Development of secondary palate requires strict regulation of ECM remodeling: sequential distribution of RECK, MMP-2, MMP-3, and MMP-9

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.

Biocompatibility evaluation of alendronate paste in rat's subcutaneous tissue

Alendronate is a known inhibitor of root resorption and the development of alendronate paste would enhance its utilization as intracanal medication. Therefore, this study aimed to investigate the biocompatibility of experimental alendronate paste in subcutaneous tissue of rats, for utilization in teeth susceptible to root resorption. The study was conducted on 15 male rats, weighing similar to 180-200 grams. The rats' dorsal regions were submitted to one incision on the median region and, laterally to the incision, the subcutaneous tissue was raised and gently dissected for introduction of two tubes, in each rat. The tubes were sealed at one end with gutta-percha and taken as control. The tubes were filled with experimental alendronate paste. The animals were killed at 7, 15 and 45 days after surgery and the specimens were processed in laboratory. The histological sections were stained with hematoxylin-eosin and analyzed by light microscopy. Scores were assigned to the in. ammatory process and statistically compared by the Tukey test (P < 0.05). Alendronate paste promoted severe inflammation process at 7 days, with statistically significant difference compared to the control (P < 0.05%). However, at 15 days, there was a regression of in. ammation and the presence of connective tissue with collagen fibers, fibroblasts and blood vessels was observed. After 45 days, it was observed the presence of well-organized connective tissue, with collagen fibers and fibroblasts, and few in. ammatory cells. No statistical difference was observed between the control and experimental paste at 15 and 45 days. The experimental alendronate paste was considered biocompatible with subcutaneous tissue of rat.

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A(2) and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents. (c) 2007 Elsevier Ltd. All rights reserved.

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)

Comprehensive Evaluation of the Effects of Enalapril on Matrix Metalloproteinases Levels in Hypertension

Angiotensin-converting enzyme inhibitors (ACEi) may downregulate matrix metalloproteinases (MMPs). We examined whether enalapril affects MMP-2, MMP-8, and MMP-9 levels and activity, and their endogenous inhibitors (tissue inhibitors of MMPs, TIMP-1 and TIMP-2) levels in hypertensive patients. Moreover, we assessed the effects of enalaprilat on MMP-9 and TIMP-1 secretion by human endothelial cells (HUVECs). Thirty-eight hypertensive patients received enalapril for 8 weeks and were compared with thirty-eight normotensive controls. Blood samples were collected at baseline and after treatment. Plasma ACE activity was determined by a fluorimetric assay. Plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were measured by ELISA and gelatin zymography. A fluorogenic peptide cleavage assay was used to measure MMP activity. HUVECs cells were stimulated by phorbol-12-myristate-13-acetate (PMA) and the effects of enalaprilat (10(-10) to 10(-6) M) on MMP-9 and TIMP-1 levels were determined. Enalapril decreased blood pressure and ACE activity in hypertensive patients (P < 0.05), but had no effects on plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 levels, or MMP activity. Enalaprilat had no effects on PMA-induced increases in MMP-9 and TIMP-1 secretion by HUVECs or on MMP activity. We show consistent evidence, both in vivo and in vitro, that enalapril does not affect MMPs and TIMPs levels in hypertensive patients.

Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice

Heart failure is accompanied by severely impaired β-adrenergic receptor (βAR) function, which includes loss of βAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of βAR function is agonist-stimulated receptor phosphorylation by the βAR kinase (βARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in βAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of βARK1 or the β2AR were mated into a genetic model of murine heart failure (MLP−/−). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP−/− and MLP−/−/β2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP−/−/βARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP−/−/βARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP−/− mice but less than controls. Importantly, heightened βAR desensitization in the MLP−/− mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the βARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal βAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit βARK1 as a novel mode of therapy.

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-l-cysteine, d-penicillamine, captopril, l-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.

Blood-borne tissue factor: Another view of thrombosis

Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to flowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused ≈70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagen-coated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VIIai, a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor Xa formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.

Cloning and characterization of cDNAs for matrix metalloproteinases of regenerating newt limbs.

Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.

Functional testicular tissue does not masculinize development of the zebra finch song system.

Current theories of sexual differentiation maintain that ovarian estrogen prevents masculine development of the copulatory system in birds, whereas estrogen derived from testicular androgens promotes masculine sexual differentiation of neuroanatomy and sexual behavior in mammals. Paradoxically, some data suggest that the neural song system in zebra finches follows the mammalian pattern with estrogenic metabolites of testicular secretions causing masculine development. To test whether the removal of estrogen from males during early development would prevent the development of masculine song systems, zebra finches were treated embryonically with an inhibitor of estrogen synthesis. In addition, this treatment in genetic female zebra finches induced both functional ovarian and testicular tissue to develop, thus allowing the assessment of the direct effects of testicular secretions on song system development. In males, the inhibition of estrogen synthesis before hatching had a small but significant effect in demasculinizing one aspect of the neural song system. In treated females, the song systems remained morphologically feminine. These results suggest that masculinization of the song system is not determined solely by testicular androgens or their estrogenic metabolites.

Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor.

The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.

Matrix Metalloproteinases And Left Ventricular Function And Structure In Spinal Cord Injured Subjects.

Subjects with spinal cord injury (SCI) exhibit impaired left ventricular (LV) diastolic function, which has been reported to be attenuated by regular physical activity. This study investigated the relationship between circulating matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and echocardiographic parameters in SCI subjects and the role of physical activity in this regard. Forty-two men with SCI [19 sedentary (S-SCI) and 23 physically-active (PA-SCI)] were evaluated by clinical, anthropometric, laboratory, and echocardiographic analysis. Plasmatic pro-MMP-2, MMP-2, MMP-8, pro-MMP-9, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay and zymography. PA-SCI subjects presented lower pro-MMP-2 and pro-MMP-2/TIMP-2 levels and improved markers of LV diastolic function (lower E/Em and higher Em and E/A values) than S-SCI ones. Bivariate analysis showed that pro-MMP-2 correlated inversely with Em and directly with E/Em, while MMP-9 correlated directly with LV mass index and LV end-diastolic diameter in the whole sample. Following multiple regression analysis, pro-MMP-2, but not physical activity, remained associated with Em, while MMP-9 was associated with LV mass index in the whole sample. These findings suggest differing roles for MMPs in LV structure and function regulation and an interaction among pro-MMP-2, diastolic function and physical activity in SCI subjects.

Jnk And Ikkβ Phosphorylation Is Reduced By Glucocorticoids In Adipose Tissue From Insulin-resistant Rats.

Peripheral insulin resistance (IR) is one of the main side effects caused by glucocorticoid (GC)-based therapies, and the molecular mechanisms of GC-induced IR are not yet fully elucidated. Thus, we aimed to investigate the effects of dexamethasone treatment on the main components of insulin and inflammatory signaling in the adipose tissue of rats. Male Wistar rats received daily injections of dexamethasone (1mg/kg body weight (b.w.), intraperitoneally (i.p.)) for 5 days (DEX), whereas control rats received saline (CTL). The metabolic status was investigated, and the epididymal fat fragments were collected for lipolysis and western blot analyses. The DEX rats became hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant, compared with the CTL rats (P<0.05). The basal glycerol release in the fat fragments was 1.5-fold higher in the DEX rats (P<0.05). The phosphorylation of protein kinase B (PKB) at ser(473) decreased by 44%, whereas, the phosphorylation of insulin receptor substrate (IRS)-1 at ser(307) increased by 93% in the adipose tissue of the DEX rats after an oral bolus of glucose (P<0.05). The basal phosphorylation of c-jun-N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B (IKKβ) proteins was reduced by 46% and 58%, respectively, in the adipose tissue of the DEX rats (P<0.05). This was paralleled with a significant reduction (47%) in the glucocorticoid receptor (GR) protein content in the adipose tissue of the DEX rats (P<0.05). The insulin-resistant status of rats induced by dexamethasone administration have PKB and IRS-1 activity attenuated in epididymal fat without increases in the phosphorylation of the proinflammatory signals JNK and IKKβ.