Evidence for the involvement of matrix metalloproteinases in the cardiovascular effects produced by nicotine

Nicotine plays a role in smoking-associated cardiovascular diseases, and may upregulate matrix metalloproteinase (MMP)-2 and MMP-9. We examined whether nicotine induces the release of MMP-2 and MMP-9 by rat smooth muscle cells (SMC), and whether doxycycline (non-selective MMP inhibitor) inhibits the vascular effects produced by nicotine. SMC were incubated with nicotine 0, 50, and 150 nM for 48 h. MMP-2 and MMP-9 levels in the cell supernatants were determined by gelatin zymography. The acute changes in mean arterial pressure caused by nicotine 2 mu mol/kg (or saline) were assessed in rats pretreated with doxycycline (or saline). We also examined whether doxcycline (30 mg/Kg, i.p., daily) modifies the effects of nicotine (10 mg/kg/day; 4 weeks) on the endothelium-dependent relaxations of rat aortic rings. Aortic MMP-2 levels were assessed by gelatin zymography. Aortic gelatinolytic activity was assessed using a gelatinolytic activity kit. MMP-2 and MMP-9 levels increased in the supernatant of SMC cells incubated with nicotine 150 nM (P<0.05) but not with 50 nM. Nicotine (2 mu mol/kg) produced lower increases in the mean arterial pressure in rats pretreated with doxycycline than those found in rats pretreated with saline (26 +/- 4 vs. 37 +/- 4 mmHg, respectively; P<0.05). Nicotine impaired of the endothelium-dependent responses to acetylcholine, and treatment with doxycycline increased the potency (pD2) by approximately 25% (P<0.05). While we found no significant differences in aortic MMP-2 levels, nicotine significantly increased gelatinolytic activity (P<0.05). These findings suggest that nicotine produces cardiovascular effects involving MMPs. It is possible that MMPs inhibition may counteract the effects produced by nicotine. (C) 2009 Elsevier B.V. All rights reserved.

Gingival crevicular fluid levels of MMP-8, MMP-9, TIMP-2, and MPO decrease after periodontal therapy

P>Background This study aimed at comparing the levels of matrix metalloproteinase (MMP)-8, tissue Inhibitor of MMPs (TIMP)-1 and TIMP-2, Myeloperoxidase (MPO), and MMP-9 in the gingival crevicular fluid (GCF) of chronic periodontitis (CP) patients and controls at baseline and 3 months after non-surgical therapy. Materials and Methods GCF was collected from one site of 15 control subjects and 27 CP patients. MMP-8, MMP-9, TIMP-1, and TIMP-2 were determined by Enzyme-linked immunoabsorbent assay; different forms of MMP-9, by gelatin zymography; and MPO, colorimetrically. Results At baseline, higher levels of MMP-8, TIMP-2, MPO, and the 87 kDa-MMP-9 were found in patients compared with controls (p < 0.001), and these molecules decreased after therapy (p < 0.03). There were no differences between the groups with respect to the higher molecular forms of MMP-9 (180, 130, 92 kDa) or total MMP-9 at baseline. No differences were observed in TIMP-1 levels. In controls, decreased levels of TIMP-2 and the higher molecular forms of MMP-9 (180, 130, 92 kDa) were found 3 months after therapy compared with baseline (p < 0.01). Conclusions Higher levels of MMP-8, TIMP-2, MPO, and 87 kDa MMP-9 were found in the GCF of patients compared with controls, and these markers decreased 3 months after periodontal therapy.

Different circulating metalloproteinases profiles in women with migraine with and without aura

Background: We compared the circulating levels of matrix metalloproteinase (MMP)-2. MMP-9, tissue inhibitors of metal loproteinase (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios in migraine patients without aura (MWA) and in migraine patients with aura (MA) with those found in healthy subjects (controls). Methods: We studied 80 migraine (40 MWA and 40 MA) women and 40 controls. Pro-MMP-2 levels were determined by zymography and MMP-9, TIMP-1, and TIMP-2 levels were determined by ELISA. Results: While we found similar TIMP-2 levels, higher plasma pro-MMP-2 and pro-MMP-2/TIMP-2 ratios were found in MWA and MA patients compared with controls (P<0.05). Higher TIMP-1 levels and lower MMP-9/TIMP-1 ratio were found in MA, but not in MWA, patients compared with controls (P<0.05). We found no significant differences when patients without headache attack were compared with patients having a headache attack (all P<0.05). Conclusions: We showed an increased net MMP-2 activity in MWA and MA. The increased MMP-9/TIMP-1 ratios in MWA patients contrast with the lower MMP-9/TIMP-1 ratios in MA patients and may reflect pathophysiological differences between these conditions. (C) 2009 Elsevier B.V. All rights reserved.

A genetic polymorphism of matrix metalloproteinase 9 (MMP-9) affects the changes in circulating MMP-9 levels induced by highly active antiretroviral therapy in HIV patients

We examined whether two functional polymorphisms (g.-1562C>T and g.-90(CA)14-24) in the matrix metalloproteinase (MMP)-9 gene or MMP-9 haplotypes affect the circulating levels of pro-MMP-9 and pro-MMP-9/TIMP-1 (tissue inhibitor of metalloproteinase-1) ratios in AIDS patients, and modulate alterations in these biomarkers after highly active antiretroviral therapy (HAART). We studied 82 patients commencing HAART. Higher pro-MMP-9 concentrations and pro-MMP-9/TIMP-1 ratios were found in CT/TT patients compared with CC patients. HAART decreased pro-MMP-9 levels and pro-MMP-9/TIMP-1 ratios in CT/TT patients, it did not modify pro-MMP-9 levels and it increased pro-MMP-9/TIMP-1 ratios in CC patients. The g.-90(CA)14-24 polymorphism, however, produced no significant effects. Moreover, we found no significant differences in HAART-induced changes in plasma pro-MMP-9, TIMP-1 and pro-MMP-9/TIMP-1 ratios when different MMP-9 haplotypes were compared. These findings suggest that the g.-1562C>T polymorphism affects pro-MMP-9 levels in patients with AIDS and modulates the alterations in pro-MMP-9 levels caused by HAART, thus possibly affecting the risk of cardiovascular complications. The Pharmacogenomics Journal (2009) 9, 265-273; doi: 10.1038/tpj.2009.13; published online 21 April 2009

In Vitro Proliferation and Osteoblastic Phenotype Expression of Cells Derived From Human Vertebral Lamina and Iliac Crest

Study Design. Osteoblastic cells derived from vertebral lamina and iliac crest were isolated and cultured under the same conditions (osteogenic medium, pH, temperature, and CO(2) levels). Objective. To compare proliferation and expression of osteoblastic phenotype of cells derived from vertebral lamina and iliac grafting. Summary of Background Data. Many factors play a role in the success of bone graft in spinal fusion including osteoblastic cell population. Two common sources of graft are vertebral lamina and iliac crest, however, differences in proliferation and osteoblastic phenotype expression between cells from these sites have not been investigated. Methods. Cells obtained from cancellous bone of both vertebral lamina and iliac crest were cultured and proliferation was evaluated by direct cell counting and viability detected by Trypan blue. Alkaline phosphatase (ALP) activity was evaluated by thymolphthalein release from thymolphthalein monophosphate and matrix mineralization by staining with alizarin red S. Gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, osteoprotegerin, and receptor activator of NF-kB ligand was analyzed by real-time PCR. All comparisons were donor-matched. Results. Proliferation was greater at days 7 and 10 in cells from vertebral lamina compared with ones from iliac crest without difference in cell viability. ALP activity was higher in cells from vertebral lamina compared with cells from iliac crest at days 7 and 10. At 21 days, mineralized matrix was higher in cells derived from vertebral lamina than from iliac crest. At day 7, gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, receptor activator of NF-kB ligand, and osteoprotegerin was higher in cells derived from vertebral lamina compared with iliac crest. Conclusion. Cell proliferation and osteoblastic phenotype development in cells derived from cancellous bone were more exuberant in cultures of vertebral lamina than of iliac crest.

Human osteoblastic cell response to a Ca- and P-enriched titanium surface obtained by anodization

The aim of the present study was to evaluate the in vitro osteogenic potential of subcultured human osteoblastic cells derived from alveolar bone on a titanium (Ti) surface produced by an anodized alkali treatment (BSP-AK). Primary osteoblastic cells were subcultured on BSP-AK and machined Ti discs (control) and grown for periods of up to 21 days under osteogenic conditions. Morphologic and biochemical methods were used to assess important parameters of in vitro bone-like tissue formation. Although no major differences were observed between the BSP-AK and the control Ti surface in terms of cell attachment and mineralized matrix formation, a significant increase in cell population, ALP activity, and collagen content was detected in cultures on BSP-AK surface. Our results demonstrate that human osteoblastic cells are sensitive to the BSP-AK-modified Ti surface during the transitional stage between the end of the proliferative phase and the onset of the differentiation /matrix maturation ones. Together with the good mechanical properties exhibited by the Ca- and P- coating, our findings suggest that BSP-AK treatment could be useful for the development of a new surface for dental and orthopedic implants. (c) 2008 Wiley Periodicals, Inc.J Biomed Mater Res 88A: 841-848, 2009

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress. we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201 +/- 5 vs. 163 +/- 7 mm Hg in diabetic rats untreated and treated with lercaniclipine, respectively; P < 0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P < 0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P < 0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P < 0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2MMP-2 expression. (C) 2008 Published by Elsevier B.V.

High concentration of residual aluminum oxide on titanium surface inhibits extracellular matrix mineralization

In the present study we characterized titanium (Ti) surfaces submitted to different treatments and evaluated the response of osteoblasts derived from human alveolar bone to these surfaces. Five different surfaces were evaluated: ground (G), ground and chemical etched (G1-HF for 60 s), sand blasted (SB-Al2O3 particles 65 pm), sand blasted and chemical etched (SLA1-HF for 60 s and SLA2-HF for 13 s). Surface morphology was evaluated under SEM and roughness parameters by contact scanning instrument. The presence of Al2O3 was detected by EDS and the amount calculated by digital analyses. Osteoblasts, were cultured on these surfaces and it was evaluated: cell adhesion, proliferation, and viability, alkaline phosphatase activity, total protein content, and matrix mineralization formation. Physical and chemical treatments produced very different surface morphologies. Al2O3 residues were detected on SB and SLA2 surfaces. Only matrix mineralization formation was affected by different surface treatments, being increased on rough surface (SLA1) and reduced on surface with high amount of Al2O3 residues (SB). On the basis of these findings, it is possible to conclude that high concentration of residual Al2O3 negatively interfere with the process of matrix mineralization formation in contact with Ti implant surfaces. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 87A: 588-597, 2008

Effects of statins on matrix metalloproteinases and their endogenous inhibitors in human endothelial cells

Statins exert anti-inflammatory effects and downregulate matrix metalloproteinases (MMPs) expression, thus contributing to restore cardiovascular homeostasis in cardiovascular diseases. We aimed at comparing the effects of different statins (simvastatin, atorvastatin, and pravastatin) on MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios released by human umbilical vein endothelial cells (HUVEC) stimulated by phorbol myristate acetate (PMA). HUVECs were incubated with statins (0.1-10 mu M) for 12 h before stimulation with PMA 100 nM. Monolayers were used to perform cell viability assays and the supernatants were collected to determine MMPs and TIMPs levels by gelatin zymography and/or enzyme immunoassay. While treatment with PMA increased MMP-9 and TIMP-1 levels (by 556% and 159%, respectively; both P < 0.05), it exerted no effects on MMP-2 and TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, attenuated PMA-induced increases in MMP-9 levels (P < 0.05). Only atorvastatin decreased baseline MMP-2 levels significantly (P < 0.05). We found no effects on TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, decreased MMP-9/TIMP-1 ratio significantly (both P < 0.05), whereas atorvastatin and pravastatin, but not simvastatin, decreased MMP-2/TIMP-2 ratio significantly (both P < 0.05). Our data support the notion that statins with different physicochemical features exert variable effects on MMP/TIMP ratios (which reflect net MMP activity). Our results suggest that more lipophilic statins (simvastatin and atorvastatin), but not the hydrophilic statin pravastatin, downregulate net MMP-9 activity. However, atorvastatin and pravastatin may downregulate net MMP-2 activity. The clinical implications of the present findings deserve further investigation.

Radiopacity Evaluation of Calcium Aluminate Cement Containing Different Radiopacifying Agents

Introduction: The aim of this study was to evaluate the radiopacity of calcium aluminate cement (Endo Binder) with 3 different radiopacifiers (bismuth oxide, zinc oxide, or zirconium oxide) in comparison with gray mineral trioxide aggregate (GMTA), white MTA, and dental structures (enamel and dentin). Methods: Eighteen test specimens of each cement with thicknesses of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mm (n = 3) were made by using a stainless steel matrix and were adapted to a standardizing device (8 x 7 cm) with a graduated aluminum stepwedge varying from 2.0-16.0 mm in thickness. To compare the radiopacity of the cements with that of dental structures, slices of first molars with a thickness increasing from 0.5-3.0 mm were obtained and placed on the standardizing device. One occlusal radiograph for each tested cement was taken, with exposure time of 0.1 seconds and focus-film distance of 20 cm. Films were processed in an automatic device, and the mean radiopacity values were obtained by using a photodensitometer. Results: Mean values showed that the thicker the specimen was, the greater was its radiopacity. Only EndoBinder + bismuth oxide (EBBO) and GMTA demonstrated radiopacity values greater than 3.0 mm of the aluminum scale for all thicknesses. When zinc oxide was used as radiopacifier agent, EndoBinder only reached the desired radiopacity with a thickness of 2.0 mm, and with zirconium oxide it was 2.5 mm. Conclusions: Bismuth oxide was the most efficient radiopacifier for EndoBinder, providing adequate radiopacity in all studied thicknesses, as recommended by ISO 6876, being similar to GMTA. (J Endod 2011;37: 67-71)

Influence of Artificial Accelerated Aging on Dimensional Stability of Acrylic Resins Submitted to Different Storage Protocols

Purpose: The aim of this study was to evaluate the influence of artificial accelerated aging on dimensional stability of two types of acrylic resins (thermally and chemically activated) submitted to different protocols of storage. Materials and Methods: One hundred specimens were made using a Teflon matrix (1.5cmx0.5mm) with four imprint marks, following the lost-wax casting method. The specimens were divided into ten groups, according to the type of acrylic resin, aging procedure, and storage protocol (30 days). GI: acrylic resins thermally activated, aging, storage in artificial saliva for 16 hours, distilled water for 8 hours; GII: thermal, aging, artificial saliva for 16 hours, dry for 8 hours; GIII: thermal, no aging, artificial saliva for 16 hours, distilled water for 8 hours, GIV: thermal, no aging, artificial saliva for 16 hours, dry for 8 hours; GV: acrylic resins chemically activated, aging, artificial saliva for 16 hours, distilled water for 8 hours; GVI: chemical, aging, artificial saliva for 16 hours, dry for 8 hours; GVII: chemical, no aging, artificial saliva for 16 hours, distilled water for 8 hours; GVIII: chemical, no aging, artificial saliva for 16 hours, dry for 8 hours GIX: thermal, dry for 24 hours; and GX: chemical, dry for 24 hours. All specimens were photographed before and after treatment, and the images were evaluated by software (UTHSCSA-Image Tool) that made distance measurements between the marks in the specimens (mm), calculating the dimensional stability. Data were submitted to statistical analysis (two-way ANOVA, Tukey test, p = 0.05). Results: Statistical analysis showed that the specimens submitted to storage in water presented the largest distance between both axes (major and minor), statistically different (p < 0.05) from control groups. Conclusions: All acrylic resins presented dimensional changes, and the artificial accelerated aging and storage period influenced these alterations.

Color stability of silorane-based composites submitted to accelerated artificial ageing-An in situ study

Objectives: To assess the in situ color stability, surface and the tooth/restoration interface degradation of a silorane-based composite (P90, 3M ESPE) after accelerated artificial ageing (AAA), in comparison with other dimethacrylate monomer-based composites (Z250/Z350, 3M ESPE and Esthet-X, Dentsply). Methods: Class V cavities (25 mm(2) x 2 mmdeep) were prepared in 48 bovine incisors, which were randomly allocated into 4 groups of 12 specimens each, according to the type of restorative material used. After polishing, 10 specimens were submitted to initial color readings (Easyshade, Vita) and 2 to analysis by scanning electronic microscopy (SEM). Afterwards, the teeth were submitted to AAA for 384 h, which corresponds to 1 year of clinical use, after which new color readings and microscopic images were obtained. The values obtained for the color analysis were submitted to statistical analysis (1-way ANOVA, Tukey, p < 0.05). Results: With regard to color stability, it was verified that all the composites showed color alteration above the clinically acceptable levels (Delta E >= 3.3), and that the silorane-based composite showed higher Delta E (18.6), with a statistically significant difference in comparison with the other composites (p < 0.05). The SEM images showed small alterations for the dimethacrylate-based composites after AAA and extensive degradation for the silorane-based composite with a rupture at the interface between the matrix/particle. Conclusion: It may be concluded that the silorane-based composite underwent greater alteration with regard to color stability and greater surface and tooth/restoration interface degradation after AAA. (C) 2011 Elsevier Ltd. All rights reserved.