitochondrial membrane potential. The enhanced expression of caspases 6, 9, BAK and HRK with downregulation of Bcl-2L1 and Ki67 further confirmed the involvement of the intrinsic apoptotic pathway. Moreover, compound 1 restricted the invasive nature of NCI-H460 cells evinced by reduced cell invasion in Boyden chamber invasion assay and downregulating the expression of metastatic markers i.e. matrix metalloproteinase 2 / 9 and VEGF. It was also found that it blocks osteopontin by negatively regulating its expression; a marker protein in cancer management. Conclusion: Conclusively, this sesquiterpene exhibited potent anticancer and anti-metastatic activity and can be explored further as possible pharmacophores

Cancer-Associated Stromal Cells Promote the Contribution of MMP2-Positive Bone Marrow-Derived Cells to Oral Squamous Cell Carcinoma Invasion

Tumor stromal components contribute to tumor development and invasion. However, the role of stromal cells in the contribution of bone marrow-derived cells (BMDCs) in oral squamous cell carcinoma (OSCC) invasion is unclear. In the present study, we created two different invasive OSCC patient-derived stroma xenografts (PDSXs) and analyzed and compared the effects of stromal cells on the relation of BMDCs and tumor invasion. We isolated stromal cells from two OSCC patients: less invasive verrucous OSCC (VSCC) and highly invasive conventional OSCC (SCC) and co-xenografted with the OSCC cell line (HSC-2) on green fluorescent protein (GFP)-positive bone marrow (BM) cells transplanted mice. We traced the GFP-positive BM cells by immunohistochemistry (IHC) and detected matrix metalloproteinase 2 (MMP2) expression on BM cells by double fluorescent IHC. The results indicated that the SCC-PDSX promotes MMP2-positive BMDCs recruitment to the invasive front line of the tumor. Furthermore, microarray analysis revealed that the expressions of interleukin 6; IL-6 mRNA and interleukin 1 beta; IL1B mRNA were higher in SCC stromal cells than in VSCC stromal cells. Thus, our study first reports that IL-6 and IL1B might be the potential stromal factors promoting the contribution of MMP2-positive BMDCs to OSCC invasion.

Carotid Artery Disease in Subjects with Type 2 Diabetes: Risk Factors and Biomarkers

Carotid atherosclerosis (CA) and, especially, carotid artery stenosis (CAS), are associated with a high risk of cardiovascular events in subjects with type 2 diabetes (T2D). In this study, we aimed to identify risk factors and biomarkers of subclinical CA and CAS in T2D individuals. High-resolution ultrasonography of carotid arteries was performed in 389 patients. Ninety-five clinical parameters were evaluated, including diabetic complications and comorbidities; antihyperglycemic, hypolipidemic, and antihypertensive therapy; indices of glycemic control and glucose variability (GV); lipid panels; estimated glomerular filtration rate (eGFR); albuminuria; blood cell count; and coagulation. Additionally, serum levels of calponin-1, relaxin, L-citrulline, and matrix metalloproteinase-2 and -3 (MMP-2, -3) were measured by ELISA. In univariate analysis, older age, male sex, diabetes duration, GV, diabetic retinopathy, chronic kidney disease, coronary artery disease, peripheral artery disease, and MMP-3 were associated with subclinical CA. In addition to these factors, long-term arterial hypertension, high daily insulin doses, eGFR, and L-citrulline were associated with CAS. In multivariate logistic regression, age, male sex, BMI, GV, and eGFR predicted CA independently; male sex, BMI, diabetes duration, eGFR, and L-citrulline were predictors of CAS. These results can be used to develop screening and prevention programs for CA and CAS in T2D subjects.

High Efficiency In Vitro Wound Healing of Dictyophora indusiata Extracts via Anti-Inflammatory and Collagen Stimulating (MMP-2 Inhibition) Mechanisms

Dictyophora indusiata or Phallus indusiatus is widely used as not only traditional medicine, functional foods, but also, skin care agents. Biological activities of the fruiting body from D. indusiata were widely reported, while the studies on the application of immature bamboo mushroom extracts were limited especially in the wound healing effect. Wound healing process composed of 4 stages including hemostasis, inflammation, proliferation, and remodelling. This study divided the egg stage of bamboo mushroom into 3 parts: peel and green mixture (PGW), core (CW), and whole mushroom (WW). Then, aqueous extracts were investigated for their nucleotide sequencing, biological compound contents, and wound healing effect. The anti-inflammatory determination via the levels of cytokine releasing from macrophages, and the collagen stimulation activity on fibroblasts by matrix metalloproteinase-2 (MMP-2) inhibitory activity were determined to serve for the wound healing process promotion in the stage 2–4 (wound inflammation, proliferation, and remodelling of the skin). All D. indusiata extracts showed good antioxidant potential, significantly anti-inflammatory activity in the decreasing of the nitric oxide (NO), interleukin-1 (IL-1), interleukin-1 (IL-6), and tumour necrosis factor-α (TNF-α) secretion from macrophage cells (p < 0.05), and the effective collagen stimulation via MMP-2 inhibition. In particular, CW extract containing high content of catechin (68.761 ± 0.010 mg/g extract) which could significantly suppress NO secretion (0.06 ± 0.02 µmol/L) better than the standard anti-inflammatory drug diclofenac (0.12 ± 0.02 µmol/L) and their MMP-2 inhibition (41.33 ± 9.44%) was comparable to L-ascorbic acid (50.65 ± 2.53%). These findings support that CW of D. indusiata could be an essential natural active ingredient for skin wound healing pharmaceutical products.

Anti-endometriotic Effects of Ceratonia Siliqua L. Pod on Endometrial Mesenchymal Stromal/stem Cells Isolated from Women with Endometriosis-associated Infertility

This study investigated the effects of carob (Ceratonia siliqua L.) pod extract (CPE) on human endometrial stem cells (ESCs) viability and to examine its impact on mRNA expression of methyltransferase (DNMT-1, DNMT-3A and DNMT-3B), histone deacetylase-1 (HDAC-1), matrix metalloproteinase-2 (MMP-2) and cyclooxygenase-2 (COX-2) in endometriotic patients. The ESCs were derived from endometrium of patients with endometrioma (OMA-ESCs) and deep infiltrative endometriosis samples of 10 women with endometriosis associated infertility (E-ESCs) were compared to the ESCs derived from endometrium of endometriosis free, normal women as control group (C-ESCs). The metabolic activity of control and case groups was evaluated by treating them with different concentrations of CPE. In the E-ESCs, treatment with 0.8 and 2 µg/mL of CPE resulted in downregulation of COX-2 and HDAC-1 compared to the control group (p = 0.02 and p = 0.02, respectively). Treatment with 0.8 µg/mL of CPE decreased MMP-2 and DNMT-3B genes expression (p = 0.02 and p = 0.03, respectively). Furthermore, COX-2 and DNMT-3A genes were significantly upregulated after treatment with 2 µg/mL of CPE. Expression of the COX-2, HDAC-1, DNMT-1, DNMT-3A, and DNMT-3B peptides decreased in E-ESCs, OMA-ESCs and C-ESCs after treatment with 0.8 and 2 µg/mL concentrations of the CPE. The GC analysis of the CPE resulted in 14 compounds with interactions with the target proteins through the docking process. In vitro CPE treatment significantly downregulated cell inflammatory pathway involved in the pathophysiology of endometriosis and may be a potential agent for treatment of endometriosis.