Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

A unique group of scabies mite pseudoproteases promotes cutaneous blood coagulation and delays plasmin-induced fibrinolysis.

BACKGROUND: Scabies, a highly contagious skin disease affecting more than 200 million people worldwide at any time, is caused by the parasitic mite Sarcoptes scabiei. In the absence of molecular markers, diagnosis requires experience making surveillance and control challenging. Superficial microthrombi in the absence of vasculitis in scabies-affected skin are a recognised, yet unexplained histopathological differential of scabies infection. This study demonstrates that a family of Scabies Mite Inactivated Cysteine Protease Paralogues (SMIPP-Cs) excreted by the mites plays a role in formation of scabies-induced superficial microthrombi. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro and ex vivo experiments involving two representative recombinant SMIPP-Cs was carried out. In the presence of SMIPP-Cs, the thrombin clotting time (TCT), fibrin formation and plasmin induced fibrinolysis were monitored in vitro. The ultrastructure of the SMIPP-C-modulated fibrin was analysed by Scanning Electron Microscopy (SEM). Immuno-histological analyses were performed ex vivo, to localise the SMIPP-C proteins within scabies infected skin biopsies. SMIPP-Cs displayed pro-coagulant properties. They bound calcium ions, reduced the thrombin clotting time, enhanced the fibrin formation rate and delayed plasmin-induced fibrinolysis. The SMIPP-Cs associated with fibrin clots during fibrinogen polymerisation and did not bind to preformed fibrin. Scanning electron microscopy revealed that the fibrin clots formed in the presence of SMIPP-Cs were aberrant and denser than normal fibrin clots. SMIPP-Cs were detected in microthrombi which are commonly seen in scabietic skin. CONCLUSIONS/SIGNIFICANCE: The SMIPP-Cs are the first scabies mite proteins found in sub-epidermal skin layers and their pro-coagulant properties promote superficial microthrombi formation in scabetic skin. Further research is needed to evaluate their potential as diagnostic or therapeutic target.

Fibrinogen function achieved through multiple covalent states.

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10-50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.

Early morphological changes in tissues when replacing abdominal wall defects by bacterial nanocellulose in experimental trials.

Experimental trials were done on five dogs to explore if an anterior abdominal wall defect could be repaired using wet (99.9%), compact BNC membranes produced by the Мedusomyces gisevii Sa-12 symbiotic culture. The abdominal wall defect was simulated by middle-midline laparotomy, and a BNC membrane was then fixed to open aponeurotic edges with blanket suture (Prolene 4-0, Ethicon). A comparative study was also done to reinforce the aponeurotic defect with both the BNC membrane and polypropylene mesh (PPM) (Ultrapro, Ethicon). The materials were harvested at 14 and 60 days postoperative to visually evaluate their location in the abdominal tissues and evaluate the presence of BNC and PPM adhesions to the intestinal loops, followed by histologic examination of the tissue response to these prosthetics. The BNC exhibited good fixation to the anterior abdominal wall to form on the 14th day a capsule of loose fibrin around the BNC. Active reparative processes were observed at the BNC site at 60 days post-surgery to generate new, stable connective-tissue elements (macrophages, giant cells, fibroblasts, fibrin) and neocapillaries. Negligible intraperitoneal adhesions were detected between the BNC and the intestinal loops as compared to the case of PPM. There were no suppurative complications throughout the postsurgical period. We noticed on the 60th day after the BNC placement that collagenous elements and new capillary vessels were actively formed in the abdominal wall tissues, generating a dense postoperative cicatrix whose intraperitoneal adhesions to the intestinal loops were insignificant compared to the PPM graft.

The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-ß) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-ß cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions.

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

Polyphosphate as a haemostatic modulator.

Platelets are small anuclear cells that play a central role in haemostasis. Platelets become activated in response to various stimuli triggering release of their granular contents into the surrounding milieu. One of these types of granules, termed dense granules, have been found to contain polyphosphate (polyP) in addition to other inorganic biomolecules, such as serotonin, ADP, ATP, PPi. Individuals deficient in dense granules exhibit bleeding tendencies, emphasizing their importance in haemostasis. Platelet polyP is of a relatively defined size, approximately 60-100 phosphate monomers in length. These linear polymers act at various points in the coagulation and fibrinolytic systems thereby modulating the haemostatic response. Due to its highly anionic nature, polyP lends itself to being a natural activator of the contact system. The contact system functions in multiple pathways including coagulation, fibrinolysis, inflammation and complement. Activation of the contact system accelerates thrombin generation, the terminal enzyme in the coagulation cascade. PolyP also modulates factors further downstream in the coagulation cascade to augment thrombin generation. The net effect is increased fibrin formation and platelet activation resulting in faster clot formation. PolyP is incorporated into the forming clot thereby modifying the structure of the resulting fibrin network and its susceptibility to degradation by certain plasminogen activators. In conclusion, release of platelet polyP at the site of injury may facilitate clot formation and augment clot stability thereby promoting wound healing.

Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome.

Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6(-/-) mice after vascular injury. HPS6(-/-) platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5'-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6(-/-) mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype.

Stationary versus agitated storage of whole blood during acute normovolemic hemodilution.

BACKGROUND: Acute normovolemic hemodilution is an intraoperative technique to reduce the number of red blood cells lost in shed blood during surgery. Standard guidelines for storage of platelets recommend constant gentle agitation to maintain gas exchange for the metabolically active platelets. The collected whole blood (WB) for acute normovolemic hemodilution remains stationary for as long as 8 hours before reinfusion. We hypothesized that gentle agitation of WB throughout storage would improve the coagulation properties of the WB at the time of reinfusion. METHODS: WB was collected from 10 volunteer donors and control samples taken. The units were split in 2 storage groups: agitated (rocked) and stationary (unrocked). Cell counts and fibrinogen levels, as well as thromboelastography (TEG®) measurements, including TEG® PlateletMapping® assays, were performed on the control sample and the test samples after 8 hours of rocked or unrocked storage at room temperature. RESULTS: Nine units WB from 9 different healthy volunteers were tested. There were no significant differences in hematocrit, hemoglobin, red blood cells counts, platelet counts, or fibrinogen levels between the control samples and the rocked and unrocked WB samples. WB coagulation as measured by TEG® was preserved during the 8-hour storage period in both the rocked and unrocked samples. There were no significant differences between the control, rocked, and unrocked samples in time to initiate clotting, time of clot formation, rate of clot formation, or maximum strength of clot values. There were also no significant differences in the fibrin contribution to clot strength between the control, rocked, and unrocked samples, and no significant difference between the platelet activation from adenosine diphosphate or arachidonic acid among any of the 3 groups. CONCLUSIONS: Given the small sample size, there is no statistical evidence on which to reject the null hypothesis of there being no difference in the changes from the baseline between coagulation function as measured by TEG® between WB that is either agitated or kept stationary for 8 hours. These findings need to be confirmed in a larger study.

Growth factor and proteinase profile of Vivostat® platelet-rich fibrin linked to tissue repair.

BACKGROUND AND OBJECTIVES: Autologous platelet-rich fibrin (PRF(®)) is prepared by the automatic Vivostat(®) system. Conflicting results with Vivostat PRF in acute wound healing prompted us to examine its cellular and biomolecular composition. Specifically, platelets, selected growth factors and matrix metalloproteinase (MMP)-9 were quantified using novel analytical methods. MATERIALS AND METHODS: Ten healthy non-thrombocytopenic volunteers donated blood for generation of intermediate fibrin-I and final PRF. Anticoagulated whole blood and serum procured in parallel served as baseline controls. Leucocyte, erythrocyte and platelet counts in whole blood and fibrin-I were determined by automated haematology analyser. Platelet concentration in PRF was quantified manually by stereologic analysis of Giemsa-stained tissue sections, and the total content of five growth factors and MMP-9 by enzyme-linked immunosorbent assays. RESULTS: The number of leucocytes and erythrocytes was reduced (P < 0·001), whereas platelets increased (P < 0·001) in fibrin-I versus whole blood. PRF contained 982 ± 206 × 10(9) platelets/l representing 3·9-fold (P < 0·001) enrichment relative to whole blood. Growth factor abundance in Vivostat PRF and serum was in descending order: transforming growth factor-ß1 [5·1-fold higher in PRF than serum, P < 0·001] > platelet-derived growth factor (PDGF)-AB [2·5-fold, P < 0·01] > PDGF-BB [1·6-fold, P < 0·05] > vascular endothelial growth factor > basic fibroblast growth factor [75-fold, P < 0·001]. MMP-9 was reduced 139-fold (P < 0·001) compared with serum, reflecting leucocyte depletion in PRF. CONCLUSION: The gained knowledge on platelet enrichment and biomolecular constituents may guide clinicians in their optimal use of Vivostat PRF for tissue regenerative applications.

CYLD enhances severe listeriosis by impairing IL-6/STAT3-dependent fibrin production.

The facultative intracellular bacterium Listeria monocytogenes (Lm) may cause severe infection in humans and livestock. Control of acute listeriosis is primarily dependent on innate immune responses, which are strongly regulated by NF-κB, and tissue protective factors including fibrin. However, molecular pathways connecting NF-κB and fibrin production are poorly described. Here, we investigated whether the deubiquitinating enzyme CYLD, which is an inhibitor of NF-κB-dependent immune responses, regulated these protective host responses in murine listeriosis. Upon high dose systemic infection, all C57BL/6 Cyld(-/-) mice survived, whereas 100% of wildtype mice succumbed due to severe liver pathology with impaired pathogen control and hemorrhage within 6 days. Upon in vitro infection with Lm, CYLD reduced NF-κB-dependent production of reactive oxygen species, interleukin (IL)-6 secretion, and control of bacteria in macrophages. Furthermore, Western blot analyses showed that CYLD impaired STAT3-dependent fibrin production in cultivated hepatocytes. Immunoprecipitation experiments revealed that CYLD interacted with STAT3 in the cytoplasm and strongly reduced K63-ubiquitination of STAT3 in IL-6 stimulated hepatocytes. In addition, CYLD diminished IL-6-induced STAT3 activity by reducing nuclear accumulation of phosphorylated STAT3. In vivo, CYLD also reduced hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 and NOX2 mRNA production as well as fibrin production in murine listeriosis. In vivo neutralization of IL-6 by anti-IL-6 antibody, STAT3 by siRNA, and fibrin by warfarin treatment, respectively, demonstrated that IL-6-induced, STAT3-mediated fibrin production significantly contributed to protection in Cyld(-/-) mice. In addition, in vivo Cyld siRNA treatment increased STAT3 phosphorylation, fibrin production, pathogen control and survival of Lm-infected WT mice illustrating that therapeutic inhibition of CYLD augments the protective NF-κB/IL-6/STAT3 pathway and fibrin production.

Iron-induced fibrin in cardiovascular disease.

Accumulating evidence within the last two decades indicates the association between cardiovascular disease (CVD) and chronic inflammatory state. Under normal conditions fibrin clots are gradually degraded by the fibrinolytic enzyme system, so no permanent insoluble deposits remain in the circulation. However, fibrinolytic therapy in coronary and cerebral thrombosis is ineffective unless it is installed within 3-5 hours of the onset. We have shown that trivalent iron (FeIII) initiates a hydroxyl radical-catalyzed conversion of fibrinogen into a fibrin-like polymer (parafibrin) that is remarkably resistant to the proteolytic dissolution and thus promotes its intravascular deposition. Here we suggest that the persistent presence of proteolysis-resistant fibrin clots causes chronic inflammation. We study the effects of certain amphiphilic substances on the iron- and thrombin-induced fibrinogen polymerization visualized using scanning electron microscopy. We argue that the culprit is an excessive accumulation of free iron in blood, known to be associated with CVD. The only way to prevent iron overload is by supplementation with iron chelating agents. However, administration of free radical scavengers as effective protection against persistent presence of fibrin-like deposits should also be investigated to contribute to the prevention of cardiovascular and other degenerative diseases.

Influence of antibiotic therapy on the level of selected angiogenic factors in patients with benign gynecologic tumors--preliminary report.

An increased fibrin level enhances the activity of proangiogenic factors and may contribute to tumor formation. Formation of new blood vessels during angiogenesis leads to neoplasm development through interaction with factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukins. The aim of this study was to investigate the influence of perioperative antibiotic therapy in women with benign gynecological tumors with regard to basic fibroblast growth factor level, fibrinogen concentration and fibrin viscosity. The influence of clindamycin plus metronidazole therapy (group I) and cephazolin therapy (group II) on fibrinogen concentration, level of bFGF and fibrin viscosity was studied in women diagnosed with nonmalignant myomas and cysts. In patients with benign gynecologic tumors, higher bFGF levels (51.40 +/- 13.72 pg/ml), fibrinogen concentration (348.26 +/- 164.74 mg/dl) and fibrin viscosity (2.63 +/- 0.36 mPa) were observed, as compared with healthy women. There were strong indications that antiangiogenic activity occurred with both clindamycin plus metronidazole and cephazolin, although the response to these particular antibiotic therapies was different. The use of various drug therapies in groups I and II resulted in faster and delayed antiangiogenic effects, respectively. Further research is essential to provide more detailed information about the mechanisms of the induction of antiangiogenic activity by perioperative adjuvant antibiotic treatment.

The cardioprotective effects of marrubiin, a diterpenoid found in Leonotis leonurus extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonotis leonurus L. (Lamiaceae) is used as a traditional medicine for a variety of ailments in South Africa. The diterpene marrubiin is the major product constituent in specimens of this plant occurring in South Africa. MATERIALS AND METHODS: Marrubiin isolated from South African specimens of L. leonurus in addition to an organic extract of L. leonurus were tested in vivo, ex vivo and in vitro for their anticoagulant, antiplatelet and anti-inflammatory activities. RESULTS: Marrubiin and the organic extract suppressed coagulation, platelet aggregation and inflammatory markers. For the coagulation markers it was found that the organic extract and marrubiin significantly prolonged activated partial thromboplastin time (APTT). Fibrin and D-dimer formation were drastically decreased. These findings were observed in an ex vivo model and an obese rat model. Chemokines enhance leukocyte recruitment to inflammatory sites. TNF-α and RANTES secretion were significantly reduced by the extract and marrubiin when determined in the obese rat model relative to the controls. Calcium mobilization and TXB(2) synthesis were suppressed by the extract and marrubiin. An in vitro model was used to elucidate the antiplatelet mechanism and it was found that the extract and marrubiin inhibited platelet aggregation by inhibiting the binding of fibrinogen to glycoprotein (GP) IIb/IIIa receptor in a concentration dependent manner. CONCLUSION: The findings reflect that marrubiin largely contributes to the extract's anticoagulant, antiplatelet and anti-inflammatory effects observed.

Hemostatic function of buffy coat platelets in additive solution treated with pathogen reduction technology.

BACKGROUND: Pathogen reduction technologies (PRTs) may influence the hemostatic potential of stored platelet (PLT) concentrates. To investigate this, buffy coat PLTs (BCPs) stored in PLT additive solution (SSP+) with or without Mirasol PRT treatment (CaridianBCT Biotechnologies) were compared by functional hemostatic assays. STUDY DESIGN AND METHODS: We performed in vitro comparison of PRT (PRT-BCP) and control pooled-and-split BCPs (CON-BCP) after 2, 3, 6, 7, and 8 days' storage. Hemostatic function was evaluated with thrombelastography (TEG) and impedance aggregometry (Multiplate), the latter also in a sample matrix (Day 2) with or without addition of red blood cells (RBCs), control plasma, and/or PRT-treated plasma. RESULTS: PRT treatment of 8-day-stored BCPs influenced clot formation (TEG) minimally, with reductions in maximum clot strength (maximum amplitude, p = 0.014) but unchanged initial fibrin formation (R), clot growth rate (α), and fibrinolysis resistance. In the absence of RBCs and plasma, PRT impaired aggregation (Multiplate) in stored BCPs, with reduced aggregation against thrombin receptor activating peptide-6 (p < 0.001), collagen (p = 0.014), adenosine 5'-diphosphate (p = 0.007), and arachidonic acid (p = 0.070). Addition of RBCs and PRT-treated or untreated plasma to PRT-BCP and CON-BCP, respectively, enhanced aggregation in both groups. CONCLUSIONS: Mirasol PRT treatment of BCPs had a minimal influence on clot formation, whereas aggregation in the absence of RBCs and plasma was significantly reduced. Addition of RBCs and plasma increased agonist-induced responses resulting in comparable aggregation between PRT-BCP and CON-BCP. The clinical relevance for PLT function in vivo of these findings will be investigated in a clinical trial.

Task-oriented modular decomposition of biological networks: trigger mechanism in blood coagulation.

Analysis of complex time-dependent biological networks is an important challenge in the current postgenomic era. We propose a middle-out approach for decomposition and analysis of complex time-dependent biological networks based on: 1), creation of a detailed mechanism-driven mathematical model of the network; 2), network response decomposition into several physiologically relevant subtasks; and 3), subsequent decomposition of the model, with the help of task-oriented necessity and sensitivity analysis into several modules that each control a single specific subtask, which is followed by further simplification employing temporal hierarchy reduction. The technique is tested and illustrated by studying blood coagulation. Five subtasks (threshold, triggering, control by blood flow velocity, spatial propagation, and localization), together with responsible modules, can be identified for the coagulation network. We show that the task of coagulation triggering is completely regulated by a two-step pathway containing a single positive feedback of factor V activation by thrombin. These theoretical predictions are experimentally confirmed by studies of fibrin generation in normal, factor V-, and factor VIII-deficient plasmas. The function of the factor V-dependent feedback is to minimize temporal and parametrical intervals of fibrin clot instability. We speculate that this pathway serves to lessen possibility of fibrin clot disruption by flow and subsequent thromboembolism.

Angiogenesis, fibrinogenesis and presence of synechiae after exeresis of a swine vocal fold mucosal microflap and use of topical mitomycin-C.

PURPOSE: To evaluate swine vocal fold healing in a period of 30 days after topical mitomycin-C application. METHODS: Twelve swine underwent exeresis of mucosal flaps from the free edge of the anterior third of both vocal folds with a cold instrument (laryngeal scissors). The animals were divided into two groups: EG (Experimental Group), consisting of 6 animals undergoing topical MMC application (4 mg/dL) on the operated area for 4 minutes; CG (Control Group), 6 animals undergoing topical saline solution application on the operated area for 4 minutes. After 30 days, the animals were sacrificed and the larynges were collected and examined for the presence of synechiae as well as a histological immunohistochemical assessment of immature and mature collagen deposition, number of blood vessels and myofibroblasts. RESULTS: Mature collagen deposition in the EG was 452.12 microm(2) and 1332.31 microm(2) in the CG; immature collagen deposition was 1511.73 microm(2) in the EG and 1020.61 microm(2) in the CG. The number of myofibroblasts was 1.556 in the EG and 3.583 in the CG. The number of blood vessels was 2.565 in EG and 6.917 in the CG. There were no synechiae in the two studied groups. CONCLUSIONS: There was an increase in immature collagen deposition in the experimental group when compared with the control group. There was a decrease in mature collagen deposition in the experimental group when compared with the control group. There was a decrease in the number of myofibroblasts in the experimental group when compared with the control group. A decrease in blood vessels was observed in the experimental group when compared with the control group. There was no synechia formation in either studied group.

Combined phacoemulsification and viscogoniosynechialysis in the management of patients with chronic angle closure glaucoma.

The purpose of this study was to evaluate the effectiveness of phacoemulsification and viscogoniosynechialysis in the management of patients with chronic angle-closure glaucoma (CACG). Fifty-six eyes of 45 recruited patients were classified into two groups: group 1 had medically controlled CACG (IOP 21 mmHg with maximum tolerated medications) including 21 eyes. All of the patients had at least one quadrant without peripheral anterior synechia in gonioscopy. After phacoemulsification, a viscoelastic agent was used for viscogoniasynecialysis. Success was defined as IOP

Angiogenesis, fibrinogenesis and presence of synechiae after exeresis of a swine vocal fold mucosal microflap and use of topical mitomycin-C / Estudo da angiogênese, fibrogênese e presença de sinéquias após exérese de fragmento de mucosa de pregas vocais de suínos utilizando instrumental frio e mitomicina-C tópica

PURPOSE: To evaluate swine vocal fold healing in a period of 30 days after topical mitomycin-C application. METHODS: Twelve swine underwent exeresis of mucosal flaps from the free edge of the anterior third of both vocal folds with a cold instrument (laryngeal scissors). The animals were divided into two groups: EG (Experimental Group), consisting of 6 animals undergoing topical MMC application (4 mg/dL) on the operated area for 4 minutes; CG (Control Group), 6 animals undergoing topical saline solution application on the operated area for 4 minutes. After 30 days, the animals were sacrificed and the larynges were collected and examined for the presence of synechiae as well as a histological immunohistochemical assessment of immature and mature collagen deposition, number of blood vessels and myofibroblasts. RESULTS: Mature collagen deposition in the EG was 452.12 μm² and 1332.31μm² in the CG; immature collagen deposition was 1511.73μm² in the EG and 1020.61μm² in the CG. The number of myofibroblasts was 1.556 in the EG and 3.583 in the CG. The number of blood vessels was 2.565 in EG and 6.917 in the CG. There were no synechiae in the two studied groups. CONCLUSIONS: There was an increase in immature collagen deposition in the experimental group when compared with the control group. There was a decrease in mature collagen deposition in the experimental group when compared with the control group. There was a decrease in the number of myofibroblasts in the experimental group when compared with the control group. A decrease in blood vessels was observed in the experimental group when compared with the control group. There was no synechia formation in either studied group.

OBJETIVO: Avaliar a cicatrização de pregas vocais de suínos utilizando mitomicina-C tópica, em 30 dias. MÉTODOS: Doze suínos foram submetidos à exérese de mucosa do bordo livre do terço anterior de ambas as pregas vocais com instrumental frio (tesoura curva) e divididos em dois grupos: Grupo E (GE), composto por seis animais com utilização tópica de mitomicina-C(4 mg/dL) na área cruenta por quatro minutos e Grupo Controle (GC), composto por seis animais com utilização tópica de soro fisiológico na área cruenta por quatro minutos. Após 30 dias os animais foram sacrificados e coletadas amostras das pregas vocais para avaliação de sinéquias e análise histológica com quantificação da deposição de colágeno maduro e imaturo, quantificação do número de vasos sanguíneos e miofibroblastos, por método imunoistoquímico. RESULTADOS: A deposição do colágeno maduro no GE foi de 452,12 μm² e 1332,31μm² no GC. A do colágeno imaturo foi de 1511,73μm² no GE e de 1020,61μm² no GC. O número de miofibroblastos foi de 1,556 no GE e de 3,583 no GC. O número de vasos sanguíneos foi de 2,565 no GE e de 6,917 no GC. Não houve formação de sinéquias nos grupo s estudados. CONCLUSÕES: No grupo experimento, quando comparado ao controle, houve significativamente aumento da deposição do colágeno imaturo e diminuição da deposição do colágeno maduro, do número de miofibroblastos e de vasos sanguíneos. Não houve formação de sinéquias em ambos os grupos.

The relationship of vascular endothelial growth factor and coagulation factor (fibrin and fibrinogen) expression in clear cell renal cell carcinoma.

OBJECTIVES: To investigate the relationship between angiogenesis and coagulation markers in tumor tissues of primary renal cell carcinoma (RCC). Tumors stimulate angiogenesis and activate the coagulation cascade. The importance of the interplay between these pathways for RCC is unknown. METHODS: In all, 69 clear cell RCC specimens were analyzed by immunohistochemical staining applied to tissue microarrays. The expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1alpha, fibrinogen and fibrin, and microvessel density were visually analyzed. Finally, staining patterns were related to clinical variables and survival. RESULTS: The VEGF expression was detected in 100% of tumors, with 68% showing a high expression, whereas hypoxia-inducible factor-1alpha staining was low (only 26% had a moderate to high staining). Fibrinogen was expressed adjacent to the tumor cells in 26% of cases, whereas in 84% it was expressed around the blood vessels. In 30% of tumors, expression of fibrin was detected. High tumor VEGF expression correlated with high fibrin staining (P = .05). From a multivariate analysis, microvessel density (P = .033) and fibrinogen adjacent to tumor cells (P = .046) were independent factors related to VEGF expression. CONCLUSIONS: In this study, we found clinical evidence for the permeability activity of VEGF as reflected by extravascular fibrinogen expression adjacent to tumor cells in the extracellular matrix. In addition, VEGF and fibrin expression were associated, indicative for concomitant activation of the coagulation cascade and angiogenesis in RCC. Taken together, these data indicate that activation of angiogenesis and coagulation are related in RCC.