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1.
Platelets ; 34(1): 2131752, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36210791

RESUMO

This study investigated the effect of L-PRF on promoting full-thickness skin grafting for the treatment of diabetic foot ulcer wounds and attempted to characterize the mechanism. In a retrospective study, we centrifugated 10-20 ml of venous blood at 1006.2 g for 20 min. The fibrin clot between the top oligocellular plasma layer and the bottom erythrocyte layer was extracted and directly used, without compression, to cover the wound after debridement. Patients who received L-PRF before skin grafting underwent surgery earlier than patients in the control group. Skin necrosis occurred in 7 patients (28%) in the L-PRF group and 16 (64%) in the control group. The difference was statistically significant, P < .05. The postoperative infection rate in the control group (56%) was significantly higher than that in the L-PRF group (24%), P < .05. During a mean follow-up of 1 year, ulcer recurrence occurred in 9 patients (36%) in the control group compared with 4 patients (16%) in the L-PRF group, P < .05. The final amputation rate was also higher in the control group (48%) than in the L-PRF group (20%). The difference is statistically significant, P < .05. The Maryland scale score and SF-36 score of the two groups of patients after treatment were significantly better than those before treatment, and the difference was statistically significant (P < .05). The L-PRF group (94.80 ± 4.14) had better foot scores at the last follow-up after treatment than the control group (88.84 ± 5.22) (P < .05). The results showed that L-PRF played a positive role in the treatment of Wagner grade 4 ulcer gangrene with free full-thickness skin grafts.


What is the context?● Diabetic foot is a serious complication in the later stage of the disease course of diabetic patients. The incidence rate is increasing year by year. In severe cases, it can lead to amputation or even death.● For diabetic ulcer wounds, dressings such as L-PRF or autologous fat are often used in the initial stage to speed up wound healing. For advanced wounds, especially patients with local tissue gangrene, simple wound dressings cannot meet the needs of wounds. People often use skin flaps or different types of skin grafts to treat advanced wounds.● Flap or skin grafting has been shown to be effective, but because of the patient's own neurovascular injury and infection, the rate of graft necrosis and ulcer recurrence is extremely high. What is new?● This study discusses the treatment of advanced wounds in diabetes. After thorough debridement and before skin grafting, we first covered the wound with L-PRF and observed the wound condition. Studies have shown that the use of L-PRF can allow the original poor wound to be reconstructed: the content of growth factors and growth-related cells is increased, blood circulation is improved and granulation tissue growth, bone and tendon exposure is improved, and infection is controlled. What is the impact?● This study provides evidence that using L-PRF to reconstruct wounds can greatly shorten the preparation time for elective surgery. Reconstructed wounds can better accept free skin grafts, and the incidence of postoperative complications and amputation (particularly, toe amputation) is also lower.


Assuntos
Diabetes Mellitus , Pé Diabético , Humanos , Pé Diabético/cirurgia , Transplante de Pele , Fibrina/uso terapêutico , Gangrena/cirurgia , Estudos Retrospectivos , Cicatrização , Leucócitos , Dedos do Pé/cirurgia
2.
Platelets ; 34(1): 2139365, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36325627

RESUMO

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.


Why was the study done? Platelets and blood coagulation system interact with each other in hemostasis and intravascular thrombosis.Direct platelet effects on fibrin formation (plasma clotting), the final stage of blood coagulation cascade, have been insufficiently studied.The work is aimed at developing a method for studying platelet participation in fibrin formation in blood plasma and investigating the influence of platelet agonists on this reaction.What is new? Platelets significantly accelerate fibrin formation and their activation with various agonists (thrombin, collagen, arachidonic acid) enhances these effects.Effects of platelets on fibrin formation correlated with their ability to stimulate thrombin generation in blood plasmaEffects of platelets on fibrin formation and thrombin generation correlated with the level of phosphatidylserine exposure on their surfaceWhat is the impact? This study provides further evidence that platelet procоagulant effects on fibrin formation should be considered in investigations of platelet involvement in hemostatic and thrombotic reactions and in the evaluation of the efficacy of antiplatelet drugs.


Assuntos
Plaquetas , Trombina , Humanos , Trombina/farmacologia , Trombina/metabolismo , Plaquetas/metabolismo , Fibrina/metabolismo , Calcimicina/metabolismo , Calcimicina/farmacologia , Ácido Araquidônico/farmacologia , Ácido Araquidônico/metabolismo , Fosfatidilserinas/metabolismo , Colágeno/farmacologia , Colágeno/metabolismo , Difosfato de Adenosina/farmacologia , Difosfato de Adenosina/metabolismo
3.
Cancer Lett ; 553: 215983, 2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36404569

RESUMO

Peritoneal metastasis is one of the most frequent causes of death in several types of advanced cancers; however, the underlying molecular mechanisms remain largely unknown. In this study, we exploited multicolor fluorescent lineage tracking to investigate the clonality of peritoneal metastasis in mouse xenograft models. When peritoneal metastasis was induced by intraperitoneal or orthotopic injection of multicolored cancer cells, each peritoneally metastasized tumor displayed multicolor fluorescence regardless of metastasis sites, indicating that it consists of multiclonal cancer cell populations. Multicolored cancer cell clusters form within the peritoneal cavity and collectively attach to the peritoneum. In vitro, peritoneal lavage fluid or cleared ascitic fluid derived from cancer patients induces cancer cell clustering, which is inhibited by anticoagulants. Cancer cell clusters formed in vitro and in vivo are associated with fibrin formation. Furthermore, tissue factor knockout in cancer cells abrogates cell clustering, peritoneal attachment, and peritoneal metastasis. Thus, we propose that cancer cells activate the coagulation cascade via tissue factor to form fibrin-mediated cell clusters and promote peritoneal attachment; these factors lead to the development of multiclonal peritoneal metastasis and may be therapeutic targets.


Assuntos
Neoplasias Peritoneais , Peritônio , Camundongos , Animais , Humanos , Peritônio/metabolismo , Tromboplastina/genética , Tromboplastina/metabolismo , Tromboplastina/uso terapêutico , Fibrinogênio , Neoplasias Peritoneais/patologia , Análise por Conglomerados , Fibrina/metabolismo , Fibrina/uso terapêutico
4.
Sci Rep ; 12(1): 18829, 2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36335251

RESUMO

Intramuscular injection of anemoside B4 (AB4) has a superior therapeutic effect on clinical mastitis in lactating cows. Here, we explored AB4's effect on milk whey in clinical mastitis-affected cows using proteomics. Among fifty clinical mastitis cows received AB4 administration (0.05 ml/kg/day, for 7 days), twelve healed cows were selected and marked as group T. Twelve clinically heathy cows received the same dose of saline for 7 days, marked as group C. Collected milk whey of group T before and after AB4 administration marked as T1 and T2, respectively. The milk whey of group C after saline injection marked as C1. Milk whey protein changes were detected using tandem mass tag-based quantitative proteomic. We identified 872 quantifiable proteins in the samples. Among them, 511 proteins between T1 and C1, and 361 proteins between T2 and T1 were significantly altered. T1 than C1 had significantly more proteins associated with inflammatory damage and trans-endothelial migration of leukocytes, whereas these proteins were reduced in T2 treated with AB4. Compared with C, proteins associated with fibrin clot degradation and complement system activation were downregulated in T1 but upregulated in T2. In summary, AB4 can exert its therapeutic effect on clinical mastitis in cows mainly by reducing inflammatory damage, activating the complement system, inhibiting trans-endothelial migration of leukocytes, and promoting degradation of milk fibrin clots.


Assuntos
Mastite Bovina , Leite , Animais , Bovinos , Feminino , Fibrina/metabolismo , Lactação , Mastite Bovina/tratamento farmacológico , Mastite Bovina/metabolismo , Leite/metabolismo , Proteínas do Leite/metabolismo , Proteômica , Soro do Leite/metabolismo , Proteínas do Soro do Leite/farmacologia , Proteínas do Soro do Leite/metabolismo
5.
Thromb Res ; 220: 131-140, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36347079

RESUMO

INTRODUCTION: Trauma induced coagulopathy (TIC) is common after severe trauma, increasing transfusion requirements and mortality among patients. TIC has several phenotypes, with primary hyperfibrinolysis being among the most lethal. We aimed to investigate the contribution of hypercoagulation, hemodilution, and fibrinolytic activation to the hyperfibrinolytic phenotype of TIC, by examining fibrin formation in a plasma-based model of TIC. We hypothesized that instabilities arising from TIC will be due primarily to increased fibrinolytic activation rather than hemodilution or tissue factor (TF) induced hypercoagulation. METHODS: The influence of TF, hemodilution, fibrinogen consumption, tissue plasminogen activator (tPA), and the antifibrinolytic tranexamic acid (TXA) on plasma clot formation and structure were examined using rheometry, optical properties, and confocal microscopy. These were then compared to plasma samples from trauma patients at risk of developing TIC. RESULTS: Combining TF-induced clot formation, 15 % hemodilution, fibrinogen consumption, and tPA-induced fibrinolysis, the clot characteristics and hyperfibrinolysis were consistent with primary hyperfibrinolysis. TF primarily increased fibrin polymerization rates and reduced fiber length. Hemodilution decreased clot optical density but had no significant effect on mechanical clot stiffness. TPA addition induced primary clot lysis as observed mechanically and optically. TXA restored mechanical clot formation but did not restore clot structure to control levels. Patients at risk of TIC showed increased clot formation, and lysis like that of our simulated model. CONCLUSIONS: This simulated TIC plasma model demonstrated that fibrinolytic activation is a primary driver of instability during TIC and that clot mechanics can be restored, but clot structure remains altered with TXA treatment.


Assuntos
Transtornos da Coagulação Sanguínea , Hemostáticos , Trombofilia , Ácido Tranexâmico , Humanos , Ativador de Plasminogênio Tecidual , Transtornos da Coagulação Sanguínea/etiologia , Hemodiluição , Tromboplastina , Fibrinogênio , Fibrina
6.
Int J Mol Sci ; 23(21)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36361742

RESUMO

Transglutaminase (TGM) isoform catalyze the cross-linking reaction of identical or different substrate proteins. Eosinophil has been recognized in chronic rhinosinusitis with nasal polyps (CRSwNP) forming tissue eosinophil in nasal polyp (NP), and TGM isoforms are suggested to be associated with a critical role in asthma and other allergic conditions. The aim of this study was to reveal the association of specific TGM isoform with both the tissue eosinophil infiltration deeply concerning with the intractable severity of CRSwNP and the fibrin polymerization ability of TGM isoform associated with the tissue eosinophil infiltration, which lead to NP formation and/or maintenance in CRSwNP. NP tissues (CRSwNP group) and uncinate process (UP) (control group) were collected from patients with CRSwNP and control subjects. We examined: (1) the expression level of TGM isoforms by using a real-time polymerase chain reaction (PCR) and the comparison to the issue eosinophil count in the CRSwNP group, (2) the location of specific TGM isoform in the mucosal tissue using immunohistochemistry, (3) the inflammatory cell showing the colocalization of specific TGM isoform in Laser Scanning Confocal Microscopy (LSCM) imaging, and (4) the fibrin polymerase activity of specific TGM isoform using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A certain level of TGM 1, 2, 3, 5 expression was present in both the CRSwNP group and the control group. Only TGM 1 expression showed a positive significant correlation with the tissue eosinophil count in the CRSwNP group. The localization of TGM 1 in NP (CRSwNP) laid mainly in a submucosal layer as inflammatory cells and was at the cytoplasm in the tissue eosinophil. Fibrin polymerase activity of TGM 1 showed the same polymerase ability of factor XIIIA. TGM 1 might influence the NP formation and/or maintenance in CRSwNP related to the tissue eosinophil infiltration, which formed fibrin mesh composing NP stroma.


Assuntos
Pólipos Nasais , Rinite , Sinusite , Humanos , Pólipos Nasais/patologia , Eosinófilos/metabolismo , Rinite/patologia , Fibrina/metabolismo , Polimerização , Sinusite/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo , Doença Crônica
7.
BMJ Case Rep ; 15(11)2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36423943

RESUMO

Acute fibrinous and organising pneumonia (AFOP) is a rare form of interstitial lung disease. It is a pathological diagnosis sharing similarities to organising pneumonia, diffuse alveolar damage and eosinophilic pneumonia, however, is histologically distinct, characterised by intra-alveolar fibrin deposition ('fibrin balls') and associated organising pneumonia. AFOP was first described in 2002, only 150 cases have been reported since. While it has been described in association with infection, autoimmune disorders, connective tissue diseases, drugs, environmental exposures and organ transplant, it can also be idiopathic in nature. AFOP follows an acute course with potential rapid fulminant respiratory failure, or a subacute trajectory with a more favourable prognosis. Corticosteroids are commonly prescribed to induce remission. While cases of relapse of AFOP during weaning or cessation of steroids have been described, there are no published cases of remote relapse of AFOP. We describe a case of idiopathic AFOP, which recurred after 12 years of good health.


Assuntos
Doenças Pulmonares Intersticiais , Pneumonia , Humanos , Pneumonia/complicações , Corticosteroides/uso terapêutico , Doenças Pulmonares Intersticiais/complicações , Doença Crônica , Recidiva , Fibrina
8.
Urologiia ; (5): 71-76, 2022 Nov.
Artigo em Russo | MEDLINE | ID: mdl-36382821

RESUMO

INTRODUCTION: The outcome of surgical treatment of renal cancer depends not only on cancer-specific survival, but also on the degree of loss of renal function, which often develops after surgery, especially radical nephrectomy. AIM: To study the features of functional changes in a solitary kidney as a compensation mechanism after radical nephrectomy for renal cancer. MATERIALS AND METHODS: The functional state of a solitary kidney in 36 patients with renal cancer who undergone to radical nephrectomy was evaluated. There were 20 and 16 women. The mean age was 59.0+/-10.8 years (from 39 to 76 years). The size of the tumor was in the range of 7.0-12.0 cm. All patients with a solitary kidney underwent a follow-up examination 3 months after surgery, including measurement of peripheral blood pressure with calculation of mean dynamic pressure, renal ultrasound, calculation of glomerular filtration rate (GFR), renal doppler ultrasound, determination of serum fibrinogen and fibrin monomers, and microscopy of the bulbar conjunctiva. Patients who had pathological abnormalities during the examination were prescribed reno-cardioprotective drugs, including perindopril in a titrated dose, apixaban 5 mg a day as thromboprophylaxis and for improvement of the flow properties of blood for a period of 3 months with re-evaluation of the above parameters. RESULTS: In 61.1% of patients after radical nephrectomy, on 2-4 postoperative days, there was a tendency to increase blood pressure compared to baseline values (p<0.05). By the seventh day after the procedure, the volume of the contralateral kidney increased on average by 16% (from 110.4+/-11.2 cm3 to 132.4+/-4.8 cm3, p<0.05). After radical nephrectomy, a decrease in GFR was detected in 33 cases (91.7%; p<0.05). Renal doppler ultrasound showed a moderate increase in linear blood flow, the resistance index in the main renal artery, and a decrease in the pulse index in the segmental and arcuate arteries. The microscopy of the bulbar conjunctiva in 83.3% of patients revealed changes in the microcirculatory bed, including narrowing of arterioles, dilation of venules, a decrease in venular and capillary blood flow. After 3 months of reno-cardioprotective therapy, it was revealed that the target values of blood pressure (<130/85 mm Hg) were achieved with an average dynamic blood pressure of 93.4+/-2.6 mm Hg. In addition, a decrease in creatinine to an average of 106.2+/-6.4, fibrinogen and fibrin monomers to subnormal values of 3.2+/-0.2 g/l and up to 8.1+/-0.5x10-2 g/l, respectively were seen. Renal hypertrophy according to ultrasound examination was preserved with a mean kidney volume 119.7+/-3.6 cm3. Disturbances in peripheral microcirculation according to the microscopy of the bulbar conjunctiva was assessed as moderate. CONCLUSION: The development of CKD in patients with a solitary kidney is accompanied by a structural reorganization of the organ with an increase in blood pressure, an increase in its volume, a decrease in function, microcirculatory disorders and hypertensive nephropathy. Considering the prognostic significance of changes in the solitary kidney, it is important not only to control the functional parameters, but also to include reno- cardioprotective therapy as a standard, since it contributes to the preservation of the renal function and prevents the rapid progression of CKD. Thus, medical and social rehabilitation of patients with a solitary kidney is required. However, it is currently cannot be considered comprehensive.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Insuficiência Renal Crônica , Rim Único , Tromboembolia Venosa , Humanos , Feminino , Pessoa de Meia-Idade , Idoso , Carcinoma de Células Renais/cirurgia , Rim Único/complicações , Rim Único/cirurgia , Microcirculação , Anticoagulantes , Estudos Retrospectivos , Tromboembolia Venosa/complicações , Tromboembolia Venosa/cirurgia , Nefrectomia/efeitos adversos , Nefrectomia/métodos , Neoplasias Renais/patologia , Rim/diagnóstico por imagem , Rim/cirurgia , Rim/patologia , Taxa de Filtração Glomerular , Insuficiência Renal Crônica/cirurgia , Fibrinogênio , Fibrina
9.
Int J Mol Sci ; 23(22)2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36430952

RESUMO

Histological structure of thrombi is a strong determinant of the outcome of vascular recanalization therapy, the only treatment option for acute ischemic stroke (AIS) patients. A total of 21 AIS patients from this study after undergoing non-enhanced CT scan and multimodal MRI were treated with mechanical stent-based and manual aspiration thrombectomy, and thromboembolic retrieved from a cerebral artery. Complementary histopathological and imaging analyses were performed to understand their composition with a specific focus on fibrin, von Willebrand factor, and neutrophil extracellular traps (NETs). Though distinct RBC-rich and platelet-rich areas were found, AIS patient thrombi were overwhelmingly platelet-rich, with 90% of thrombi containing <40% total RBC-rich contents (1.5 to 37%). Structurally, RBC-rich areas were simple, consisting of tightly packed RBCs in thin fibrin meshwork with sparsely populated nucleated cells and lacked any substantial von Willebrand factor (VWF). Platelet-rich areas were structurally more complex with thick fibrin meshwork associated with VWF. Plenty of leukocytes populated the platelet-rich areas, particularly in the periphery and border areas between platelet-rich and RBC-rich areas. Platelet-rich areas showed abundant activated neutrophils (myeloperoxidase+ and neutrophil-elastase+) containing citrullinated histone-decorated DNA. Citrullinated histone-decorated DNA also accumulated extracellularly, pointing to NETosis by the activated neutrophils. Notably, NETs-containing areas showed strong reactivity to VWF, platelets, and high-mobility group box 1 (HMGB1), signifying a close interplay between these components.


Assuntos
Armadilhas Extracelulares , AVC Isquêmico , Acidente Vascular Cerebral , Trombose , Humanos , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Fator de von Willebrand/metabolismo , Histonas , Acidente Vascular Cerebral/patologia , Trombose/patologia , Fibrina/metabolismo , DNA
10.
Clin Appl Thromb Hemost ; 28: 10760296221126172, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36217728

RESUMO

OBJECTIVES: Preeclampsia (PE) is a serious complication of pregnancy. The fibrinolytic system play crucial roles regarding placentation and evolution of PE. AIM: To study comprehensively components of the fibrinolytic system and fibrin lysability in women with PE. DESIGN AND METHODS: 117 women with PE and matched controls were included. Tissue type plasminogen activator (t-PA), plasminogen, PAI-1, plasmin inhibitor (PI), D-dimer, the fibrinolytic potential of dextran sulphate euglobulin fraction (DEF), PAI-2, polymere PAI-2, fibrin clot lysability, thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen were assessed. RESULTS: Women with PE had significantly increased concentrations of t-PA and PAI-1, whereas the plasma concentration of PAI-2 was significantly lower compared to controls, p < 0.0001. Polymere PAI-2 was detected in both groups. DEF, TAFI and fibrinogen were not different between the groups. D-dimer was significantly increased and plasminogen/PI together with fibrin clot lysability time decreased in the PE-group, p = 0.0004 p = 0.04, p = 0.03, p < 0.0001 respectively. CONCLUSION: This study demonstrates that PE is associated with an affected t-PA/PAI-1 system, decreased PAI-2 and increased fibrin lysability. Furthermore, PAI-2 has the potential to polymerize during pregnancy.


Assuntos
Antifibrinolíticos , Carboxipeptidase B2 , Pré-Eclâmpsia , Trombose , Sulfato de Dextrana/farmacologia , Feminino , Fibrina , Fibrinogênio/farmacologia , Fibrinólise , Humanos , Organotiofosfatos , Plasminogênio/farmacologia , Inibidor 1 de Ativador de Plasminogênio , Inibidor 2 de Ativador de Plasminogênio/farmacologia , Gravidez , Ativador de Plasminogênio Tecidual
11.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232775

RESUMO

Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Diferenciação Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ciclina D1/metabolismo , Cisteína/metabolismo , Fibrina/metabolismo , Humanos , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteocalcina/metabolismo , Osteonectina/metabolismo , Osteopontina/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte
12.
Int J Mol Sci ; 23(20)2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36293273

RESUMO

INTRODUCTION: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this. METHODS: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified. RESULTS: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles. CONCLUSIONS: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.


Assuntos
Preservação da Fertilidade , Neoplasias , Humanos , Feminino , Ovário/metabolismo , Preservação da Fertilidade/métodos , Trombina/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Bioengenharia , Neoplasias/metabolismo
13.
Int J Mol Sci ; 23(20)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36293367

RESUMO

Integrin αIIbß3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbß3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbß3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbß3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Trombose , Camundongos , Animais , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Inibidores da Agregação Plaquetária/metabolismo , Actomiosina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Plaquetas/metabolismo , Trombose/metabolismo , Fibrinogênio/metabolismo , Proteína Quinase C/metabolismo , Difosfato de Adenosina/metabolismo , Fibrina/metabolismo , Fosfatidilinositóis/metabolismo
14.
Int J Mol Sci ; 23(20)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36293401

RESUMO

Age-related macular degeneration (AMD) is a global leading cause of visual impairment in older populations. 'Wet' AMD, the most common subtype of this disease, occurs when pathological angiogenesis infiltrates the subretinal space (choroidal neovascularization), causing hemorrhage and retinal damage. Gold standard anti-vascular endothelial growth factor (VEGF) treatment is an effective therapy, but the long-term prevention of visual decline has not been as successful. This warrants the need to elucidate potential VEGF-independent pathways. We generated blood out-growth endothelial cells (BOECs) from wet AMD and normal control subjects, then induced angiogenic sprouting of BOECs using a fibrin gel bead assay. To deconvolute endothelial heterogeneity, we performed single-cell transcriptomic analysis on the sprouting BOECs, revealing a spectrum of cell states. Our wet AMD BOECs share common pathways with choroidal neovascularization such as extracellular matrix remodeling that promoted proangiogenic phenotype, and our 'activated' BOEC subpopulation demonstrated proinflammatory hallmarks, resembling the tip-like cells in vivo. We uncovered new molecular insights that pathological angiogenesis in wet AMD BOECs could also be driven by interleukin signaling and amino acid metabolism. A web-based visualization of the sprouting BOEC single-cell transcriptome has been created to facilitate further discovery research.


Assuntos
Neovascularização de Coroide , Degeneração Macular Exsudativa , Humanos , Neovascularização de Coroide/tratamento farmacológico , Transcriptoma , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Degeneração Macular Exsudativa/tratamento farmacológico , Fatores de Crescimento do Endotélio Vascular , Interleucinas/uso terapêutico , Aminoácidos , Fibrina , Inibidores da Angiogênese/uso terapêutico
15.
PLoS One ; 17(10): e0275956, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36301961

RESUMO

Fibrin clot structure/function contributes to cardiovascular disease. We examined sulfur-containing metabolites as determinants of fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to outcomes in coronary artery disease (CAD) patients. Effects of B-vitamin/folate therapy on CLT and Absmax were studied. Plasma samples were collected from 1,952 CAD patients randomized in a 2 x 2 factorial design to (i) folic acid, vitamins B12, B6; (ii) folic acid, vitamin B12; (iii) vitamin B6; (iv) placebo for 3.8 years in the Western Norway B-Vitamin Intervention Trial. Clot lysis time (CLT) and maximum absorbance (Absmax) were determined using a validated turbidimetric assay. Acute myocardial infarction (AMI) and mortality were assessed during a 7-year follow-up. Data were analyzed using bivariate and multiple regression. Survival free of events was studied using Kaplan Mayer plots. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional hazards models. Baseline urinary homocysteine (uHcy)-thiolactone and plasma cysteine (Cys) were significantly associated with CLT while plasma total Hcy was significantly associated with Absmax, independently of fibrinogen, triglycerides, vitamin E, glomerular filtration rate, body mass index, age, sex plasma creatinine, CRP, HDL-C, ApoA1, and previous diseases. B-vitamins/folate did not affect CLT and Absmax. Kaplan-Meier analysis showed associations of increased baseline CLT and Absmax with worse outcomes. In Cox regression analysis, baseline CLT and Absmax (>cutoff) predicted AMI (CLT: HR 1.58, 95% CI 1.10-2.28; P = 0.013. Absmax: HR 3.22, CI 1.19-8.69; P = 0.021) and mortality (CLT: HR 2.54, 95% CI 1.40-4.63; P = 0.002. Absmax: 2.39, 95% CI 1.17-4.92; P = 0.017). After adjustments for other prognostic biomarkers these associations remained significant. Cys and uHcy-thiolactone, but not tHcy, were significant predictors of AMI in Cox regression models that included CLT. Conclusions uHcy-thiolactone and plasma Cys are novel determinants of CLT, an important predictor of adverse CAD outcomes. CLT and Absmax were not affected by B-vitamin/folate therapy, which could account for the lack of efficacy of such therapy in CAD. Trial registration: URL: http://clinicaltrials.gov. Identifier: NCT00354081.


Assuntos
Doença da Artéria Coronariana , Infarto do Miocárdio , Trombose , Complexo Vitamínico B , Humanos , Fibrina/metabolismo , Prognóstico , Vitamina B 12 , Ácido Fólico , Homocisteína , Infarto do Miocárdio/tratamento farmacológico , Trombose/tratamento farmacológico , Fatores de Risco
16.
Biomolecules ; 12(10)2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36291710

RESUMO

A peritoneal adhesion (PA) is a fibrotic tissue connecting the abdominal or visceral organs to the peritoneum. The formation of PAs can induce a variety of clinical diseases. However, there is currently no effective strategy for the prevention and treatment of PAs. Damage to peritoneal mesothelial cells (PMCs) is believed to cause PAs by promoting inflammation, fibrin deposition, and fibrosis formation. In the early stages of PA formation, PMCs undergo mesothelial-mesenchymal transition and have the ability to produce an extracellular matrix. The PMCs may transdifferentiate into myofibroblasts and accelerate the formation of PAs. Therefore, the aim of this review was to understand the mechanism of action of PMCs in PAs, and to offer a theoretical foundation for the treatment and prevention of PAs.


Assuntos
Matriz Extracelular , Peritônio , Epitélio , Miofibroblastos , Fibrina
17.
Int J Implant Dent ; 8(1): 39, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36184700

RESUMO

PURPOSE: To compare the release of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-I) and interleukin 1ß (IL-1ß) of plasma rich in growth factors (PRGF) and leucocyte platelet-rich fibrin (L-PRF) and to evaluate their biological implication in osteoblasts. METHODS: Blood from 3 healthy volunteers was processed into PRGF, immediate L-PRF (L-PRF 0') and L-PRF 30 min after collection (L-PRF-30') and a control group. Growth factors release were analyzed at 7 times by ELISA. Cell proliferation, collagen-I synthesis and alkaline phosphatase activity were assessed in primary cultures of human osteoblasts. RESULTS: A slower controlled release of IGF-I, VEGF and PDGF was observed in the PRGF group at day 14. A higher synthesis of type I collagen was also quantified in PRGF. L-PRF released significantly higher amounts of IL-1ß, that was almost absent in the PRGF. CONCLUSIONS: The addition of leukocytes dramatically increases the secretion of proinflammatory cytokines, which are likely to negatively influence the synthesis of type I collagen and alkaline phosphatase (ALP) by osteoblasts.


Assuntos
Fibrina Rica em Plaquetas , Fosfatase Alcalina/metabolismo , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Preparações de Ação Retardada/metabolismo , Fibrina/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-1beta/metabolismo , Leucócitos , Osteoblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Neuropharmacology ; 221: 109277, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36223864

RESUMO

Ischemic stroke is characterized by the loss of cerebral blood flow, which frequently leads to neurological deficits. Tissue plasminogen activator is the only therapeutic agent approved to treat ischemic stroke but increases the risk of intracranial hemorrhage and mortality. The fibrinogen-depleting agent lumbrokinase has been used to improve myocardial perfusion in symptomatic stable angina and to prevent secondary ischemic stroke. Lumbrokinase is highly fibrin-specific and only active in the presence of fibrin. Therefore, lumbrokinase has a low risk of hemorrhage due to excessive fibrinolysis. In this study, we aimed to clarify the neuroprotection of lumbrokinase in mice subjected to permanent middle cerebral artery occlusion. Lumbrokinase significantly attenuated infarct volume and improved neurological dysfunction. Lumbrokinase dramatically decreased the expressions of the endoplasmic reticulum (ER) transmembrane receptor protein inositol-requiring enzyme-1 (IRE1) and its downstream transcription factor, XBP-1, caspase-12, and NF-κB activity, thereby significantly inhibiting apoptosis and autophagy and decreasing the NLRP3 inflammasome. Our evidence indicates that post-stroke treatment with lumbrokinase protects against ischemic stroke, thereby regulating ER stress through the collective inhibitory effect of the IRE1 signaling pathways to decrease apoptosis, autophagy, and inflammatory responses. We suggest that lumbrokinase is potential as an adjuvant treatment for ischemic stroke.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Animais , Camundongos , Ativador de Plasminogênio Tecidual/uso terapêutico , Estresse do Retículo Endoplasmático , Infarto da Artéria Cerebral Média/tratamento farmacológico , Proteínas Serina-Treonina Quinases , Apoptose , Proteínas de Membrana/metabolismo , Fibrina/farmacologia , Fibrina/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo
19.
Tissue Eng Regen Med ; 19(6): 1169-1184, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36201158

RESUMO

BACKGROUND: The production of tissue-engineered vascular graft (TEVG) usually involves a prolonged bioreactor cultivation period of up to several weeks to achieve maturation of extracellular matrix and sufficient mechanical strength. Therefore, we aimed to substantially shorten this conditioning time by combining a TEVG textile scaffold with a recently developed copolymer reinforced fibrin gel as a cell carrier. We further implemented our grafts with magnetic resonance imaging (MRI) contrast agents to allow the in-vitro monitoring of the TEVG's remodeling process. METHODS: Biodegradable polylactic-co-glycolic acid (PLGA) was electrospun onto a non-degradable polyvinylidene fluoride scaffold and molded along with copolymer-reinforced fibrin hydrogel and human arterial cells. Mechanical tests on the TEVGs were performed both instantly after molding and 4 days of bioreactor conditioning. The non-invasive in vitro monitoring of the PLGA degradation and the novel imaging of fluorinated thermoplastic polyurethane (19F-TPU) were performed using 7T MRI. RESULTS: After 4 days of close loop bioreactor conditioning, 617 ± 85 mmHg of burst pressure was achieved, and advanced maturation of extracellular matrix (ECM) was observed by immunohistology, especially in regards to collagen and smooth muscle actin. The suture retention strength (2.24 ± 0.3 N) and axial tensile strength (2.45 ± 0.58 MPa) of the TEVGs achieved higher values than the native arteries used as control. The contrast agents labeling of the TEVGs allowed the monitorability of the PLGA degradation and enabled the visibility of the non-degradable textile component. CONCLUSION: Here, we present a concept for a novel textile-reinforced TEVG, which is successfully produced in 4 days of bioreactor conditioning, characterized by increased ECM maturation and sufficient mechanical strength. Additionally, the combination of our approach with non-invasive imaging provides further insights into TEVG's clinical application.


Assuntos
Meios de Contraste , Engenharia Tecidual , Humanos , Engenharia Tecidual/métodos , Prótese Vascular , Fibrina , Têxteis
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