RESUMEN
BACKGROUND: Dysfunction of the nitric oxide pathway is implicated in peripheral arterial disease. Nitric oxide synthase (NOS) isoforms and NOS activity were studied in muscle from patients with critical leg ischaemia (CLI). Alterations in NOS during revascularization surgery were also assessed. METHODS: Muscle biopsies were taken from patients with CLI undergoing amputation and also from patients undergoing femorodistal bypass at the start of surgery, after arterial clamping and following reperfusion. The presence of NOS within muscle sections was confirmed using reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. NOS isoform distribution was studied by immunohistochemistry. NOS mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blotting. NOS activity was assessed with the citrulline assay. RESULTS: All three NOS isoforms were found in muscle, associated with muscle fibres and microvessels. NOS I and III protein expression was increased in CLI (P = 0.041). During revascularization, further ischaemia and reperfusion led to a rise in NOS III protein levels (P = 0.008). NOS activity was unchanged. CONCLUSION: Alterations in NOS I and III occurred in muscle from patients with CLI and further changes occurred during bypass surgery.
Asunto(s)
Puente de Arteria Coronaria , Enfermedad de la Arteria Coronaria/enzimología , Isquemia/enzimología , Pierna/irrigación sanguínea , Músculo Esquelético/irrigación sanguínea , Óxido Nítrico Sintasa/metabolismo , Anciano , Anciano de 80 o más Años , Biopsia , Western Blotting , Femenino , Humanos , Inmunohistoquímica , Isoenzimas/metabolismo , Masculino , Persona de Mediana Edad , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Tourniquet-induced ischemia is often used in orthopedic and reconstructive procedures. This is associated with muscle damage and dysfunction, which limits tourniquet application time. Endothelin-1 (ET-1) is a potent vasoconstrictor, which has been implicated in ischemic conditions and ischemia-reperfusion injury. This study aimed to investigate the role of ET-1 in human skeletal muscle subjected to tourniquet-induced acute ischemia and reperfusion. Thirteen patients undergoing total knee replacement were studied. Plasma and muscle ET-1 concentrations were measured at the start of surgery, after an hour of acute ischemia, and 15 minutes following reperfusion. ET-1 receptor binding was also studied by use of autoradiography, and ET-1 mRNA expression investigated by use of real-time polymerase chain reaction (RT-PCR). Tissue ET-1 increased following the period of acute ischemia and persisted during reperfusion. ET-1 was associated with microvessels and macrophages in the muscle. No changes in circulating ET-1 levels, ET-1 mRNA expression, or ET-1 receptor binding were found. It is concluded that the ET-1 pathway is involved in acute ischemia and reperfusion and it may contribute to the muscle injury that occurs during surgical procedures.
Asunto(s)
Endotelina-1/fisiología , Isquemia/fisiopatología , Pierna/irrigación sanguínea , Músculo Esquelético/irrigación sanguínea , Torniquetes , Enfermedad Aguda , Anciano , Anciano de 80 o más Años , Artroplastia de Reemplazo de Rodilla , Autorradiografía , Interpretación Estadística de Datos , Endotelina-1/análisis , Endotelina-1/antagonistas & inhibidores , Endotelina-1/sangre , Endotelina-1/genética , Endotelina-1/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Isquemia/metabolismo , Macrófagos/fisiología , Masculino , Microcirculación , Persona de Mediana Edad , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Receptores de Endotelina/metabolismo , Reperfusión , Factores de TiempoRESUMEN
BACKGROUND: endogenous vasoconstrictor peptides may play a role in the pathophysiology of critical limb ischaemia (CLI). This study investigated endothelin-1 (ET-1) and urotensin-II (U-II) mRNA expression, peptide distribution and ET receptor subtype binding in chronically ischaemic muscle. METHODS: open muscle biopsies were taken from patients undergoing amputations for CLI and from patients undergoing coronary artery bypass surgery (controls). ET-1 and U-II mRNA expression in muscle biopsies was studied using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). ET-1 and U-II immunohistochemistry was performed on muscle sections and ET receptor binding studied using in vitro autoradiography. RESULTS: ET-1 mRNA expression was significantly increased in CLI compared to controls (p<0.05) whilst no significant change in U-II expression occurred. ET-1 immunoreactivity was also increased in CLI with no difference in U-II immunostaining observed. ET(B) receptor binding was significantly increased in CLI (median 4, range 1-8 vs 2, range 1-3, dpm x 10(3)/mm(2), p=0.01, Mann-Whitney test) whilst ET(A) receptor binding was not significantly raised. Binding was associated with microvessels and macrophages. CONCLUSIONS: in CLI, the ET-1 pathway is upregulated but U-II is unaffected. ET-1 may vasoconstrict microvessels and mediate inflammation in chronically ischaemic muscle. ET-1 binding to ET(B) receptors in particular may play an important role in the pathophysiology of CLI underscoring the therapeutic potential of ET(B) receptor antagonists in the management of CLI.
Asunto(s)
Endotelina-1/metabolismo , Isquemia/metabolismo , Pierna/irrigación sanguínea , Urotensinas/metabolismo , Anciano , Anciano de 80 o más Años , Amputación Quirúrgica , Autorradiografía , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Humanos , Técnicas para Inmunoenzimas , Isquemia/cirugía , Masculino , Persona de Mediana Edad , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Regulación hacia ArribaRESUMEN
BACKGROUND: Ischaemia-induced angiogenesis occurs in critical leg ischaemia (CLI) and endothelin (ET) 1 may be involved in this process. The aim of this study was to quantify microvessels and study ET receptor expression and distribution in critically ischaemic leg muscle. METHODS: Leg muscle biopsies were taken from 12 patients with CLI and 12 patients with no leg ischaemia. Microvessels were identified immunohistochemically on muscle sections, and the number of immunopositive cells was quantified. ETA and ETB receptor messenger RNA (mRNA) expression was studied using real-time quantitative reverse transcriptase-polymerase chain reaction, and receptor binding was localized and assessed by in vitro autoradiography. RESULTS: The number of microvessels in CLI muscle biopsies was 2.6 times higher than that in controls (P < 0.01). ETB receptor mRNA expression and binding were significantly increased in CLI tissue (P < 0.05), while ETA receptor levels were not significantly raised. High-resolution autoradiography showed that ET receptor binding was associated with microvessels. CONCLUSION: Angiogenesis occurs in CLI and raised ETB receptors within the muscle were associated with microvessels, suggesting that ET-1 may mediate angiogenesis via these receptors in critically ischaemic muscle.
Asunto(s)
Endotelina-1/fisiología , Isquemia/metabolismo , Pierna/irrigación sanguínea , Músculo Esquelético/irrigación sanguínea , Neovascularización Patológica/etiología , Receptores de Endotelina/metabolismo , Anciano , Anciano de 80 o más Años , Autorradiografía , Biopsia/métodos , Femenino , Humanos , Inmunohistoquímica , Isquemia/patología , Masculino , Microcirculación , Persona de Mediana EdadRESUMEN
Despite early identification and aggressive modification of atherosclerotic risk factors, many patients still require surgical revascularisation for established atherosclerotic vascular disease. However, bypass surgery is hampered by a high incidence of vein graft failure. New strategies are being introduced to improve these results, with early data suggesting that improved patency rates are possible. These vary from the use of adjuvant pharmacological agents and local gene transfer strategies to the modification of vein harvesting techniques in order to reduce vascular damage to all layers of the graft. Advances in vascular biology have resulted in new insights into the role of the endothelium and adventitia in vein graft remodelling. Although recent pharmacological adjuvant therapy and molecular techniques have been described that may be used to reduce the incidence of vein graft occlusion a more desirable approach for improved graft patency rates may be achieved simply by using atraumatic surgical techniques aimed at minimising vascular damage during vessel harvesting and subsequent anastamoses during bypass surgery.