RESUMO
BACKGROUND: Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described. METHODS: Fibrin gels were polymerized and incubated with recombinant human cathepsins (cat) K, L, or S, or plasmin, for dose-dependent and time-dependent studies. Dissolution of fibrin gels was imaged. SDS-PAGE was used to resolve cleaved fragments released from fibrin gels and remnant insoluble fibrin gel that was solubilized prior to electrophoresis to assess fibrin α, ß, and γ polypeptide hydrolysis by cathepsins. Multiplex cathepsin zymography determined active amounts of cathepsins remaining. RESULTS: There was significant loss of α and ß fibrin polypeptides after incubation with cathepsins, with catS completely dissolving fibrin gel by 24â¯h. Binding to fibrin stabilized catL active time; it associated with cleaved fibrin fragments of multiple sizes. This was not observed for catK or S. CatS also remained active for longer times during fibrin incubation, but its association/binding did not withstand SDS-PAGE preparation. CONCLUSIONS: Human cathepsins K, L, and S are fibrinolytic, and specifically can degrade the α and ß fibrin polypeptide chains, generating fragments unique from plasmin. GENERAL SIGNIFICANCE: Demonstration of cathepsins K, L, and S fibrinolytic activity leads to further investigation of contributory roles in disrupting vascular hemostasis, or breakdown of fibrin-based engineered vascular constructs where non-plasmin mediated fibrinolysis must be considered.
Assuntos
Catepsina K/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Fibrina/metabolismo , Fibrinolisina/metabolismo , Fibrinólise , Géis , Hemostasia , HumanosRESUMO
The purpose of this study was to evaluate elasticity and viscoelasticity in the anterior and deeper stromal regions of the cornea after cross linking with three different protocols using atomic force microscopy (AFM) through indentation. A total of 40 porcine corneas were used in this study and were divided into 4 groups (10 corneas per group): control (no treatment), Dresden (corneal epithelial debridement, riboflavin pretreatment for 30 min and a 3mw/cm(2) for 30 min UVA irradiation), accelerated (corneal epithelial debridement, riboflavin pretreatment for 30 min and a 30mw/cm(2) for 3 min UVA irradiation), and genipin (corneal epithelial debridement and submersion of anterior surface in a 1% genipin solution for 4 h). Elasticity and viscoelasticity were quantified using AFM through indentation for all corneas, for the anterior stroma and at a depth of 200 µm. For the control, Dresden, accelerated, and genipin groups, respectively, the average Young's modulus for the anterior stromal region was 0.60 ± 0.58 MPa, 1.58 ± 1.04 MPa, 0.86 ± 0.46 MPa, and 1.71 ± 0.51 MPa; the average for the 200 µm stromal depth was 0.08 ± 0.06 MPa, 0.08 ± 0.04 MPa, 0.08 ± 0.04 MPa, and 0.06 ± 0.01 MPa. Corneas crosslinked with the Dresden protocol and genipin were significantly stiffer than controls (p < 0.05) in the anterior region only. For the control, Dresden, Accelerated, and genipin groups, respectively, the average calculated apparent viscosity for the anterior stroma was 88.2 ± 43.7 kPa-s, 8.3 ± 7.1 kPa-s, 8.1 ± 2.3 kPa-s, and 9.5 ± 3.8 kPa-s; the average for the 200 µm stromal depth was 35.0 ± 3.7 kPa-s, 49.6 ± 35.1 kPa-s, 42.4 ± 17.6 kPa-s, and 41.8 ± 37.6 kPa-s. All crosslinking protocols resulted in a decrease in viscosity in the anterior region only (p < 0.05). The effects of cross-linking seem to be limited to the anterior corneal stroma and do not extend to the deeper stromal region. Additionally, the Dresden and genipin protocols seem to produce a stiffer anterior corneal stroma when compared to the accelerated protocol.
Assuntos
Substância Própria/fisiologia , Reagentes de Ligações Cruzadas/farmacologia , Técnicas de Imagem por Elasticidade/métodos , Elasticidade/fisiologia , Microscopia de Força Atômica/métodos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Animais , Colágeno/metabolismo , Paquimetria Corneana , Substância Própria/efeitos dos fármacos , Riboflavina/farmacologia , Suínos , Raios Ultravioleta , ViscosidadeRESUMO
Sickle cell disease (SCD) is the most common hereditary blood disorder in the United States. SCD is frequently associated with osteonecrosis, osteoporosis, osteopenia, and other bone-related complications such as vaso-occlusive pain, ischemic damage, osteomyelitis, and bone marrow hyperplasia known as sickle bone disease (SBD). Previous SBD models have failed to distinguish the age- and sex-specific characteristics of bone morphometry. In this study, we use the Townes mouse model of SCD to assess the pathophysiological complications of SBD in both SCD and sickle cell trait. Changes in bone microarchitecture and bone development were assessed by using high-resolution quantitative micro-computed tomography and the three-dimensional reconstruction of femurs from male and female mice. Our results indicate that SCD causes bone loss and sex-dependent anatomical changes in bone. SCD female mice in particular are prone to trabecular bone loss, whereas cortical bone degradation occurs in both sexes. We also describe the impact of genetic knockdown of cathepsin K- and E-64-mediated cathepsin inhibition on SBD.
Assuntos
Anemia Falciforme , Doenças Ósseas Metabólicas , Osteoporose , Anemia Falciforme/patologia , Animais , Doenças Ósseas Metabólicas/etiologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Osteoporose/etiologia , Microtomografia por Raio-XRESUMO
Throughout the process of vascular growth and remodeling, the extracellular matrix (ECM) concurrently undergoes significant changes due to proteolytic activity-regulated by both endothelial and surrounding stromal cells. The role of matrix metalloproteinases has been well-studied in the context of vascular remodeling, but other proteases, such as cysteine cathepsins, could also facilitate ECM remodeling. To investigate cathepsin-mediated proteolysis in vascular ECM remodeling, and to understand the role of shear flow in this process, in vitro microvessels were cultured in previously designed microfluidic chips and assessed by immunostaining, zymography, and western blotting. Primary human vessels (HUVECs and fibroblasts) were conditioned by continuous fluid flow and/or small molecule inhibitors to probe cathepsin expression and activity. Luminal flow (in contrast to static culture) decreases the activity of cathepsins in microvessel systems, despite a total protein increase, due to a concurrent increase in the endogenous inhibitor cystatin C. Observations also demonstrate that cathepsins mostly co-localize with fibroblasts, and that fibrin (the hydrogel substrate) may stabilize cathepsin activity in the system. Inhibitor studies suggest that control over cathepsin-mediated ECM remodeling could contribute to improved maintenance of in vitro microvascular networks; however, further investigation is required. Understanding the role of cathepsin activity in in vitro microvessels and other engineered tissues will be important for future regenerative medicine applications.
RESUMO
Sickle cell disease is caused by the amino acid substitution of glutamic acid to valine, which leads to the polymerization of deoxygenated sickle hemoglobin (HbS) into long strands. These strands are responsible for the sickling of red blood cells (RBCs), making blood hyper-coagulable leading to an increased chance of vaso-occlusive crisis. The conformational changes in sickled RBCs traveling through narrow blood vessels in a highly viscous fluid are critical in understanding; however, there are few studies that investigate the origins of the molecular mechanical behavior of sickled RBCs. In this work, we investigate the molecular mechanical properties of HbS molecules. A mechanical model was used to estimate the directional stiffness of an HbS molecule and the results were compared to adult human hemoglobin (HbA). The comparison shows a significant difference in strength between HbS and HbA, as well as anisotropic behavior of the hemoglobin molecules. The results also indicated that the HbS molecule experienced more irreversible mechanical behavior than HbA under compression. Further, we have characterized the elastic and compressive properties of a double stranded sickle fiber using six HbS molecules, and it shows that the HbS molecules are bound to each other through strong inter-molecular forces.
Assuntos
Hemoglobina Falciforme/química , Fenômenos Mecânicos , Simulação de Dinâmica Molecular , Conformação Proteica , Algoritmos , Fenômenos Químicos , Humanos , Modelos Teóricos , TemperaturaRESUMO
Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin-based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis. To further explore this, we used two independent computational technqiues, molecular docking and bioinformatics sequence analysis (PACMANS), to predict potential binding interactions and sites of hydrolysis between cathepsins K, L, and S and fibrinogen. By comparing the results from these two objective, computational methods, it was determined that cathepsins K, L, and S do bind and cleave fibrinogen α, ß, and γ chains at similar and unique sites. These differences were visualized experimentally by the unique cleaved fibrinogen banding patterns after incubation with each of the cathepsins, separately. In conclusion, human cysteine cathepsins K, L, and S are a new class of proteases that should be considered during fibrin(ogen) degradation studies both for disease processes where coagulation is a concern, and also in the implementation and design of bioengineered systems.
Assuntos
Catepsinas/metabolismo , Biologia Computacional/métodos , Fibrinogênio/química , Fibrinogênio/metabolismo , Sítios de Ligação , Catepsina K/química , Catepsina K/metabolismo , Catepsina L/química , Catepsina L/metabolismo , Catepsinas/química , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , ProteóliseRESUMO
A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, the endothelial cells (ECs) forming the vasculature can exhibit a number of distinct functional subphenotypes like arterial or venous ECs, as well as angiogenic tip and stalk ECs. In this study, we investigate the in vitro differentiation of EC subphenotypes from embryonic stem cells (ESCs). Using our staged induction methods and chemically defined mediums, highly angiogenic EC subpopulations, as well as less proliferative and less migratory EC subpopulations, are derived. Furthermore, the EC subphenotypes exhibit distinct surface markers, gene expression profiles, and positional affinities during sprouting. While both subpopulations contained greater than 80% VE-cad+/CD31+ cells, the tip/stalk-like EC contained predominantly Flt4+/Dll4+/CXCR4+/Flt-1- cells, while the phalanx-like EC was composed of higher numbers of Flt-1+ cells. These studies suggest that the tip-specific EC can be derived in vitro from stem cells as a distinct and relatively stable EC subphenotype without the benefit of its morphological positioning in the sprouting vessel.