cytochrome-c-t and Thrombosis

The periosteum is a membrane that surrounds bones, providing essential cellular and biological components for fracture healing and bone repair. Tissue engineered scaffolds able to function as periosteum substitutes can significantly improve bone regeneration in severely injured tissues. Efforts to develop more bioactive and tunable periosteal substitutes are required to improve the success of this tissue engineering approach. In this work, a chemical modification was performed in chitosan, a polysaccharide with osteoconductive properties, by introducing phosphate groups to its structure. The phosphorylated polymer (Chp) was used to produce chitosan-xanthan-based scaffolds for periosteal tissue engineering. Porous and mechanically reinforced matrices were obtained with addition of the surfactant Kolliphor® P188 and the silicone rubber Silpuran® 2130A/B. Scaffolds properties, such as large pore sizes (850-1097 μm), micro-roughness and thickness (0.7-3.5 mm in culture medium), as well as low thrombogenicity compared to standard implantable materials, extended degradation time and negligible cytotoxicity, enable their application as periosteum substitutes. Moreover, the higher adsorption of bone morphogenetic protein mimic (cytochrome C) by Chp-based formulations suggests improved osteoinductivity of these materials, indicating that, when used in vivo, the material would be able to concentrate native BMPs and induce osteogenesis. The scaffolds produced were not toxic to adipose tissue-derived stem cells, however, cell adhesion and proliferation on the scaffolds surfaces can be still further improved. The mineralization observed on the surface of all formulations indicates that the materials studied have promising characteristics for the application in bone regeneration.

Topics: Adipose Tissue; Adsorption; Alkaline Phosphatase; Calcium; Cell Death; Cells, Cultured; Chitosan; Cytochromes c; Elastic Modulus; Humans; L-Lactate Dehydrogenase; Muramidase; Osseointegration; Osteogenesis; Periosteum; Phosphorylation; Polysaccharides, Bacterial; Porosity; Spectroscopy, Fourier Transform Infrared; Stem Cells; Stress, Mechanical; Thrombosis; Tissue Engineering; Tissue Scaffolds

Hypoxia preconditioning has been proven to be an effective method to enhance the therapeutic action of mesenchymal stem cells (MSCs). However, the beneficial effects of hypoxic MSCs in ischemia/reperfusion (I/R) lung injury have yet to be investigated. In this study, we hypothesized that the administration of hypoxic MSCs would have a positive therapeutic impact on I/R lung injury at molecular, cellular, and functional levels.. I/R lung injury was induced in isolated and perfused rat lungs. Hypoxic MSCs were administered in perfusate at a low (2.5×105 cells) and high (1×106 cells) dose. Rats ventilated with a low tidal volume of 6 ml/kg served as controls. Hemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined.. I/R induced permeability pulmonary edema with capillary leakage and increased levels of reactive oxygen species (ROS), pro-inflammatory cytokines, adhesion molecules, cytosolic cytochrome C, and activated MAPK, NF-κB, and apoptotic pathways. The administration of a low dose of hypoxic MSCs effectively attenuated I/R pathologic lung injury score by inhibiting inflammatory responses associated with the generation of ROS and anti-apoptosis effect, however this effect was not observed with a high dose of hypoxic MSCs. Mechanistically, a low dose of hypoxic MSCs down-regulated P38 MAPK and NF-κB signaling but upregulated glutathione, prostaglandin E2, IL-10, mitochondrial cytochrome C and Bcl-2. MSCs infused at a low dose migrated into interstitial and alveolar spaces and bronchial trees, while MSCs infused at a high dose aggregated in the microcirculation and induced pulmonary embolism.. Hypoxic MSCs can quickly migrate into extravascular lung tissue and adhere to other inflammatory or structure cells and attenuate I/R lung injury through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. However, the dose of MSCs needs to be optimized to prevent pulmonary embolism and thrombosis.

Topics: Animals; Antioxidants; Apoptosis; Biomarkers; Bronchoalveolar Lavage Fluid; Capillaries; Caspase 3; Cell Hypoxia; Cytochromes c; Cytosol; Glutathione; Hemodynamics; Hydrogen Peroxide; Intercellular Adhesion Molecule-1; Leukocyte Count; Lung Injury; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mitogen-Activated Protein Kinases; NF-kappa B; Organ Size; Peroxidase; Protein Carbonylation; Proto-Oncogene Proteins c-bcl-2; Pulmonary Embolism; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Thiobarbituric Acid Reactive Substances; Thrombosis; Vascular Cell Adhesion Molecule-1

Water-pipe tobacco smoking is becoming prevalent in all over the world including Western countries. There are limited data on the cardiovascular effects of water-pipe smoke (WPS), in particular following chronic exposure. Here, we assessed the chronic cardiovascular effects of nose-only WPS exposure in C57BL/6 mice. The duration of the session was 30 minutes/day, 5 days/week for 6 consecutive months. Control mice were exposed to air. WPS significantly increased systolic blood pressure. The relative heart weight and plasma concentrations of troponin-I and B-type natriuretic peptide were increased in mice exposed to WPS. Arterial blood gas analysis showed that WPS caused a significant decrease in [Formula: see text] and an increase in [Formula: see text] WPS significantly shortened the thrombotic occlusion time in pial arterioles and venules and increased the number of circulating platelet. Cardiac lipid peroxidation, measured as thiobarbituric acid-reactive substances, was significantly increased, while superoxide dismutase activity, total nitric oxide activity, and glutathione concentration were reduced by WPS exposure. Likewise, immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome

Topics: Animals; Arterioles; Blood Gas Analysis; Blood Pressure; Carbon Dioxide; Carboxyhemoglobin; Comet Assay; Cytochromes c; DNA Damage; Fibrosis; Glutathione; Heart; Immunohistochemistry; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Myocardium; Natriuretic Peptide, Brain; Nicotiana; Nitric Oxide; Nitric Oxide Synthase Type II; Oxygen; Partial Pressure; Pia Mater; Platelet Count; Smoke; Smoking; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Thrombosis; Time Factors; Troponin I; Venules

It has been shown, both experimentally and clinically, that water-pipe smoke (WPS) exposure adversely affects the cardiovascular system (CVS) through the generation of oxidative stress and inflammation. Betaine, a naturally occurring compound in common foods, has antioxidant and anti-inflammatory actions. However, its potential to mitigate the adverse effect of WPS on the CVS has never been reported before. This is the subject of this study in mice.. Mice were exposed daily for 30 min to either normal air (control), or to WPS for two consecutive weeks. Betaine was administered daily by gavage at a dose of 10mg/kg, 1h before either air or WPS exposure.. Betaine mitigated the in vivo prothrombotic effect of WPS in pial arterioles and venules. Moreover, it reversed the WPS-induced decrease in circulating platelets. Likewise, betaine alleviated platelet aggregation in vitro, and the shortening of activated partial thromboplastin time and prothrombin time induced by WPS. Betaine reduced the increase of plasminogen activator inhibitor-1 and fibrinogen concentrations in plasma induced by WPS. Betaine also diminished the WPS-induced increase of plasma concentrations of interleukin 6 and tumor necrosis factor α, and attenuated the increase of lipid peroxidation and superoxide dismutase. Immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome C by cardiomyocytes of the WPS-exposed mice. These effects were averted by betaine.. Our findings suggest that betaine treatment significantly mitigated WPS-induced hypercoagulability, and inflammation, as well as systemic and cardiac oxidative stress.

Topics: Administration, Oral; Animals; Antioxidants; Betaine; Blood Platelets; Cytochromes c; Fibrinogen; Gene Expression; Interleukin-6; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Nitric Oxide Synthase Type II; Oxidative Stress; Partial Thromboplastin Time; Plasminogen Activator Inhibitor 1; Platelet Aggregation; Primary Cell Culture; Prothrombin Time; Smoking; Superoxide Dismutase; Thrombocytopenia; Thrombosis; Tumor Necrosis Factor-alpha

In spite of the growing popularity of herbal medicines and natural food supplements, their effects on cardiovascular homeostasis remain largely unknown, especially regarding pro-thrombotic risks.. In the present study, 21 herbal tea extracts were screened for the procoagulant activities on platelets, an important promoter of thrombosis to examine if herbal medicines or natural products may have prothrombotic risks. We discovered that Dipsacus asper (DA), known to have analgesic and anti-inflammatory effects, potently induced procoagulant activities in platelets. We tried to identify the active ingredient and elucidate the underlying mechanism.. Among 10 major ingredients of DA, dipsacus saponin C (DSC) was identified as a key active ingredient in DA-induced procoagulant activities. DSC-induced procoagulant activities were achieved by the exposure of phosphatidylserine (PS) and PS-bearing microparticle generation that were caused by the alteration in the activities of phospholipid translocases: scramblase and flippase. These events were initiated by increased intracellular calcium and ATP depletion. Notably, DSC induced a series of apoptotic events including the disruption of mitochondrial membrane potential, translocation of Bax and Bak, cytochrome c release and caspase-3 activation. The key roles of apoptotic pathway and caspase activation were demonstrated by the reversal of DSC-induced PS exposure and procoagulant activities with the pretreatment of caspase inhibitors. Interestingly, EGTA reversed DSC-induced procoagulant activities and apoptotic events suggesting that an intracellular calcium increase may play a central role. These results were also confirmed in vivo where platelets of the rats exposed to DSC or DA exhibited PS exposure. Most importantly, DSC or DA administration led to increased thrombus formation.. These results demonstrate that herbal medicines or natural products such as DA or DSC might have prothrombotic risks through procoagulant activation of platelets.

Topics: Adenosine Triphosphate; Adolescent; Adult; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blood Coagulation; Blood Platelets; Calcium; Caspase 3; Cell-Derived Microparticles; Chelating Agents; Coagulants; Cytochromes c; Dipsacaceae; Dose-Response Relationship, Drug; Egtazic Acid; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Oleanolic Acid; Partial Thromboplastin Time; Phosphatidylserines; Phospholipid Transfer Proteins; Plant Preparations; Plant Roots; Platelet Activation; Prothrombin Time; Rats; Rats, Sprague-Dawley; Risk Assessment; Risk Factors; Saponins; Thrombosis; Time Factors; Young Adult

Thrombotic risk associated with chemotherapy including doxorubicin (DOX) has been frequently reported; yet, the exact mechanism is not fully understood. Here, we report that DOX can induce procoagulant activity in platelets, an important contributor to thrombus formation. In human platelets, DOX increased phosphatidylserine (PS) exposure and PS-bearing microparticle (MP) generation. Consistently, DOX-treated platelets and generated MPs induced thrombin generation, a representative marker for procoagulant activity. DOX-induced PS exposure appeared to be from intracellular Ca²⁺ increase and ATP depletion, which resulted in the activation of scramblase and inhibition of flippase. Along with this, apoptosis was induced by DOX as determined by the dissipation of mitochondrial membrane potential (Δψ), cytochrome c release, Bax translocation, and caspase-3 activation. A Ca²⁺ chelator ethylene glycol tetraacetic acid, caspase inhibitor Q-VD-OPh, and antioxidants (vitamin C and trolox) can attenuate DOX-induced PS exposure and procoagulant activity significantly, suggesting that Ca²⁺, apoptosis, and reactive oxygen species (ROS) were involved in DOX-enhanced procoagulant activity. Importantly, rat in vivo thrombosis model demonstrated that DOX could manifest prothrombotic effects through the mediation of platelet procoagulant activity, which was accompanied by increased PS exposure and Δψ dissipation in platelets.

Topics: Adolescent; Adult; Animals; Antibiotics, Antineoplastic; bcl-2-Associated X Protein; Blood Platelets; Caspase 3; Cells, Cultured; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Doxorubicin; Humans; Male; Membrane Potential, Mitochondrial; Microscopy, Confocal; Platelet Activation; Platelet Aggregation; Protein Transport; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Thrombosis; Time Factors; Young Adult