TIMP-1 Gene Expression Research Articles

Enhancer of zeste homolog 2 (EZH2) gene inhibition via 3-Deazaneplanocin A (DZNep) in human liver cells and it is relation with fibrosis

Abstract Objectives Hepatic fibrosis is a complex and dynamic process, such as “wound healing”. The effect of 3-deazaneplanocin A (DZNep) via enhancer of zeste homolog 2 (EZH2) inhibition on transforming growth factor, matrix metalloproteinases 2-9 (MMP2, MMP9) and matrix metalloproteinases inhibitor 3 (TIMP3), alpha-smooth muscle actin (α-SMA), collagen 1A1(COL1A1), and collagen 3A1 (COL3A1) genes/proteins in human hepatic stellate cell line were examined. Inhibition effect of EZH2 on colony formation and migration were investigated. Methods 5 μM DZNep was treated to LX2 cells for 3 days. Real time PCR and Western blot method were used for analyses. Results DZNep was shown to reduce colony formation and migration. It increased the α-SMA only in gene expression level but decreased at the protein levels. It down-regulated both gene expression and protein levels of EZH2, while up-regulated gene expression and protein levels of TGF-β. DZNep induced gene expression and protein levels of MMP2 and TIMP3. However, DZNep induced COL1A1 and COL3A1 gene expression levels but reduced protein levels only in COL3A1 but not COL1A1. Conclusion DZNep has a positive anti-fibrotic effect by reducing α-SMA and COL3A1 protein levels in the LX2 cell line, however, we have found that DZNep may also has fibrotic effect because it increases TGF-β both expression and protein levels.

Therapeutic Potential of Dental Pulp Stem Cells and Leukocyte- and Platelet-Rich Fibrin for Osteoarthritis.

Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13, TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects.

Towards Understanding Therapeutic Failures in Masquelet Surgery: First Evidence that Defective Induced Membrane Properties are Associated with Clinical Failures.

The two-stage Masquelet induced-membrane technique (IMT) consists of cement spacer-driven membrane induction followed by an autologous cancellous bone implantation in this membrane to promote large bone defect repairs. For the first time, this study aims at correlating IMT failures with physiological alterations of the induced membrane (IM) in patients. For this purpose, we compared various histological, immunohistochemical and gene expression parameters obtained from IM collected in patients categorized lately as successfully (Responders; n = 8) or unsuccessfully (Non-responders; n = 3) treated with the Masquelet technique (6 month clinical and radiologic post-surgery follow-up). While angiogenesis or macrophage distribution pattern remained unmodified in non-responder IM as compared to responder IM, we evidenced an absence of mesenchymal stem cells and reduced density of fibroblast-like cells in non-responder IM. Furthermore, non-responder IM exhibited altered extracellular matrix (ECM) remodeling parameters such as a lower expression ratio of metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinases (TIMP-1) mRNA as well as an important collagen overexpression as shown by picrosirius red staining. In summary, this study is the first to report evidence that IMT failure can be related to defective IM properties while underlining the importance of ECM remodeling parameters, particularly the MMP-9/TIMP-1 gene expression ratio, as early predictive biomarkers of the IMT outcome regardless of the type of bone, fracture or patient characteristics.

Interaction of 8-oxoguanine DNA Glycosylase-1 with DNA: RNA Hybrid -Epigenetic Regulation of TIMP1 Gene Expression

Interaction of 8-oxoguanine DNA Glycosylase-1 with DNA: RNA Hybrid -Epigenetic Regulation of TIMP1 Gene Expression

Global transcriptome analysis of rat hypothalamic arcuate nucleus demonstrates reversal of hypothalamic gliosis following surgically and diet induced weight loss

The central mechanisms underlying the marked beneficial metabolic effects of bariatric surgery are unclear. Here, we characterized global gene expression in the hypothalamic arcuate nucleus (Arc) in diet-induced obese (DIO) rats following Roux-en-Y gastric bypass (RYGB). 60 days post-RYGB, the Arc was isolated by laser-capture microdissection and global gene expression was assessed by RNA sequencing. RYGB lowered body weight and adiposity as compared to sham-operated DIO rats. Discrete transcriptome changes were observed in the Arc following RYGB, including differential expression of genes associated with inflammation and neuropeptide signaling. RYGB reduced gene expression of glial cell markers, including Gfap, Aif1 and Timp1, confirmed by a lower number of GFAP immunopositive astrocyte profiles in the Arc. Sham-operated weight-matched rats demonstrated a similar glial gene expression signature, suggesting that RYGB and dietary restriction have common effects on hypothalamic gliosis. Considering that RYGB surgery also led to increased orexigenic and decreased anorexigenic gene expression, this may signify increased hunger-associated signaling at the level of the Arc. Hence, induction of counterregulatory molecular mechanisms downstream from the Arc may play an important role in RYGB-induced weight loss.

Staphylococcus aureus on the effect of expression of MMPs/TIMPs and uPA system in bovine mammary fibroblasts.

Bovine mammary fibrosis is characteristic of chronic in injury in response to diverse pathogens. Staphylococcus aureus (S.aureus) is a frequent cause of mastitis in bovine and is prone to persistent infection. Diverse studies have shown MMPs/TIMPs and uPA system as a potent target for the treatment of fibrosis. However, pathogenesis of S. aureus-induced mammary fibrosis has not been completely defined. BMFBs treated with heat-inactivated S. aureus (105, 106, and 108CFU/mL) for 6, 12, 24, and 48h. Total RNA and protein were isolated from the treatments and controls of BMFBs samples. MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, COL Ⅰ, uPA, uPAR and PAI-1 gene and protein expression were examined by RT-qPCR and Western blot analysis. Gelatin zymography assay was performed to assess the levels of MMP-2 and MMP-9 enzyme secreted. BMFBs treated with heat-inactivated S. aureus increased mRNA and protein expression levels of MMP-1, MMP-2, MMP-3, MMP-9 and MMP-13, and heat-inactivated S. aureus induced TIMP-1, TIMP-2 and COL Ⅰ expression. There was a clear activation of MMP-2 in the presence of heat-inactivated S. aureus in the conditioned medium from the BMFBs, whereas MMP-9 was no significantly altered. Moreover, uPA system was activated in BMFBs to S. aureus. Activation of the uPA system together with its impact on the MMPs levels could play a significant role in S. aureus-induced BMFBs with mechanism of ECM metabolism, MMPs/TIMPs and uPA system could participate in bovine mammary fibrosis.

Carbon Nanomaterials Stimulate HMGB1 Release From Macrophages and Induce Cell Migration and Invasion.

Carbon nanomaterials (CNMs) are widely used in industrial and medical sectors. The increasing exposure of CNMs necessitates the studies of their potential environmental and health effects. High-mobility group box-1 (HMGB1) is a nuclear DNA-binding protein, but when released from cells, may cause sustained inflammatory response and promote cell migration and invasion. In this work, we found that 7-day exposure of 2.5 mg/kg/day CNMs, including C60, single-walled carbon nanotubes, and graphene oxides significantly elevated the level of HMGB1 in blood and lung lavage fluids in C57BL/6 mice. Subsequently, cellular effects and underlying mechanism were explored by using Raw264.7. The results showed that noncytotoxic CNMs enhanced HMGB1 intracellular translocation and release via activating P2X7 receptor. Released HMGB1 further activated receptor for advanced glycation endproducts (RAGE) and downstream signaling pathway by upregulating RAGE and Rac1 expression. Simultaneously, CNMs prepared the cells for migration and invasion by modulating MMP2 and TIMP2 gene expression as well as cytoskeleton reorganization. Intriguingly, released HMGB1 from macrophages promoted the migration of nearby lung cancer cell, which can be efficiently inhibited by neutralizing antibodies against HMGB1 and RAGE. Taken together, our work demonstrated that CNMs stimulated HMGB1 release and cell migration/invasion through P2X7R-HMGB1-RAGE pathway. The revealed mechanisms might facilitate a better understanding on the inflammatory property and subsequent cell functional alteration of CNMs.

Morus alba Stem Extract Suppresses Matrix Metalloproteinases (MMP)-1, MMP-9, and Tissue Inhibitors of Metalloproteinase (TIMP)-1 Expression via Inhibition of IκBα Degradation Induced by Porphyromonas gingivalis LPS Signal in THP-1 Cells.

Objectives The aim of this study is to evaluate the inhibitory effects of M. alba stem extract (MSE) on the expression of matrix metalloproteinases (MMP)-1, MMP-9, and tissue inhibitors of metalloproteinase (TIMP)-1 in Porphyromonas gingivalis lipopolysaccharide (LPS)-activated-acute monocytic leukemia cell line (THP-1). Materials and Methods THP-1 cells were treated with noncytotoxic concentrations of MSE combined with 1 µg/mL of P. gingivalis LPS. The mRNA levels of MMP-1, MMP-9, and TIMP-1 were evaluated via quantitative real-time polymerase chain reaction. The secreted proteins in the culture media were detected by enzyme-linked immunosorbent assay. The degradation of inhibitor of kappa B-alpha (IκBα) protein was tracked by Western blotting. Statistical Analysis Comparisons in experiments were analyzed with analysis of variance followed by Tukey honestly significant difference comparison test. Results Twenty and 40 µg/mL of MSE significantly downregulated MMP-1 and MMP-9 genes and protein expression but upregulated the gene expression of TIMP-1 ( p < 0.05). P. gingivalis LPS induced degradation of IκBα, while addition of MSE (20 and 40 µg/mL) increased IκBα cytosolic levels. MSE was able to suppress the P. gingivalis LPS-induced MMPs expression and also increased the gene expression of TIMP-1 via the inhibition of the cytoplasmic IκBα degradation in THP-1 cells. Conclusions The present observations suggest that MSE exerted a positive effect on the regulatory mechanism between MMPs and TIMP, which is an important implication for the therapeutic potential of MSE in periodontitis.

The effect of chrysin–curcumin-loaded nanofibres on the wound-healing process in male rats

Aim The aim of the present study was to produce chrysin–curcumin-loaded PCL-PEG nanofibres by an electrospinning technique and to evaluate the biological activity of the chrysin–curcumin-loaded PCL-PEG fibres for wound healing and its related genes using in vivo methods. Materials and methods The electrospinning method was carried out for the preparation of the chrysin, curcumin and chrysin–curcumin-loaded PCL-PEG nanofibres with different concentrations. FTIR and SEM were performed to characterize the chemical structures and morphology of the nanofibres. In vitro drug release, as well as in vivo wound-healing studies were investigated in male rats. The expressions of genes related to the wound-healing process were also evaluated by real-time PCR. Results Our study showed that the chrysin–curcumin-loaded nanofibres have anti-inflammatory properties in several stages of the wound-healing process by affecting the IL-6, MMP-2, TIMP-1, TIMP-2 and iNOS gene expression. Our results demonstrated that the effect of the chrysin-loaded nanofibre, the curcumin-loaded nanofibre and the chrysin–curcumin-loaded nanofibre in the wound-healing process is dose dependent and in accordance with the obtained results in that it might affect the inflammation phase more than the other stages of the wound-healing process. Conclusion We have introduced chrysin–curcumin-loaded PCL-PEG nanofibres as a novel compound for shortening the duration of the wound-healing process.

Effects of proteasome inhibitors on cytokines, metalloproteinases and their inhibitors and collagen type-I expression in periprosthetic tissues and fibroblasts from loose arthroplasty endoprostheses

ABSTRACTObjective: Aseptic loosening is a major problem in total joint replacement. Implant wear debris provokes a foreign body host response and activates cells to produce a variety of mediators and ROS, leading to periprosthetic osteolysis. Elevated ROS levels can harm proteasome function. Proteasome inhibitors have been reported to alter the secretory profile of cells involved in inflammation and also to induce ROS production. In this work, we aimed to document the effects of proteasome inhibitors MG-132 and Epoxomicin, on the production of factors involved in aseptic loosening, in periprosthetic tissues and fibroblasts, and investigate the role of proteasome impairment in periprosthetic osteolysis.Materials and methods: IL-6 levels in tissue cultures were determined by sandwich ELISA. MMP-1, -3, -13, -14 and TIMP-1 levels in tissue or cell cultures were determined by indirect ELISA. Results for MMP-1 and TIMP-1 in tissue cultures were confirmed by Western blotting. MMP-2 and MMP-9 levels were determined by gelatin zymography. Gene expression of IL-6, MMP-1,-3,-14, TIMP-1 and collagen type-I was determined by RT-PCR.Results: Results show that proteasome inhibition induces the expression of ΜΜΡ-1, -2, -3, -9 and suppresses that of IL-6, MMP-14, -13, TIMP-1 and collagen type I, enhancing the collagenolytic and gelatinolytic activity already present in periprosthetic tissues, as documented in various studies.Conclusions: These findings suggest that proteasome impairment could be a contributing factor to aseptic loosening. Protection and enhancement of proteasome efficacy could thus be considered as an alternative strategy toward disease treatment.

Identification of signaling pathways mediating human chondrocyte degeneration induced by the chemokine CCL-2

Purpose: Increased expression of chemokines in cartilage, synovial membrane and subchondral bone are believed to be linked to osteoarthritis (OA). The chemokine receptor CCR2 and its major human circulating ligand CCL2 (a.k.a. MCP-1), have been recognized as important potential targets in OA and have been shown also to mediate OA pain. In previous clinical studies, we determined that CCL2 serum levels at baseline were significantly associated with radiographic knee OA progression and joint space narrowing at 5-year follow up. Our findings in a murine model of OA based on the destabilization of the medial meniscus (DMM), showed that the articular cartilage of OA knees had increased protein levels of the chemokine CCL12, a murine CCL2 homologue that binds the same CCR2 receptor, while it was undetectable in controls; CCL12 protein levels increased progressively with OA severity. Interestingly, systemic blockage of the CCR2 at early OA stages, led to decreased levels of matrix metalloproteinases (MMPs) in articular cartilage, accompanied by reduced joint damage and pain. The aim of the present study was to analyze downstream mediators of CCL2/CCR2 signaling in human articular chondrocytes that lead to the activation of genes involved in cartilage degradation. Methods: We used human articular chondrocytes from normal cartilage obtained from tissue donors to determine the signaling pathways activated by CCL2 (through CCR2) that lead to production of MMPs. Human chondrocyte primary cultures were treated with or without recombinant CCL2 (20ng/ml) in serum free medium for 5, 10 and 15 minutes. Cell lysates were collected and subjected to Immunoblotting (IB) using antibodies against phospho-ERK, phospho-p38 and phospho-JNK. To determine whether CCL2 treatments led to changes in the expression of genes involved in cartilage degeneration, human articular chondrocytes with or without recombinant CCL2 were incubated in serum free medium for mRNA collection; two different incubations were performed, a short treatment (6h) and a long treatment (18-24h). Quantitative RT-PCR analyses were performed to quantify the gene expression of mmp-1, mmp-3, mmp-13, comp and timp-1 using TaqMan method. Gene expression for the ccr2 receptor was also assessed. In some experiments, human articular chondrocytes were pre-incubated with the ERK inhibitor U0126 (10 μM) for 1 hour prior to CCL2 treatment, and then IB (for phospho-Erk) or qRT-PCR analyses (for mmp-13 and mmp-3) were performed. Results: CCL2 treatments of human chondrocytes led to activation of phospho-ERK and phospho-p38, but not phospho-JNK. When gene expression was assessed by RT-PCR, both short (6h) and long (18-24h) CCL2 treatments led to an upregulation of ccr2 expression compared to untreated controls, indicating a CCL2 positive feedback on its receptor. Both short and long CCL2 treatments also led to an upregulation in the expression of mmp’s genes (mmp-1, mmp-3, and mmp-13), as well as an increased expression of the tissue inhibitor of metalloproteinases-1 (timp-1) and comp, which has been demonstrated to be a biomarker for cartilage breakdown. Inhibition of ERK signaling by U0126 abolished the CCL-2-induced upregulation of mmp-13, at both the short and long incubation times (Figure 1). Interestingly, inhibition of ERK signaling did not significantly affect mmp-3 upregulation by CCL-2. Conclusions: Our data suggest that CCL2 stimulation of articular cartilage chondrocytes acts as positive feedback on its CCR2 receptor leading to cartilage degradation. The triggering of the CCL2/CCR2 axis leads to the activation of specific MAPK signaling pathways, such as ERK and p38 signaling. In particular, our results suggest that ERK signaling mediates the CCL-2-induced mmp-13 activation but may not be involved, or only partially involved, in the mmp-3 upregulation. Additional studies are currently in progress in our laboratory to determine whether inhibition of ERK and p38 is correlated to the expression of different genes involved in cartilage metabolism and degradation (such as mmp-1, timp-1, comp), following short or long CCL-2 treatments.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CDK8 Selectively Promotes the Growth of Colon Cancer Metastases in the Liver by Regulating Gene Expression of TIMP3 and Matrix Metalloproteinases.

: Unresectable hepatic metastases of colon cancer respond poorly to existing therapies and are a major cause of colon cancer lethality. In this study, we evaluated the therapeutic viability of targeting the mediator kinase CDK8, an early clinical stage drug target, as a means to suppress metastasis of colon cancer. CDK8 was amplified or overexpressed in many colon cancers and CDK8 expression correlated with shorter patient survival. Knockdown or inhibition of CDK8 had little effect on colon cancer cell growth but suppressed metastatic growth of mouse and human colon cancer cells in the liver. This effect was due in part to inhibition of already established hepatic metastases, indicating therapeutic potential of CDK8 inhibitors in the metastatic setting. In contrast, knockdown or inhibition of CDK8 had no significant effect on the growth of tumors implanted subcutaneously, intrasplenically, or orthotopically in the cecum. CDK8 mediated colon cancer growth in the liver through downregulation of matrix metalloproteinase (MMP) inhibitor TIMP3 via TGFβ/SMAD-driven expression of a TIMP3-targeting microRNA, miR-181b, along with induction of Mmp3 in murine or MMP9 in human colon cancer cells via Wnt/β-catenin-driven transcription. These findings reveal a new mechanism for negative regulation of gene expression by CDK8 and a site-specific role for CDK8 in colon cancer hepatic metastasis. Our results indicate the utility of CDK8 inhibitors for the treatment of colon cancer metastases in the liver and suggest that CDK8 inhibitors may be considered in other therapeutic settings involving TGFβ/SMAD or Wnt/β-catenin pathway activation. SIGNIFICANCE: These findings demonstrate that inhibition of the transcription-regulating kinase CDK8 exerts a site-specific tumor-suppressive effect on colon cancer growth in the liver, representing a unique therapeutic opportunity for the treatment of advanced colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/23/6594/F1.large.jpg.

Molecularly targeted anti-cancer drugs inhibit the invasion and metastasis of hepatocellular carcinoma by regulating the expression of MMP and TIMP gene families

To investigate the effect of multi-kinase kinase inhibitors (sorafenib; regorafenib; lenvatinib) on the invasion and metastasis of human hepatocellular carcinoma (HCC) cells, and the outcome of this effect on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), yet unclarified. Cells were subjected to four different treatments: blank control group, sorafenib (10 μmol/L) treatment group, regorafenib (20 mmol/L) treatment group, and lenvatinib (4 μmol/L) treatment group. Anti-invasion and anti-metastasis effects were tested using the wound-healing assay and transwell invasion assay. Real-time PCR and Western blot analyses were used to determine the impact of sorafenib, regorafenib, and lenvatinib on the gene expression of MMPs and TIMPs in the two HCC lines (Hep3B and SMMC-7721). Results from the wound-healing and transwell invasion assays showed the three tested anti-cancer drugs to have a significant inhibitory effect on the metastasis and invasion of HCC cells. Real-time PCR and western blot analyses revealed that sorafenib down-regulated the expressions of MMP-7,10,16 and up-regulated those of TIMP-1,3,4, regorafenib down-regulated the expression of MMP-1 and up-regulated TIMP-3 gene expression, and lenvatinib down-regulated the expressions of MMP-1,2,7,9,10,16 and up-regulated those of TIMP-1,3,4. However, these three targeted anti-cancer drugs seem to have no significant regulatory effect on the expressions of other MMPs and TIMPs family genes. In conclusion, sorafenib, regorafenib, and lenvatinib inhibit the invasion and metastasis of HCC cells by regulating MMPs/TIMPs expression levels.

Perubahan Rasio Ekspresi mRNA MMP-8/TIMP-1 Setelah Aplikasi Gel Ekstrak Bunga Rosella 10% pada Pasien dengan Gingivitis Setelah Pemasangan Mahkota Tiruan Akrilik

The acrylic-resin crown is still used as a choice in the manufacture of artificial crown because it is easy in the procedure. However accumulation plaque around cervical of the acrylic-resin crown can lead to the occurrence of gingivitis. Some research found that degradation of ECM by MMP-8 and TIMP-1 as a inhibitor during gingivitis. Roselle as a herbal plant in the treatment began to increase. Roselle is rich in Phytochemical compound which has been shown to reduce inflammation. This research determined effectiveness of roselle extract gel 10% in reducing gingivitis in patients using acrylic-resin crown and related to changes in mRNA MMP-8/TIMP-1 gene expression ratio. The subjects were 9 patients who has gingivitis post resin-acrylic crown insertion and divided in to three groups treatment with roselle extract gel 10%, negative control (base gel), and positive control (Povidon iodine). GCF samples were taken in cervical area of acrylic-resin crown with paper strips before and seven days after application of roselle extract gel 10%. The change of ratio MMP-8 and TIMP-1 m RNA gene expression is tested using Real Time-Polymerase Chain Reaction. Paired- T test shows on negative control group (p=0,301, p&lt;0,05) does not show any change ratio of mRNA MMP- 8/TIMP-1, on positive control, there is changing ratio but not significant(p =0,060). The treatment group shows significant change in ratio of mRNA MMP-8 /TIMP-1 gene expression(p=0,018) Conclusion, Extract Roselle gel 10% can reduce the gingivitis and change the ratio of MMP-8 / TIMP 1 mRNA.

Expression profiles of selected genes in tumors and matched surgical margins in oral cavity cancer: Do we have to pay attention to the molecular analysis of the surgical margins?

Head and neck squamous cell carcinomas (HNSCCs) are associated with an interplay between genetics and the environment; they account for 3% of all diagnosed malignant tumors in men and 2% of those in women. The aim of the study was to analyze the significance of TIMP3, SFRP1, SFRP2, CDH1, RASSF1, RORA, and DAPK1 gene expression in head and neck squamous cell carcinoma tumors, and in matching surgical margin samples. We also analyzed the association between clinical parameters and the expression of the selected genes. Following surgical resection, 56 primary HNSCC tumors and matching surgical margin samples were collected from patients at the Clinic of Oncological and Reconstructive Surgery of Maria Skłodowska-Curie Memorial Cancer Center and the Institute of Oncology in Gliwice, Poland. The gene expression levels were analyzed by quantitative reverse transcription (qRT)-PCR. SFRP1 gene expression was statistically significantly lower in the tumor samples than in the surgical margins (0.30 ±0.36 vs 0.62 ±0.36; p < 0.01). No correlation was found between gene expression and clinical parameters, except DAPK1, where low expression correlated with alcohol abuse (0.85 ±1.19 vs 1.97 ±3.22; p = 0.074). Moreover, patients with G3 grade tumors, i.e., poorly differentiated tumors, had significantly higher values of DAPK1 gene expression than the G1 (well-differentiated tumors) and G2 (moderately differentiated) groups. There are many different reasons and concepts for altered gene expression in tumors and surgical margin tissue. Tumor heterogeneity and its microenvironment are undoubtedly linked to the biology of HNSCC. In order to understand specific tumor behavior and the microenvironment, further studies are needed. To find markers connected with cancer development and to provide insight into the earliest stages of cancer development, attention should also be focused on molecular analysis of the surgical margins.

Angiotensin receptor neprilysin inhibition provides superior cardioprotection compared to angiotensin converting enzyme inhibition after experimental myocardial infarction

BackgroundAngiotensin receptor neprilysin inhibitor (ARNi) enhances beneficial natriuretic peptides by inhibiting their breakdown through neprilysin. Although the first-in-class ARNi sacubitril/valsartan (LCZ696) reduced mortality and morbidity in heart failure (HF) with reduced ejection fraction (EF) compared to angiotensin converting enzyme inhibitor (ACEi), mechanistic data on ARNi are scarce. ARNi may be superior to ACEi in attenuating adverse cardiac remodeling and dysfunction post-myocardial infarction (MI). MethodsRats randomized at 1 week post-MI were administered LCZ696 (60 mg/kg, N = 12), the ACEi perindopril (2 mg/kg, N = 11) or vehicle (corn oil, N = 13), orally for 4 weeks. Sham rats received vehicle (corn oil, N = 9). Echocardiography was assessed before and after treatment, prior to invasive hemodynamics using pressure-volume analysis. Hypertrophy and fibrosis was evaluated by histochemical staining, and analysis of myocardial gene and protein expression using real-time quantitative PCR and Western blot. ResultsCompared to Sham, MI groups had large infarcts (>40%) and reduced left ventricular (LV) EF. LCZ696 improved LVEF and end systolic pressure-volume relationship compared to perindopril (P < 0.05). LCZ696 but not perindopril reduced lung weight and LV filling pressures post-MI. Reductions in cardiac hypertrophy and fibrosis were similar, however gene expression of hypertrophic markers, ANP and βMHC were reduced with LCZ696 versus perindopril. LCZ696 versus perindopril reduced myocardial TIMP2 gene expression with a trend (P = 0.067) to lowering collagen I. ConclusionLCZ696 attenuated adverse cardiac remodeling and dysfunction and reduced pulmonary congestion and hypertrophic markers after MI compared to perindopril. This study supports clinical evaluation of ARNi versus ACEi in targeting post-MI cardiac dysfunction and remodeling.

Glucocorticoid sensitivity and inflammatory status of peripheral blood mononuclear cells in patients with coronary artery disease

Objective: Mechanisms behind sustained inflammation in patients with coronary artery disease (CAD) are not clarified but hypothalamus-pituitary-adrenal (HPA) axis dysfunction may have a role. Here, we investigated whether inflammatory status of peripheral blood mononuclear cells (PBMCs) was associated with altered glucocorticoid sensitivity in CAD patients.Methods: In 55 CAD patients and 30 controls, mRNA levels of GR-α, GR-β, NF-κB, IκBα, MMP-9 and TIMP-1 were measured in PBMCs. Suppressive effects of dexamethasone on GR-α, GR-β, NF-κB, IκBα, MMP-9 and TIMP-1 mRNA levels were assessed in PBMCs ex vivo. Salivary cortisol was repeatedly measured over 3 days.Results: GR-α mRNA levels were higher in CAD patients than in controls, 0.50 (0.38–0.59) versus 0.26 (0.18–0.37), p < .001, while GR-β mRNA levels were equally low in both groups. GR-α mRNA expression was associated with inflammatory gene expression and, also, with flatter diurnal cortisol rhythm. In both patients and controls, dexamethasone suppressed gene expression of NF-κB, IκBα, MMP-9 and TIMP-1 (p < .001). Dexamethasone also reduced GR-α mRNA levels (p < .001), while LPS increased it (p < .001).Conclusions: PBMCs from CAD patients displayed an inflammatory gene expression profile. This was not explained by reduced glucocorticoid sensitivity. Instead, inflammation was associated with increased expression of GR-α mRNA, suggesting a hypocortisolemic state.Key messages • Peripheral blood mononuclear cells from patients with coronary artery disease (CAD) display an inflammatory gene expression profile. • This inflammatory state cannot be explained by reduced glucocorticoid sensitivity in CAD patients. • Instead, the inflammatory gene expression profile is associated with upregulated levels of glucocorticoid receptor-α mRNA, suggesting a hypocortisolemic state.

Expression of tissue inhibitor of metalloproteinases and matrix metalloproteinases in the ischemic brain of photothrombosis model mice.

Middle cerebral artery occlusion (MCAO) is the most widely used animal model of ischemic stroke. This model well recapitulates the pathological features of most human cases; however, MCAO is technically difficult to achieve in mice and has some disadvantages for investigating the molecular mechanisms of pathological progression in stroke. The recently developed photothrombosis model may be more suitable for research on the molecular mechanisms of ischemic stroke in mice. Yet, similarities and differences between the photothrombosis and MCAO models are not well characterized. In the present study, we examined the expression of tissue inhibitor of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) in the brains of photothrombosis model mice. Our results indicated that the gene expression of TIMP-1 was upregulated in endothelial cells in the pathological area surrounding the infarction, similar to the MCAO model. Yet, pathologically induced changes in TIMP-1 were not affected by treatment with aspirin or etodolac. Whereas MMP-2 and MMP-8 mRNA were upregulated after infarction in both models, MMP-9 expression, which is induced in the infarct area in the MCAO model, was unchanged in the photothrombosis model. These findings suggest that the expression patterns of TIMP-1 and MMP-9 are regulated independently in photothrombosis model mice.

Variability of MMP/TIMP and TGF-β1 Receptors throughout the Clinical Progression of Chronic Venous Disease.

Chronic venous disease (CVeD) is a prevalent condition with a significant socioeconomic burden, yet the pathophysiology is only just beginning to be understood. Previous studies concerning the dysregulation of matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases (TIMPs)) within the varicose vein wall are inconsistent and disregard clinical progression. Moreover, it is highly plausible that MMP and TIMP expression/activity is affected by transforming growth factor (TGF)-β1 and its signaling receptors (TGFβRs) expression/activity in the vein wall. A case–control study was undertaken to analyze genetic and immunohistochemical differences between healthy (n = 13) and CVeD (early stages: n = 19; advanced stages: n = 12) great saphenous vein samples. Samples were grouped based on anatomic harvest site and subjected to quantitative polymerase chain reaction for MMP1, MMP2, MMP8, MMP9, MMP12, MMP13, TIMP1, TIMP2, TIMP3, TIMP4, TGFβR1, TGFβR2, and TGFβR3 gene expression analysis, and then to immunohistochemistry for immunolocalization of MMP2, TIMP2, and TGFβR2. Decreased gene expression of MMP12, TIMP2, TIMP3, TIMP4, and TGFβR2 was found in varicose veins when compared to controls. Regarding CVeD clinical progression, two facts arose: results across anatomical regions were uneven; decreased gene expression of MMP9 and TGFβR3 and increased gene expression of MMP2 and TIMP3 were found in advanced clinical stages. Most immunohistochemistry results for tunica intima were coherent with qPCR results. In conclusion, decreased expression of TGFβRs might suggest a reduction in TGF-β1 participation in the MMP/TIMP imbalance throughout CVeD progression. Further studies about molecular events in the varicose vein wall are required and should take into consideration the venous anatomical region and CVeD clinical progression.