Halting Tumor Progression via Novel Non-Hydroxamate Triazole-Based Mannich Bases MMP-2/9 Inhibitors; Design,Microwave-Assisted Synthesis,and Biological Evaluation

Matrix metalloproteinases (MMPs) are key signaling modulators in the tumor microenvironment. Among MMPs, MMP-2 and MMP-9 are receiving renewed interest as validated druggable targets for halting different tumor progression events. Over the last decades, a diverse range of MMP-2/9 inhibitors has been identified starting from the early hydroxamic acid-based peptidomimetics to the next generation non-hydroxamates. Herein, focused 1,2,4-triazole-1,2,3-triazole molecular hybrids with varying lengths and decorations, mimicking the thematic features of non-hydroxamate inhibitors, were designed and synthesized using efficient protocols and were alkylated with pharmacophoric amines to develop new Mannich bases. After full spectroscopic characterization the newly synthesized triazoles tethering Mannich bases were subjected to safety assessment via MTT assay against normal human fibroblasts, then evaluated for their potential anticancer activities against colon (Caco-2) and breast (MDA-MB 231) cancers. The relatively lengthy bis-Mannich bases 15 and 16 were safer and more potent than 5-fluorouracil with sub-micromolar IC₅₀ and promising selectivity to the screened cancer cell lines rather than normal cells. Both compounds upregulated p53 (2–5.6-fold) and suppressed cyclin D expression (0.8–0.2-fold) in the studied cancers, and thus, induced apoptosis. 15 was superior to 16 in terms of cytotoxic activities, p53 induction, and cyclin D suppression. Mechanistically, both were efficient MMP-2/9 inhibitors with comparable potencies to the reference prototype hydroxamate-based MMP inhibitor NNGH at their anticancer IC₅₀ concentrations. 15 (IC₅₀ = 0.143 µM) was 4-fold more potent than NNGH against MMP-9 with promising selectivity (3.27-fold) over MMP-2, whereas 16 was comparable to NNGH. Concerning MMP-2, 16 (IC₅₀ = 0.376 µM) was 1.2-fold more active than 15. Docking simulations predicted their possible binding modes and highlighted the possible structural determinants of MMP-2/9 inhibitory activities. Computational prediction of their physicochemical properties, ADMET, and drug-likeness metrics revealed acceptable drug-like criteria.     

MMP-9 Deletion Attenuates Arteriovenous Fistula Neointima through Reduced Perioperative Vascular Inflammation

Matrix metalloproteinase 9 (MMP-9) expression is upregulated in vascular inflammation and participates in vascular remodeling, including aneurysm dilatation and arterial neointima development. Neointima at the arteriovenous (AV) fistula anastomosis site primarily causes AV fistula stenosis and failure; however, the effects of MMP-9 on perioperative AV fistula remodeling remain unknown. Therefore, we created AV fistulas (end-to-side anastomosis) in wild-type (WT) and MMP-9 knockout mice with chronic kidney disease to further clarify this. Neointima progressively developed in the AV fistula venous segment of WT mice during the four-week postoperative course, and MMP-9 knockout increased the lumen area and attenuated neointima size by reducing smooth muscle cell and collagen components. Early perioperative AV fistula mRNA sequencing data revealed that inflammation-related gene sets were negatively enriched in AV fistula of MMP-9 knockout mice compared to that in WT mice. qPCR results also showed that inflammatory genes, including tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), were downregulated. In addition, Western blot results showed that MMP-9 knockout reduced CD44 and RAC-alpha serine/threonine-protein kinase (Akt) and extracellular signal-regulated kinases (ERK) phosphorylation. In vitro, MMP-9 addition enhanced IL-6 and MCP-1 expression in vascular smooth muscle cells, as well as cell migration, which was reversed by an MMP-9 inhibitor. In conclusion, MMP-9 knockout attenuated AV fistula stenosis by reducing perioperative vascular inflammation.     

Association of MMP-2 and MMP-9 Polymorphisms with Diabetes and Pathogenesis of Diabetic Complications

Type 2 diabetes mellitus (T2D) affects millions of people around the world, and its complications have serious health consequences. In addition to external factors, the causes of morbidity and increased risk were also sought in the variability of the human genome. A phenomenon that can answer these questions is the occurrence of single-nucleotide polymorphisms (SNP). They constitute a field for research into genetic determinants responsible for the increase in the risk of the discussed metabolic disease. This article presents the outline of two enzymes: metalloproteinases 2 and 9 (MMP-2, MMP-9), their biological activity and the effect caused by differences in individual alleles in the population, as well as the reports on the importance of these DNA sequence variations in the occurrence of diabetes mellitus type 2 and associated conditions. The results of the conducted research indicate a relationship between two MMP-2 polymorphisms (rs243865, rs243866) and two MMP-9 polymorphisms (rs3918242, rs17576) and the presence of T2D. This could offer a promising possibility to use them as predictive and diagnostic markers. However, due to the low number of reports, more research is needed to clearly confirm the link between these SNPs and diabetes.     

Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.     

Matrix Metalloproteinases MMP-2 and MMP-9, Their Inhibitors TIMP-1 and TIMP-2, Vascular Endothelial Growth Factor and sVEGFR-2 as Predictive Markers of Ischemic Retinopathy in Patients with Systemic Sclerosis—Case Series Report

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder associated with multiple organ involvement. The aim of the study was to present two SSc patients who were diagnosed with ischemic retinopathy in both eyes. As a background to our case study, we decided to investigate the imbalance of angiogenesis factors in 25 SSc patients in relation to 25 healthy controls. Assays of matrix metalloproteinases-2 and -9 (MMP-2, MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1) and -2 (TIMP-2), vascular endothelial growth factor (VEGF), and soluble VEGF receptor-2 (sVEGFR-2) in blood serum and tears were performed. A significantly increased levels of MMP-9 in serum and tears, (p = 0.0375 and p < 0.001, respectively) as well as VEGF/sVEGFR-2 ratio in tears (p < 0.001) were found in the whole SSc patients group compared with controls, while reduced levels of these parameters in patients with ischemic sclerodermic retinopathy were noted. We also observed decreased level MMP-2 in tears and increased levels of TIMP-2 in blood serum and tears of SSc patients with retinal ischemic changes. MMP-9, MMP-2, TIMP-2, and VEGF/sVEGFR-2 may play a crucial role in ischemic retinal degeneration or retinal reorganization in SSc.     

Sinulariolide Suppresses Human Hepatocellular Carcinoma Cell Migration and Invasion by Inhibiting Matrix Metalloproteinase-2/-9 through MAPKs and PI3K/Akt Signaling Pathways

Sinulariolide is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigate the migration and invasion effects of sinulariolide in hepatocellular carcinoma cell HA22T. Sinulariolide inhibited the migration and invasion effects of hepatocellular carcinoma cells in a concentration-dependent manner. The results of zymography assay showed that sinulariolide suppressed the activities of matrix metalloproteinase (MMP)-2 and MMP-9. Moreover, protein levels of MMP-2, MMP-9, and urokinase-type plasminogen activator (uPA) were reduced by sinulariolide in a concentration-dependent manner. Sinulariolide also exerted an inhibitory effect on phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, Focal adhesion kinase (FAK), growth factor receptor-bound protein 2 (GRB2). Taken together, these results demonstrated that sinulariolide could inhibit hepatocellular carcinoma cell migration and invasion and alter HA22T cell metastasis by reduction of MMP-2, MMP-9, and uPA expression through the suppression of MAPKs, PI3K/Akt, and the FAK/GRB2 signaling pathway. These findings suggest that sinulariolide merits further evaluation as a chemotherapeutic agent for human hepatocellular carcinoma.     

健康牙齿和龋齿成牙本质细胞中MMP-2和MMP-9的活性

目的比较健康牙齿和龋齿成牙本质细胞中基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的活性,以探讨MMP-2和MMP-9在龋病发病机制中的作用。方法培养健康牙齿和龋齿成牙本质细胞,利用酶谱分析细胞培养上清中MMP-2和MMP-9的活性。结果龋齿组中MMP-2和MMP-9的活性显著高于健康组。结论MMP-2和MMP-9在健康牙齿和龋齿成牙本质细胞中的活性不同,提示MMPs可能在龋病的进展过程中发挥作用。     

Lewis (y) Antigen Overexpression Increases the Expression of MMP-2 and MMP-9 and Invasion of Human Ovarian Cancer Cells

Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable cell line constitutively expressing Lewis (y) antigen (RMG-1-hFUT) by transfecting the cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1, and investigated whether Lewis (y) antigen regulates the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and tissue inhibitors of metalloproteinases (TIMP-1) and TIMP-2. We found that RMG-1-hFUT cells exhibited higher invasive capacities than their control cells. In addition, expression of TIMP-1 and TIMP-2 was down-regulated and expression of MMP-2 and MMP-9 was up-regulated. Anti-Lewis (y) antigen antibody treatment significantly reversed the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9. Taken together, we provide the first evidence that down-regulation of TIMP-1 and TIMP-2 and up-regulation of MMP-2 and MMP-9 represents one of the mechanisms by which Lewis (y) antigen promotes cell invasion.     

Adipsin Serum Concentrations and Adipose Tissue Expression in People with Obesity and Type 2 Diabetes

(1) Adipsin is an adipokine that may link increased fat mass and adipose tissue dysfunction to obesity-related cardiometabolic diseases. Here, we investigated whether adipsin serum concentrations and adipose tissue (AT) adipsin mRNA expression are related to parameters of AT function, obesity and type 2 diabetes (T2D). (2) Methods: A cohort of 637 individuals with a wide range of age and body weight (Age: 18–85 years; BMI: 19–70 kg/m²) with (n = 237) or without (n = 400) T2D was analyzed for serum adipsin concentrations by ELISA and visceral (VAT) and subcutaneous (SAT) adipsin mRNA expression by RT-PCR. (3) Results: Adipsin serum concentrations were significantly higher in patients with T2D compared to normoglycemic individuals. We found significant positive univariate relationships of adipsin serum concentrations with age (r = 0.282, p < 0.001), body weight (r = 0.264, p < 0.001), fasting plasma glucose (r = 0.136, p = 0.006) and leptin serum concentrations (r = 0.362, p < 0.001). Neither VAT nor SAT adipsin mRNA expression correlated with adipsin serum concentrations after adjusting for age, sex and BMI. Independent of T2D status, we found significantly higher adipsin expression in SAT compared to VAT (4) Conclusions: Our data suggest that adipsin serum concentrations are strongly related to obesity and age. However, neither circulating adipsin nor adipsin AT expression reflects parameters of impaired glucose or lipid metabolism in patients with obesity with or without T2D.     

Salidroside, 8(E)-Nuezhenide,and Ligustroside from Ligustrum japonicum Fructus Inhibit Expressions of MMP-2 and -9 in HT 1080 Fibrosarcoma

A phenyl ethanoid, salidroside (SAL), and two secoiridoids, 8(E)-nuezhenide (NZD) and ligustroside (LIG), were isolated from fruits of Ligustrum japonicum, used as traditional folk medicine, and their chemical structures were elucidated by the comparison of spectral data with published literature. Matrix metalloproteinases (MMPs) are major enzymes that play crucial roles in the metastasis and invasive behavior of tumors. In particular, MMP-2 and MMP-9, regulated by the MAPK signaling pathways, including p38, ERK and JNK, are known to play a key role in the degradation of the basement membrane. In the present study, the effects of SAL, NZD and LIG on the expression of MMP-2 and -9 were examined in phorbol 12-myristate 13-acetate (PMA)-induced HT 1080 cells. All the compounds significantly lowered the amount of MMP-2 and MMP-9 released, as determined by gelatin zymography and ELISA. In addition, the mRNA and protein expression levels of MMP-2 and MMP-9 were significantly suppressed, as measured by RT-PCR and Western blotting. According to the Western blotting assay, SAL and LIG effectively reduced the expression of MMP-2 in a dose-dependent manner. NZD lowered the expression of MMP-9 in a similar way. The phosphorylation of p38, ERK and JNK was also significantly suppressed by these compounds. These findings suggest that all the compounds regulate the release and expression of MMP-2 and MMP-9 via MAPK signaling pathways.     

Pre-Treatment of Platinum Resistant Ovarian Cancer Cells with an MMP-9/MMP-2 Inhibitor Prior to Cisplatin Enhances Cytotoxicity as Determined by High Content Screening

Platinum resistance is a major cause of treatment failure in ovarian cancer. We previously identified matrix metalloproteinase 9 (MMP-9) as a potential therapeutic target of chemoresistant disease. A2780cis (cisplatin-resistant) and A2780 (cisplatin-sensitive) ovarian carcinoma cell lines were used. The cytotoxic effect of MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) alone or in combination with cisplatin was determined using high content screening. Protein expression was examined using immunohistochemistry and ELISA. Co-incubation of cisplatin and an MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) resulted in significantly greater cytotoxicity as compared to either treatment alone in a cisplatin resistant MMP-9 overexpressing cell line; A2780cis. In addition, pre-incubating with MMP-9i prior to cisplatin further enhances the cytotoxic effect. No significant difference was observed in MMP-9 protein in tissue but a trend towards increased MMP-9 was observed in recurrent serum. We propose that MMP-9/MMP-2i may be utilized in the treatment of recurrent/chemoresistant ovarian cancers that overexpress MMP-9 mRNA but its role in vivo remains to be evaluated.     

Lupinus albus Protein Components Inhibit MMP-2 and MMP-9 Gelatinolytic Activity In Vitro and In Vivo

Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are regarded as important clinical targets due to their nodal-point role in inflammatory and oncological diseases. Here, we aimed at isolating and characterizing am MMP-2 and-9 inhibitor (MMPI) from Lupinus albus and at assessing its efficacy in vitro and in vivo. The protein was isolated using chromatographic and 2-D electrophoretic procedures and sequenced by using MALDI-TOF TOF and MS/MS analysis. In vitro MMP-2 and 9 inhibitions were determined on colon adenocarcinoma (HT29) cells, as well as by measuring the expression levels of genes related to these enzymes. Inhibitory activities were also confirmed in vivo using a model of experimental TNBS-induced colitis in mice, with oral administrations of 15 mg·kg⁻¹. After chromatographic and electrophoretic isolation, the L. albus MMP-9 inhibitor was found to comprise a large fragment from δ-conglutin and, to a lower extent, small fragments of β-conglutin. In vitro studies showed that the MMPI successfully inhibited MMP-9 activity in a dose-dependent manner in colon cancer cells, with an IC50 of 10 µg·mL⁻¹ without impairing gene expression nor cell growth. In vivo studies showed that the MMPI maintained its bioactivities when administered orally and significantly reduced colitis symptoms, along with a very significant inhibition of MMP-2 and -9 activities. Overall, results reveal a novel type of MMPI in lupine that is edible, proteinaceous in nature and soluble in water, and effective in vivo, suggesting a high potential application as a nutraceutical or a functional food in pathologies related to abnormally high MMP-9 activity in the digestive system.     

COVID-19: Immunohistochemical Analysis of TGF-β Signaling Pathways in Pulmonary Fibrosis

Acute respiratory distress syndrome (ARDS) followed by repair with lung remodeling is observed in COVID-19. These findings can lead to pulmonary terminal fibrosis, a form of irreversible sequelae. There is evidence that TGF-β is intimately involved in the fibrogenic process. When activated, TGF-β promotes the differentiation of fibroblasts into myofibroblasts and regulates the remodeling of the extracellular matrix (ECM). In this sense, the present study evaluated the histopathological features and immunohistochemical biomarkers (ACE-2, AKT-1, Caveolin-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 tissue expression) involved in the TGF-β1 signaling pathways and pulmonary fibrosis. The study consisted of 24 paraffin lung samples from patients who died of COVID-19 (COVID-19 group), compared to 10 lung samples from patients who died of H1N1pdm09 (H1N1 group) and 11 lung samples from patients who died of different causes, with no lung injury (CONTROL group). In addition to the presence of alveolar septal fibrosis, diffuse alveolar damage (DAD) was found to be significantly increased in the COVID-19 group, associated with a higher density of Collagen I (mature) and III (immature). There was also a significant increase observed in the immunoexpression of tissue biomarkers ACE-2, AKT-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 in the COVID-19 group. A significantly lower expression of Caveolin-1 was also found in this group. The results suggest the participation of TGF-β pathways in the development process of pulmonary fibrosis. Thus, it would be plausible to consider therapy with TGF-β inhibitors in those patients recovered from COVID-19 to mitigate a possible development of pulmonary fibrosis and its consequences for post-COVID-19 life quality.     

Substituted Syndecan-2-Derived Mimetic Peptides Show Improved Antitumor Activity over the Parent Syndecan-2-Derived Peptide

We previously showed that a synthetic peptide (S2-P) corresponding to a portion of the human syndecan-2 (SDC2) sequence can bind to the pro-domain of matrix metalloproteinase-7 (MMP-7) to inhibit colon cancer activities. Since S2-P had a relatively weak binding affinity for the MMP-7 pro-domain, we herein modified the amino acid sequence of S2-P to improve the anticancer potential. On the basis of the interaction structure of S2-P and MMP-7, four peptides were generated by replacing amino acids near Tyr 51, which is critical for the interaction. The SDC2-mimetic peptides harboring an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-D) or with an Ala-to-Phe substitution at the N-terminal side of Tyr 51 and an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-FE) showed improved interaction affinities for the MMP-7 pro-domain. Compared to S2-P, S2-FE was better able to inhibit the SDC2–MMP-7 interaction, the cell surface localization of MMP-7, the gelatin degradation activity of MMP-7, and the cancer activities (cell migration, invasion, and colony-forming activity) of human HCT116 colon cancer cells in vitro. In vivo, S2-FE inhibited the primary tumor growth and lung metastasis of CT26 mouse colon cancer cells in a xenograft mouse model. Together, these data suggest that S2-FE could be useful therapeutic anticancer peptides for colon cancer.     

Estrogen Receptor Subtypes Elicit a Distinct Gene Expression Profile of Endothelial-Derived Factors Implicated in Atherosclerotic Plaque Vulnerability

In the presence of established atherosclerosis, estrogens are potentially harmful. MMP-2 and MMP-9, their inhibitors (TIMP-2 and TIMP-1), RANK, RANKL, OPG, MCP-1, lysyl oxidase (LOX), PDGF-β, and ADAMTS-4 play critical roles in plaque instability/rupture. We aimed to investigate (i) the effect of estradiol on the expression of the abovementioned molecules in endothelial cells, (ii) which type(s) of estrogen receptors mediate these effects, and (iii) the role of p21 in the estrogen-mediated regulation of the aforementioned factors. Human aortic endothelial cells (HAECs) were cultured with estradiol in the presence or absence of TNF-α. The expression of the aforementioned molecules was assessed by qRT-PCR and ELISA. Zymography was also performed. The experiments were repeated in either ERα- or ERβ-transfected HAECs and after silencing p21. HAECs expressed only the GPR-30 estrogen receptor. Estradiol, at low concentrations, decreased MMP-2 activity by 15-fold, increased LOX expression by 2-fold via GPR-30, and reduced MCP-1 expression by 3.5-fold via ERβ. The overexpression of ERα increased MCP-1 mRNA expression by 2.5-fold. In a low-grade inflammation state, lower concentrations of estradiol induced the mRNA expression of MCP-1 (3.4-fold) and MMP-9 (7.5-fold) and increased the activity of MMP-2 (1.7-fold) via GPR-30. Moreover, p21 silencing resulted in equivocal effects on the expression of the abovementioned molecules. Estradiol induced different effects regarding atherogenic plaque instability through different ERs. The balance of the expression of the various ER subtypes may play an important role in the paradoxical characterization of estrogens as both beneficial and harmful.     

Adiponectin and Leptin Exert Antagonizing Effects on HUVEC Tube Formation and Migration Modulating the Expression of CXCL1, VEGF,MMP-2 and MMP-9

Adiponectin and leptin are two abundant adipokines with different properties but both described such as potent factors regulating angiogenesis. AdipoRon is a small-molecule that, binding to AdipoRs receptors, acts as an adiponectin agonist. Here, we investigated the effects of AdipoRon and leptin on viability, migration and tube formation on a human in vitro model, the human umbilical vein endothelial cells (HUVEC) focusing on the expression of the main endothelial angiogenic factors: hypoxia-inducible factor 1-alpha (HIF-1α), C-X-C motif chemokine ligand 1 (CXCL1), vascular endothelial growth factor A (VEGF-A), matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9). Treatments with VEGF-A were used as positive control. Our data revealed that, at 24 h treatment, proliferation of HUVEC endothelial cells was not influenced by AdipoRon or leptin administration; after 48 h longer exposure time, the viability was negatively influenced by AdipoRon while leptin treatment and the combination of AdipoRon+leptin produced no effects. In addition, AdipoRon induced a significant increase in complete tubular structures together with induction of cell migration while, on the contrary, leptin did not induce tube formation and inhibited cell migration; interestingly, the co-treatment with both AdipoRon and leptin determined a significant decrease of the tubular structures and cell migration indicating that leptin antagonizes AdipoRon effects. Finally, we found that the effects induced by AdipoRon administration are accompanied by an increase in the expression of CXCL1, VEGF-A, MMP-2 and MMP-9. In conclusion, our data sustain the active role of adiponectin and leptin in linking adipose tissue with the vascular endothelium encouraging the further deepening of the role of adipokines in new vessel’s formation, to candidate them as therapeutic targets.     

Serum Level of MMP-3 and MMP-9 in Patients with Diabetes Mellitus Type 2 Infected with Epstein-Barr Virus

Diabetes mellitus type 2 (DM2) has recently become one of the most important health problems in the world. Patients with DM2 with long-term glycaemia are more likely to become infected than the healthy population. Matrix metalloproteinases (MMPs) play a key role in tissue remodeling during various physiological processes. However, it has been reported that certain MMPs are overexpressed during the development of various human diseases. In this study, we analyzed the levels of MMP-3 and MMP-9 in the serum of DM2 patients with and without Epstein-Barr virus (EBV) infection. The study included 115 patients with DM2 hospitalized in the Internal Ward of the Masovian Specialist Hospital in Radom, Poland, who were divided into two groups: EBV-positive and EBV-negative. The levels of MMP-3 and MMP-9 were tested in the serum of patients using the ELISA method, while the presence of EBV in saliva was tested by polymerase chain reaction (PCR). The presented studies showed a significant difference in the concentration of both MMPs in diabetic patients additionally infected with EBV compared to the group of non-infected individuals. It seems that MMPs may be useful biomarkers in the diagnosis, prognosis, and monitoring of diabetes associated with EBV infection.     

Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.     

ADAM8-Dependent Extracellular Signaling in the Tumor Microenvironment Involves Regulated Release of Lipocalin 2 and MMP-9

The metalloprotease-disintegrin ADAM8 is critically involved in the progression of pancreatic cancer. Under malignant conditions, ADAM8 is highly expressed and could play an important role in cell–cell communication as expression has been observed in tumor and immune cells of the tumor microenvironment (TME) such as macrophages. To analyze the potential role of ADAM8 in the TME, ADAM8 knockout PDAC tumor cells were generated, and their release of extracellular vesicles (EVs) was analyzed. In EVs, ADAM8 is present as an active protease and associated with lipocalin 2 (LCN2) and matrix metalloprotease 9 (MMP-9) in an ADAM8-dependent manner, as ADAM8 KO cells show a lower abundance of LCN2 and MMP-9. Sorting of ADAM8 occurs independent of TSG101, even though ADAM8 contains the recognition motif PTAP for the ESCRTI protein TSG101 within the cytoplasmic domain (CD). When tumor cells were co-cultured with macrophages (THP-1 cells), expression of LCN2 and MMP-9 in ADAM8 KO cells was induced, suggesting that macrophage signaling can overcome ADAM8-dependent intracellular signaling in PDAC cells. In co-culture with macrophages, regulation of MMP-9 is independent of the M1/M2 polarization state, whereas LCN2 expression is preferentially affected by M1-like macrophages. From these data, we conclude that ADAM8 has a systemic effect in the tumor microenvironment, and its expression in distinct cell types has to be considered for ADAM8 targeting in tumors.