RESUMO
Cellular metabolism influences immune cell function, with mitochondrial fatty acid ß-oxidation and oxidative phosphorylation required for multiple immune cell phenotypes. Carnitine palmitoyltransferase 1a (Cpt1a) is considered the rate-limiting enzyme for mitochondrial metabolism of long-chain fatty acids, and Cpt1a deficiency is associated with infant mortality and infection risk. This study was undertaken to test the hypothesis that impairment in Cpt1a-dependent fatty acid oxidation results in increased susceptibility to infection. Screening the Cpt1a gene for common variants predicted to affect protein function revealed allele rs2229738_T, which was associated with pneumonia risk in a targeted human phenome association study. Pharmacologic inhibition of Cpt1a increases mortality and impairs control of the infection in a murine model of bacterial pneumonia. Susceptibility to pneumonia is associated with blunted neutrophilic responses in mice and humans that result from impaired neutrophil trafficking to the site of infection. Chemotaxis responsible for neutrophil trafficking requires Cpt1a-dependent mitochondrial fatty acid oxidation for amplification of chemoattractant signals. These findings identify Cpt1a as a potential host determinant of infection susceptibility and demonstrate a requirement for mitochondrial fatty acid oxidation in neutrophil biology.
Assuntos
Carnitina O-Palmitoiltransferase , Metabolismo dos Lipídeos , Neutrófilos , Animais , Humanos , Lactente , Camundongos , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Neutrófilos/metabolismoRESUMO
Sepsis is a devastating disease with high morbidity and mortality and no specific treatments. The pathophysiology of sepsis involves a hyperinflammatory response and release of damage-associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), from activated and dying cells. Purinergic receptors activated by ATP have gained attention for their roles in sepsis, which can be pro- or anti-inflammatory depending on the context. Current data regarding the role of ATP-specific purinergic receptor P2X7 (P2X7R) in vascular function and inflammation during sepsis are conflicting, and its role on the endothelium has not been well characterized. In this study, we hypothesized that the P2X7R antagonist AZ 10606120 (AZ106) would prevent endothelial dysfunction during sepsis. As proof of concept, we first demonstrated the ability of AZ106 (10 µM) to prevent endothelial dysfunction in intact rat aorta in response to IL-1ß, an inflammatory mediator upregulated during sepsis. Likewise, blocking P2X7R with AZ106 (10 µg/g) reduced the impairment of endothelial-dependent relaxation in mice subjected to intraperitoneal injection of cecal slurry (CS), a model of polymicrobial sepsis. However, contrary to our hypothesis, AZ106 did not improve microvascular permeability or injury, lung apoptosis, or illness severity in mice subjected to CS. Instead, AZ106 elevated spleen bacterial burden and circulating inflammatory markers. In conclusion, antagonism of P2X7R signaling during sepsis appears to disrupt the balance between its roles in inflammatory, antimicrobial, and vascular function.
Assuntos
Receptores Purinérgicos P2X7 , Sepse , Trifosfato de Adenosina , Animais , Inflamação , Camundongos , Ratos , Sepse/microbiologia , Transdução de SinaisRESUMO
Subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm leads to vasospasm resulting in delayed cerebral ischemia. Therapeutic options are currently limited to hemodynamic optimization and nimodipine, which have marginal clinical efficacy. Nitric oxide (NO) modulates cerebral blood flow through activation of the cGMP-Protein Kinase G (PKG) pathway. Our hypothesis is that SAH results in downregulation of signaling components in the NO-PKG pathway which could explain why treatments for vasospasm targeting this pathway lack efficacy and that treatment with a cell permeant phosphopeptide mimetic of downstream effector prevents delayed vasospasm after SAH. Using a rat endovascular perforation model, reduced levels of NO-PKG pathway molecules were confirmed. Additionally, it was determined that expression and phosphorylation of a PKG substrate: Vasodilator-stimulated phosphoprotein (VASP) was downregulated. A family of cell permeant phosphomimetic of VASP (VP) was wasdesigned and shown to have vasorelaxing property that is synergistic with nimodipine in intact vascular tissuesex vivo. Hence, treatment targeting the downstream effector of the NO signaling pathway, VASP, may bypass receptors and signaling elements leading to vasorelaxation and that treatment with VP can be explored as a therapeutic strategy for SAH induced vasospasm and ameliorate neurological deficits.
Assuntos
Fosfopeptídeos/uso terapêutico , Hemorragia Subaracnóidea/tratamento farmacológico , Vasodilatadores/uso terapêutico , Vasoespasmo Intracraniano/tratamento farmacológico , Animais , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Regulação para Baixo , Desenho de Fármacos , Sinergismo Farmacológico , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Mimetismo Molecular , Nimodipina/farmacologia , Óxido Nítrico/metabolismo , Fosfopeptídeos/farmacocinética , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Hemorragia Subaracnóidea/metabolismo , Suínos , Vasodilatadores/farmacocinéticaRESUMO
Vascular injury leads to membrane disruption, ATP release, and endothelial dysfunction. Increases in the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and decreases in the phosphorylation of Niban, a protein implicated in ER stress and apoptosis, are associated with vascular injury. A cell permeant phosphopeptide mimetic of Niban (NiPp) was generated. The effects of NiPp in restoring endothelial function were determined ex vivo using intact rat aortic tissue (RA) after pharmacological activation of p38 MAPK and also in multiple clinically relevant injury models. Anisomycin (Aniso) increased p38 MAPK phosphorylation and reduced endothelial-dependent relaxation in RA. Treatment with NiPp prevented Ansio-induced reduction in endothelial function and increases in p38 MAPK phosphorylation. NiPp treatment also restored endothelial function after stretch injury (subfailure stretch), treatment with acidic Normal Saline (NS), and P2X7R activation with 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Aged, diseased, human saphenous vein (HSV) remnants obtained from patients undergoing coronary bypass surgical procedures have impaired endothelial function. Treatment of these HSV segments with NiPp improved endothelial-dependent relaxation. Kinome screening experiments indicated that NiPp inhibits p38 MAPK. These data demonstrate that p38 MAPK and Niban signaling have a role in endothelial function, particularly in response to injury. Niban may represent an endogenous regulator of p38 MAPK activation. The NiPp peptide may serve as an experimental tool to further elucidate p38 MAPK regulation and as a potential therapeutic for endothelial dysfunction.
Assuntos
Aorta/efeitos dos fármacos , Biomarcadores Tumorais/química , Biomimética , Endotélio Vascular/efeitos dos fármacos , Proteínas de Neoplasias/química , Fosfopeptídeos/farmacologia , Lesões do Sistema Vascular/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Aorta/lesões , Aorta/metabolismo , Apoptose , Células Cultivadas , Endotélio Vascular/lesões , Endotélio Vascular/metabolismo , Humanos , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Increased endothelial permeability is central to the pathogenesis of sepsis and leads to organ dysfunction and death but the endogenous mechanisms that drive increased endothelial permeability are not completely understood. We previously reported that cell-free hemoglobin (CFH), elevated in 80% of patients with sepsis, increases lung microvascular permeability in an ex vivo human lung model and cultured endothelial cells. In this study, we augmented a murine model of polymicrobial sepsis with elevated circulating CFH to test the hypothesis that CFH increases microvascular endothelial permeability by inducing endothelial apoptosis. Mice were treated with an intraperitoneal injection of cecal slurry with or without a single intravenous injection of CFH. Severity of illness, mortality, systemic and lung inflammation, endothelial injury and dysfunction and lung apoptosis were measured at selected time points. We found that CFH added to CS increased sepsis mortality, plasma inflammatory cytokines as well as lung apoptosis, edema and inflammation without affecting large vessel reactivity or vascular injury marker concentrations. These results suggest that CFH is an endogenous mediator of increased endothelial permeability and apoptosis in sepsis and may be a promising therapeutic target.
Assuntos
Apoptose , Permeabilidade Capilar , Hemoglobinas/metabolismo , Pulmão/irrigação sanguínea , Pulmão/patologia , Sepse/metabolismo , Sepse/patologia , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Sepse/microbiologiaRESUMO
Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1ß (IL-1ß) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1ß led to impaired endothelial relaxation. IL-1ß treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses.
Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Inflamação/metabolismo , Solução Salina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Receptores Purinérgicos P2X7/metabolismo , Veia Safena/efeitos dos fármacos , Veia Safena/metabolismo , Suínos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
IMPACT STATEMENT: Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to traditional vasodilators, and inhibition of vasospasm after SAH remains a large unmet clinical need. SAH causes changes in the phosphorylation state of the small heat shock proteins (HSPs), HSP20 and HSP27, in the vasospastic vessels. In this study, the levels of HSP27 and HSP20 were manipulated using nanotechnology to mimic the intracellular phenotype of SAH-induced vasospasm, and the effect of this manipulation was tested on vasomotor responses in intact tissues. This work provides insight into potential therapeutic targets for the development of more effective treatments for SAH induced vasospasm.
Assuntos
Vasos Sanguíneos/fisiologia , Nanotecnologia/métodos , Transdução de Sinais , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Inativação Gênica , Proteínas de Choque Térmico/metabolismo , Humanos , Micelas , Contração Muscular , Músculo Liso/fisiologia , Nanopartículas/química , Peptídeos/química , Peptídeos/metabolismo , Polimerização , RNA Interferente Pequeno/metabolismo , Ratos , Eletricidade EstáticaRESUMO
A viable vascular endothelial layer prevents vasomotor dysfunction, thrombosis, inflammation, and intimal hyperplasia. Injury to the endothelium occurs during harvest and "back table" preparation of human saphenous vein prior to implantation as an arterial bypass conduit. A subfailure overstretch model of rat aorta was used to show that subfailure stretch injury of vascular tissue leads to impaired endothelial-dependent relaxation. Stretch-induced impaired relaxation was mitigated by treatment with purinergic P2X7 receptor (P2X7R) inhibitors, brilliant blue FCF (FCF) and A740003, or apyrase, an enzyme that catalyzes the hydrolysis of ATP. Alternatively, treatment of rat aorta with exogenous ATP or 2'(3')-O-(4-Benzoyl benzoyl)-ATP (BzATP) also impaired endothelial-dependent relaxation. Treatment of human saphenous vein endothelial cells (HSVEC) with exogenous ATP led to reduced nitric oxide production which was associated with increased phosphorylation of the stress activated protein kinase, p38 MAPK. ATP- stimulated p38 MAPK phosphorylation of HSVEC was inhibited by FCF and SB203580. Moreover, ATP inhibition of nitric oxide production in HSVEC was prevented by FCF, SB203580, L-arginine supplementation and arginase inhibition. Finally, L-arginine supplementation and arginase inhibition restored endothelial dependent relaxation after stretch injury of rat aorta. These results suggest that vascular stretch injury leads to ATP release, activation of P2X7R and p38 MAPK resulting in endothelial dysfunction due to arginase activation. Endothelial function can be restored in both ATP treated HSVEC and intact stretch injured rat aorta by P2X7 receptor inhibition with FCF or L-arginine supplementation, implicating straightforward therapeutic options for treatment of surgical vascular injury.
Assuntos
Endotélio Vascular/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Procedimentos Cirúrgicos Vasculares/métodos , Animais , Endotélio Vascular/fisiopatologia , Feminino , Óxido Nítrico/biossíntese , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Human saphenous veins used for arterial bypass undergo stretch injury at the time of harvest and preimplant preparation. Vascular injury promotes intimal hyperplasia, the leading cause of graft failure, but the molecular events leading to this response are largely unknown. This study investigated adenosine triphosphate (ATP) as a potential molecular mediator in the vascular response to stretch injury, and the downstream effects of the purinergic receptor, P2X7R, and p38 MAPK activation. MATERIALS AND METHODS: A subfailure stretch rat aorta model was used to determine the effect of stretch injury on release of ATP and vasomotor responses. Stretch-injured tissues were treated with apyrase, the P2X7R antagonist, A438079, or the p38 MAPK inhibitor, SB203580, and subsequent contractile forces were measured using a muscle bath. An exogenous ATP (eATP) injury model was developed and the experiment repeated. Change in p38 MAPK phosphorylation after stretch and eATP tissue injury was determined using Western blotting. Noninjured tissue was incubated in the p38 MAPK activator, anisomycin, and subsequent contractile function and p38 MAPK phosphorylation were analyzed. RESULTS: Stretch injury was associated with release of ATP. Contractile function was decreased in tissue subjected to subfailure stretch, eATP, and anisomycin. Contractile function was restored by apyrase, P2X7R antagonism, and p38-MAPK inhibition. Stretch, eATP, and anisomycin-injured tissue demonstrated increased phosphorylation of p38 MAPK. CONCLUSIONS: Taken together, these data suggest that the vascular response to stretch injury is associated with release of ATP and activation of the P2X7R/P38 MAPK pathway, resulting in contractile dysfunction. Modulation of this pathway in vein grafts after harvest and before implantation may reduce the vascular response to injury.
Assuntos
Trifosfato de Adenosina/metabolismo , Aorta Abdominal/lesões , Receptores Purinérgicos P2X7/metabolismo , Lesões do Sistema Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/fisiopatologia , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Western Blotting , Feminino , Contração Muscular/fisiologia , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Lesões do Sistema Vascular/fisiopatologiaRESUMO
While the pathophysiology and clinical significance of arterial calcifications have been studied extensively, minimal focus has been placed on venous calcification deposition. In this study, we evaluated the association between calcium deposition in human saphenous vein (HSV), endothelial function, and patient demographic risk factors. Fifty-four HSV segments were collected at the time of coronary artery bypass graft (CABG) surgery. The presence or absence of calcium deposits was visualized using the Von Kossa staining method. Endothelial function was determined by measuring muscle tissue contraction with phenylephrine and relaxation with carbachol in a muscle bath. Additional segments of vein underwent histologic evaluation for preexisting intimal thickness and extracellular matrix (ECM) deposition. Patient demographics data were obtained through our institution's electronic medical record, with patient consent. Calcium was present in 16 of 54 samples (29.6%). Veins with calcium deposits had significantly greater intimal-to-medial thickness ratios (p = 0.0058) and increased extracellular collagen deposition (p = 0.0077). Endothelial relaxation was significantly compromised in calcified veins vs. those without calcium (p = 0.0011). Significant patient risk factors included age (p = 0.001) and preoperative serum creatinine (p = 0.017). Calcified veins can be characterized as having endothelial dysfunction with increased basal intimal thickness and increased ECM deposition. Patient risk factors for calcium deposits in veins were similar to those for arteries, namely, advanced age and kidney disease. Further studies are needed to determine the effect of preexisting vein calcification on short- and long-term graft patency.
RESUMO
BACKGROUND: Intimal hyperplasia remains the primary cause of vein graft failure for the 1 million yearly bypass procedures performed using human saphenous vein (HSV) grafts. This response to injury is caused in part by the harvest and preparation of the conduit. The use of Brilliant Blue FCF (FCF) restores injury-induced loss of function in vascular tissues possibly via inhibition of purinergic receptor signaling. This study investigated whether pretreatment of the vein graft with FCF prevents intimal hyperplasia. METHODS: Cultured rat aortic smooth muscle cells (A7r5) were used to determine the effect of FCF on platelet-derived growth factor-mediated migration and proliferation, cellular processes that contribute to intimal hyperplasia. The effectiveness of FCF treatment during the time of explantation on preventing intimal hyperplasia was evaluated in a rabbit jugular-carotid interposition model and in an organ culture model using HSV. RESULTS: FCF inhibited platelet-derived growth factor-induced migration and proliferation of A7r5 cells. Treatment with FCF at the time of vein graft explantation inhibited the subsequent development of intimal thickening in the rabbit model. Pretreatment with FCF also prevented intimal thickening of HSV in organ culture. CONCLUSIONS: Incorporation of FCF as a component of vein graft preparation at the time of explantation represents a potential therapeutic approach to mitigate intimal hyperplasia, reduce vein graft failure, and improve outcome of the autologous transplantation of HSV.
Assuntos
Benzenossulfonatos/farmacologia , Movimento Celular/efeitos dos fármacos , Corantes/farmacologia , Veias Jugulares/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima , Veia Safena/efeitos dos fármacos , Coleta de Tecidos e Órgãos/efeitos adversos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Hiperplasia , Veias Jugulares/metabolismo , Veias Jugulares/patologia , Veias Jugulares/transplante , Modelos Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Técnicas de Cultura de Órgãos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Coelhos , Ratos , Receptores Purinérgicos P2X7/efeitos dos fármacos , Receptores Purinérgicos P2X7/metabolismo , Veia Safena/metabolismo , Veia Safena/patologia , Veia Safena/transplante , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
OBJECTIVE: Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. APPROACH AND RESULTS: HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 µM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. CONCLUSIONS: These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm.
Assuntos
Actinas/metabolismo , Cadeias Leves de Miosina/metabolismo , Papaverina/farmacologia , Veia Safena/efeitos dos fármacos , Veia Safena/metabolismo , Cálcio/metabolismo , Humanos , Immunoblotting , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fosforilação/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Traditional methods of intraoperative human saphenous vein preparation for use as bypass grafts can be deleterious to the conduit. The purpose of this study was to characterize acute graft preparation injury, and to mitigate this harm via an improved preparation technique. Porcine saphenous veins were surgically harvested (unprepared controls, UnP) and prepared using traditional (TraP) and improved preparations (ImP). The TraP used unregulated radial distension, marking with a surgical skin marker and preservation in heparinized normal saline. ImP used pressure-regulated distension, brilliant blue FCF-based pen marking and preservation in heparinized Plasma-Lyte A. Rings from each preparation were suspended in a muscle bath for characterization of physiologic responses to vasoactive agents and viscoelasticity. Cellular viability was assessed using the methyl thiazolyl tetrazolium (MTT) assay and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptosis. Contractile responses to potassium chloride (110 mM) and phenylephrine (10 µM), and endothelial-dependent and independent vasodilatory responses to carbachol (0.5 µM) and sodium nitroprusside (1 µM), respectively, were decreased in TraP tissues compared to both UnP and ImP tissues (p ⩽ 0.05). TraP tissues demonstrated diminished viscoelasticity relative to UnP and ImP tissues (p ⩽ 0.05), and reduced cellular viability relative to UnP control (p ⩽ 0.01) by the MTT assay. On the TUNEL assay, TraP tissues demonstrated a greater degree of apoptosis relative to UnP and ImP tissues (p ⩽ 0.01). In conclusion, an improved preparation technique prevents vascular graft smooth muscle and endothelial injury observed in tissues prepared using a traditional approach.
Assuntos
Preservação de Órgãos/métodos , Veia Safena/transplante , Coleta de Tecidos e Órgãos/métodos , Vasoconstrição , Vasodilatação , Animais , Anticoagulantes/farmacologia , Apoptose , Sobrevivência Celular , Elasticidade , Eletrólitos/farmacologia , Heparina/farmacologia , Modelos Animais , Preservação de Órgãos/efeitos adversos , Soluções para Preservação de Órgãos/farmacologia , Veia Safena/efeitos dos fármacos , Veia Safena/patologia , Veia Safena/fisiopatologia , Cloreto de Sódio/farmacologia , Sus scrofa , Fatores de Tempo , Coleta de Tecidos e Órgãos/efeitos adversos , Rigidez Vascular , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , ViscosidadeRESUMO
Severe bronchospasm refractory to ß-agonists is a challenging aspect of asthma therapy, and novel therapeutics are needed. ß-agonist-induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of the small heat shock-related protein (HSP) 20. We hypothesized that a transducible phosphopeptide mimetic of HSP20 (P20 peptide) causes relaxation of human ASM (HASM) by interacting with target(s) downstream of the ß2-adrenergic receptor (ß2AR) pathway. The effect of the P20 peptide on ASM contractility was determined in human and porcine ASM using a muscle bath. The effect of the P20 peptide on filamentous actin dynamics and migration was examined in intact porcine ASM and cultured primary HASM cells. The efficacy of the P20 peptide in vivo on airway hyperresponsiveness (AHR) was determined in an ovalbumin (OVA) sensitization and challenge murine model of allergic airway inflammation. P20 peptide caused dose-dependent relaxation of carbachol-precontracted ASM and blocked carbachol-induced contraction. The ß2AR inhibitor, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551), abrogated isoproterenol but not P20 peptide-mediated relaxation. The P20 peptide decreased filamentous actin levels in intact ASM, disrupted stress fibers, and inhibited platelet-derived growth factor-induced migration of HASM cells. The P20 peptide treatment reduced methacholine-induced AHR in OVA mice without affecting the inflammatory response. These results suggest that the P20 peptide decreased airway constriction and disrupted stress fibers through regulation of the actin cytoskeleton downstream of ß2AR. Thus, the P20 peptide may be a potential therapeutic for asthma refractory to ß-agonists.
Assuntos
Proteínas de Choque Térmico HSP20/metabolismo , Pulmão/fisiopatologia , Contração Muscular/efeitos dos fármacos , Peptídeos/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Actinas/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Alérgenos/efeitos adversos , Animais , Hiper-Reatividade Brônquica/patologia , Carbacol , Movimento Celular/efeitos dos fármacos , Constrição , Humanos , Inflamação/patologia , Pulmão/efeitos dos fármacos , Camundongos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ovalbumina/efeitos adversos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Sus scrofaRESUMO
BACKGROUND: Injury to saphenous vein grafts during surgical preparation may contribute to the subsequent development of intimal hyperplasia, the primary cause of graft failure. Surgical skin markers currently used for vascular marking contain gentian violet and isopropanol, which damage tissue and impair physiologic functions. Brilliant blue FCF (FCF) is a nontoxic dye alternative that may also ameliorate preparation-induced injury. METHODS: Porcine saphenous vein (PSV) was used to evaluate the effect of FCF on physiologic responses in a muscle bath. Cytotoxicity of FCF was measured using human umbilical venous smooth muscle cells. Effect of FCF on the development of intimal hyperplasia was evaluated in organ culture using PSV. Intracellular calcium fluxes and contractile responses were measured in response to agonists and inhibitors in rat aorta and human saphenous vein. RESULTS: Marking with FCF did not impair smooth muscle contractile responses and restored stretch injury-induced loss in smooth muscle contractility of PSV. Gentian violet has cytotoxic effects on human umbilical venous smooth muscle cells, whereas FCF is nontoxic. FCF inhibited intimal thickening in PSV in organ culture. Contraction induced by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate and intracellular calcium flux were inhibited by FCF, oxidized adenosine triphosphate, KN-62, and brilliant blue G, suggesting that FCF may inhibit the purinergic receptor P2X7. CONCLUSIONS: Our studies indicated that FCF is a nontoxic marking dye for vein grafts that ameliorates vein graft injury and prevents intimal thickening, possibly due to P2X7 receptor inhibition. FCF represents a nontoxic alternative for vein graft marking and a potentially therapeutic approach to enhance outcome in autologous transplantation of human saphenous vein into the coronary and peripheral arterial circulation.
Assuntos
Benzenossulfonatos/farmacologia , Corantes/farmacologia , Veia Safena/efeitos dos fármacos , Enxerto Vascular/métodos , Lesões do Sistema Vascular/prevenção & controle , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Citoproteção , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Hiperplasia , Técnicas In Vitro , Masculino , Neointima , Técnicas de Cultura de Órgãos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/efeitos dos fármacos , Receptores Purinérgicos P2X7/metabolismo , Veia Safena/lesões , Veia Safena/fisiopatologia , Veia Safena/transplante , Suínos , Enxerto Vascular/efeitos adversos , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Lesões do Sistema Vascular/fisiopatologia , Vasoconstrição/efeitos dos fármacosRESUMO
INTRODUCTION: Recent clinical data suggest intraoperative preservation of human saphenous vein (HSV) in normal saline is associated with vein graft failure. We evaluated the influence of several preservation media on acute physiologic function and cellular viability of HSV conduit. METHODS: Unprepared (UP) HSV obtained from coronary artery bypass graft patients was characterized on a muscle bath after 2-hour storage in 6 solutions: Plasma-Lyte A, 0.9% NaCl (normal saline), University of Wisconsin solution, Celsior solution, autologous whole blood, or glutathione-ascorbic acid L-arginine (GALA) solution. Vascular smooth muscle contractility was assessed after exposure to depolarizing KCl and phenylephrine. The relaxation of phenylephrine-precontracted HSV to sodium nitroprusside and carbachol (endothelial-independent and -dependent relaxation, respectively) was also assessed. Cellular viability was determined via the methyl thiazolyl tetrazolium (MTT) assay. Rat aortae were used to assess the effect of pH during graft preservation on endothelial-dependent relaxation. RESULTS: Preservation of HSV in normal saline and autologous whole blood impaired contractile responses to KCl relative to UP tissues, whereas preservation in University of Wisconsin solution and Celsior solution enhanced contractile responses (P < .05). Relative to UP tissues, responses to phenylephrine were decreased with preservation in normal saline, whereas preservation in University of Wisconsin solution, Celsior solution, and GALA all potentiated these responses (P < .05). Only preservation in normal saline impaired endothelial-independent relaxation (P = .005). Preservation in Plasma-Lyte A (P = .02), normal saline (P = .002), and University of Wisconsin solution (P = .02) impaired endothelial-dependent relaxation. Normal saline preservation decreased MTT viability index relative to UP tissues (0.02 ± 0.002 mg(-1)0.5 mL(-1) vs 0.033 ± 0.005 mg(-1)0.5 mL(-1); P = .03). Endothelial function was impaired by acidic pH in rat aorta. CONCLUSION: Preservation of HSV in normal saline causes graft injury leading to impaired physiologic function and decreased viability of the HSV. This harm is mitigated by the use of buffered salt solutions as preservation media.
Assuntos
Soluções para Preservação de Órgãos , Preservação de Órgãos/métodos , Veia Safena/transplante , Enxerto Vascular , Animais , Sobrevivência Celular , Sobrevivência de Enxerto , Humanos , Músculo Liso Vascular/fisiologia , Avaliação de Processos e Resultados em Cuidados de Saúde , Ratos , VasodilataçãoRESUMO
IMPORTANCE: Surgical skin markers are used off-label to mark human saphenous veins (HSVs) to maintain orientation before implantation as aortocoronary or peripheral arterial bypass grafts. These surgical skin markers impair functional responses of the HSV tissue. OBJECTIVES: To investigate the effect of brilliant blue dye 1 (brilliant blue FCF [for food coloring]; hereinafter, FCF) as a nontoxic alternative marking dye and to determine whether FCF has pharmacological properties. DESIGN, SETTING, AND PARTICIPANTS: Segments of HSVs were collected in university hospitals from patients undergoing coronary artery bypass grafting procedures immediately after harvest (unmanipulated) or after typical intraoperative surgical graft preparation (after manipulation). Rat inferior venae cavae were used to determine the pharmacological properties and cellular targets of FCF. Endothelial and smooth muscle functional responses were determined in a muscle bath, and intimal thickening in HSVs was determined after 14 days in organ culture. MAIN OUTCOMES AND MEASURES: Contractile responses were measured in force and converted to stress. Smooth muscle function was expressed as maximal responses to potassium chloride depolarization contractions. Endothelial function was defined as the percentage of relaxation of maximal agonist-induced contraction. Neointimal thickness was measured by histomorphometric analysis. RESULTS: Human saphenous veins stored in the presence of FCF had no loss of endothelial or smooth muscle function. Unmanipulated HSVs preserved in the presence of FCF demonstrated a significant increase in endothelial-dependent relaxation (mean [SEM], 25.2% [6.4%] vs 30.2% [6.7%]; P = .02). Application of FCF to functionally nonviable tissue significantly enhanced the smooth muscle responses (mean [SEM], 0.018 [0.004] × 105 N/m² vs 0.057 [0.016] × 105 N/m²; P = .05). Treatment with FCF reduced intimal thickness in organ culture (mean [SEM], -17.5% [2.1%] for unmanipulated HSVs vs -27.9% [3.7%] for HSVs after manipulation; P < .001). In rat inferior venae cavae, FCF inhibited the contraction induced by the P2X7 receptor agonist 2'(3')-O-(4-benzoyl)benzoyl-adenosine-5'-triphosphate (mean [SEM], 14.8% [2.2%] vs 6.5% [1.8%]; P = .02) to an extent similar to the P2X7 receptor antagonist oxidized adenosine triphosphate (mean [SEM], 5.0% [0.9%]; P < .02 vs control) or the pannexin hemichannel inhibitor probenecid (mean [SEM], 7.3% [1.6%] and 4.7% [0.9%] for 0.5mM and 2mM, respectively; P < .05). CONCLUSIONS AND RELEVANCE: Treatment with FCF did not impair endothelial or smooth muscle function in HSVs. Brilliant blue FCF enhanced endothelial-dependent relaxation, restored smooth muscle function, and prevented intimal hyperplasia in HSVs in organ culture. These pharmacological properties of FCF may be due to P2X7 receptor or pannexin channel inhibition. Brilliant blue FCF is an alternative, nontoxic marking dye that may improve HSV conduit function and decrease intimal hyperplasia.
Assuntos
Benzenossulfonatos/toxicidade , Corantes/toxicidade , Disfunção Primária do Enxerto/induzido quimicamente , Disfunção Primária do Enxerto/fisiopatologia , Veia Safena/efeitos dos fármacos , Análise de Variância , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Veia Safena/patologia , Veia Safena/fisiopatologia , Veia Safena/transplante , Veia Cava Inferior/efeitos dos fármacos , Veia Cava Inferior/patologia , Veia Cava Inferior/fisiopatologiaRESUMO
IMPORTANCE: Long-term patency of human saphenous veins (HSVs) used as autologous conduits for coronary artery bypass grafting (CABG) procedures remains limited because of vein graft failure (VGF). Vein graft failure has been reported to be as high as 45% at 12 to 18 months after surgery and leads to additional surgery, myocardial infarction, recurrent angina, and death. Preparation of HSVs before implantation leads to conduit injury, which may promote VGF. OBJECTIVES: To investigate whether pressure distension during vein graft preparation leads to endothelial injury and intimal thickening and whether limiting intraluminal pressure during pressure distension by using a pressure release valve (PRV) preserves endothelial function and prevents neointima thickening. DESIGN, SETTING, AND PARTICIPANTS: Segments of HSVs were collected in a university hospital from 13 patients undergoing CABG procedures immediately after harvest (unmanipulated [UM]), after pressure distension (after distension [AD]), and after typical intraoperative surgical graft preparation (after manipulation [AM]). Porcine saphenous veins (PSVs) from 7 healthy research animals were subjected to manual pressure distension with or without an in-line PRV that prevents pressures of 140 mm Hg or greater. Endothelial function of the HSVs and PSVs was determined in a muscle bath, endothelial integrity was assessed, and intimal thickening in PSVs was evaluated after 14 days in organ culture. MAIN OUTCOMES AND MEASURES: Endothelial function was measured in force, converted to stress, and defined as the percentage relaxation of maximal phenylephrine-induced contraction. Endothelial integrity was assessed by immunohistologic examination. Neointimal thickness was measured by histomorphometric analysis. RESULTS: Pressure distension of HSVs led to decreased mean (SEM) endothelial-dependent relaxation (5.3% [2.3%] for AD patients vs 13.7% [2.5%] for UM patients; P < .05) and denudation. In the AM group, the function of the conduits was further decreased (-3.2% [3.2%]; P < .05). Distension of the PSVs led to reduced endothelial-dependent relaxation (7.6% [4.4%] vs 61.9% [10.2%] in the control group; P < .05), denudation, and enhanced intimal thickening (15.0 [1.4] µm vs 2.2 [0.8] µm in the control group; P < .05). Distension with the PRV preserved endothelial-dependent relaxation (50.3% [9.6%]; P = .32 vs control), prevented denudation, and reduced intimal thickening (3.4 [0.8] µm; P = .56 vs controls) in PSVs. CONCLUSIONS AND RELEVANCE: Use of a PRV during graft preparation limits intraluminal pressure generated by manual distension, preserves endothelial integrity, and reduces intimal hyperplasia. Integration of this simple device may contribute to improved long-term vein graft patency.
Assuntos
Ponte de Artéria Coronária/métodos , Endotélio Vascular/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Pressão/efeitos adversos , Veia Safena/patologia , Veia Safena/transplante , Animais , Fenômenos Biomecânicos , Endotélio Vascular/lesões , Humanos , Técnicas Imunoenzimáticas , Técnicas de Cultura de Órgãos , Estudos Retrospectivos , Veia Safena/lesões , Suínos , Fatores de Tempo , Vasoconstrição/fisiologiaRESUMO
INTRODUCTION: Human saphenous vein (HSV) is the most widely used bypass conduit for peripheral and coronary vascular reconstructions. However, outcomes are limited by a high rate of intimal hyperplasia (IH). HSV undergoes a series of ex vivo surgical manipulations prior to implantation, including hydrostatic distension, marking, and warm ischemia in solution. We investigated the impact of surgical preparation on HSV cellular function and development of IH in organ culture. We hypothesized that oxidative stress is a mediator of HSV dysfunction. METHODS: HSV was collected from patients undergoing vascular bypass before and after surgical preparation. Smooth muscle and endothelial function were measured using a muscle bath. Endothelial preservation was assessed with immunohistochemical staining. An organ culture model was used to investigate the influence of surgical preparation injury on the development of IH. Superoxide levels were measured using a high-performance liquid chromatography-based assay. The influence of oxidative stress on HSV physiologic responses was investigated by exposing HSV to hydrogen peroxide (H2O2). RESULTS: Surgical vein graft preparation resulted in smooth muscle and endothelial dysfunction, endothelial denudation, diminished endothelial nitric oxide synthase staining, development of increased IH, and increased levels of reactive oxygen species. Experimental induction of oxidative stress in unmanipulated HSV by treatment with H2O2 promoted endothelial dysfunction. Duration of storage time in solution did not contribute to smooth muscle or endothelial dysfunction. CONCLUSIONS: Surgical vein graft preparation causes dysfunction of the smooth muscle and endothelium, endothelial denudation, reduced endothelial nitric oxide synthase expression, and promotes IH in organ culture. Moreover, increased levels of reactive oxygen species are produced and may promote further vein graft dysfunction. These results argue for less injurious means of preparing HSV prior to autologous transplantation into the arterial circulation.