RESUMEN
Chronic arterial occlusion leads to growth of collaterals - a process termed arteriogenesis, in which macrophages play a prominent role in remodelling and growth. However, a detailed analysis which of distinct macrophage subpopulations involved in arteriogenesis has never been performed. In the present study the temporal and spatial distribution of macrophage subtypes during arteriogenesis in a rat model with chronically elevated fluid shear stress (FSS) is investigated. Local macrophage subpopulations were histologically immuno-phenotyped using CD68 (a ubiquitous macrophage marker) and CD163, a specific M2 macrophage marker. Without occlusion few M2-macrophages reside in the perivascular space. Early after occlusion (12h) the number of M2 macrophages increases strongly and M1 macrophages begin emerging into the collateral. After 3 days they appear in the perivascular space. Both macrophage subtypes increase until 28d after treatment, whereas M2 macrophages dominate at the site of collateral growth. The local distribution of the subpopulations changes during the arteriogenic process. Whereas M1 macrophages are detected directly adjacent to the media, M2 macrophages are present in the most outer perivascular region of the growing collateral vessel. Systemic alterations of blood leucocytes in mice after femoral artery ligature (FAL) were investigated by FACS analysis of serial blood samples. During collateral remodelling histological changes were not reflected in circulating monocytes in the peripheral blood. The activation state of macrophages in mice with FAL was modulated by injections of either dexamethasone or the interleukins IL10 or IL3/IL14. The arteriogenic response was assessed by hind limb perfusion with laser Doppler measurements after 3, 7 and 14d. Suppressing inflammatory monocyte subtypes (M1) with dexamethasone led to impaired perfusion recovery after FAL in mice, whereas IL10 or IL4/IL13 application significantly increased perfusion recovery. This investigation demonstrates that a forced shift towards M2 macrophages improves the arteriogenic response. The distinct early increase and spatial distribution of M2 macrophages support the idea that this subtype plays a predominant role during collateral remodelling.
Asunto(s)
Circulación Colateral/fisiología , Arteria Femoral/fisiología , Macrófagos/fisiología , Animales , Arteria Femoral/metabolismo , Interleucinas/metabolismo , Leucocitos/metabolismo , Leucocitos/fisiología , Ligadura/métodos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Ratas , Ratas Sprague-Dawley , Resistencia al Corte/fisiología , Análisis Espacio-TemporalRESUMEN
To evaluate the role of neuronal nitric oxides synthase (nNOS) in collateral artery growth (arteriogenesis), we analyzed the expression pattern of nNOS at distinct time points on RNA and protein levels in a rabbit and a murine model of peripheral arteriogenesis. In the rabbit model, Northern blot analyses revealed a significant upregulation of nNOS at 6 h (1.6-fold), 12 h (2.2-fold) and 24 h (2.0-fold) after induction of arteriogenesis via femoral artery ligation, when compared to the sham operated side. In mice, an upregulation of nNOS was also detected using Northern blot (at 6 h, 12 h) and qRT-PCR (12 h: 2.4-fold). On the protein level, nNOS was found to be upregulated 24 h after femoral artery ligation. Immunohistochemical staining showed that nNOS was localized in endothelial and smooth muscle cells of collateral arteries, as well as in skeletal muscle and nerves. In summary, our data provide evidence that nNOS is not constitutively expressed, but is induced during arteriogenesis, playing a role in supplying reactive oxygen species such as H2O2 and low levels of NO.
Asunto(s)
Arterias/crecimiento & desarrollo , Circulación Colateral , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Conejos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVES: This study aimed to determine the importance of the shear-stress-sensitive calcium channels Trpc1, Trpm7, Trpp2, Trpv2 (transient receptor potential cation channel, subfamily V, member 2) and Trpv4 for cerebral arteriogenesis. The expression profiles were analysed, comparing the stimulation of collateral growth by target-specific drugs to that achieved by maximum increased fluid shear stress (FSS). DESIGN: A prospective, controlled study wherein rats were subjected to bilateral carotid artery ligature (BCL), or BCL + arteriovenous fistula, or BCL + drug application. METHODS: Messenger RNA (mRNA) abundance and protein expression were determined in FSS-stimulated cerebral collaterals by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Drugs were applied via osmotic mini pumps and arteriogenesis was evaluated by post-mortem angiograms and Ki67 immunostaining. RESULTS: Trpv4 was the only mechanosensitive Trp channel showing significantly increased mRNA abundance and protein expression after FSS stimulation. Activation of Trpv4 by 4α-phorbol-12,13-didecanoate caused significantly enhanced collateral growth (length: 4.43 ± 0.20 mm and diameter: 282.6 ± 8.1 µm) compared with control (length: 3.80 ± 0.06 mm and diameter: 237.3 ± 5.3 µm). Drug application stimulated arteriogenesis to almost the same extent as did maximum FSS stimulation (length: 4.61 ± 0.07 mm and diameter: 327.4 ± 12.6 µm). CONCLUSIONS: Trpv4 showed significantly increased expression in FSS-stimulated cerebral collaterals. Pharmacological Trpv4 activation enhanced cerebral arteriogenesis, pinpointing Trpv4 as a possible candidate for the development of new therapeutic concepts.
Asunto(s)
Circulación Cerebrovascular/fisiología , Circulación Colateral/fisiología , Regulación de la Expresión Génica , Arteriosclerosis Intracraneal/etiología , Forboles/efectos adversos , ARN Mensajero/genética , Canales Catiónicos TRPV/genética , Animales , Circulación Cerebrovascular/efectos de los fármacos , Circulación Colateral/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inmunohistoquímica , Arteriosclerosis Intracraneal/genética , Arteriosclerosis Intracraneal/metabolismo , Masculino , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/metabolismo , Presión Osmótica , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/biosíntesis , Canales Catiónicos TRPV/efectos de los fármacosRESUMEN
We investigated the effect of pharmacological activation of the Ca(2+)-channel transient receptor potential cation channel, subfamily V, member 4 (TRPV4) on collateral growth in a pig hind limb-ischemia model thereby identifying subcellular mechanisms. Domestic pigs received femoral artery ligature and were randomly assigned to one of the following groups (each n=6): (1) 4alpha-phorbol 12,13-didecanoate (4alphaPDD) treatment; (2) treatment with an arterio-venous shunt (AV-shunt) distal to the occlusion; or (3) implantation of NaCl-filled minipump. Six sham-operated pigs acted as controls. Aortic and peripheral mean arterial pressure (MAP) measurements were performed to assess the collateral flow index (CFI). Tissue was isolated from M. quadriceps for immunohistochemistry and from isolated collateral arteries for quantitative real time PCR (qRT-PCR). Shortly after ligature the CFI dropped from 0.96+/-0.02 to 0.21+/-0.02 in all ligature-treated groups. In ligature-only-treated pigs CFI increased to 0.56+/-0.03 after 7days. Treatment with 4alphaPDD led to an enhancement of CFI compared with ligature alone (0.73+/-0.03). CD31-staining showed improved arteriolar density. Increased Ki67 staining in collaterals indicated proliferation. qRT-PCR and Western blot analysis showed upregulation or modulation of Ca(2+)-dependent transcription factors nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), Kv channel interacting protein 3, calsenilin (KCNIP3/CSEN/DREAM), and myocyte enhancer factor 2C (MEF2C) in 4alphaPDD- and AV-shunt-treated pigs compared with controls. Improved CFI after 4alphaPDD treatment identifies TRPV4 as an initial fluid shear-stress sensor and collateral remodelling and growth trigger. Subcellularly, modulation of Ca(2+)-dependent transcription factors indicates a pivotal role for Ca(2+)-signalling during arteriogenesis.
Asunto(s)
Miembro Posterior/irrigación sanguínea , Isquemia/fisiopatología , Animales , Aorta/metabolismo , Aorta/fisiopatología , Arterias/metabolismo , Arterias/fisiopatología , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatología , Señalización del Calcio , Arteria Femoral/metabolismo , Arteria Femoral/fisiopatología , Arteria Femoral/cirugía , Miembro Posterior/metabolismo , Miembro Posterior/fisiopatología , Isquemia/metabolismo , Extremidad Inferior/irrigación sanguínea , Extremidad Inferior/fisiopatología , Masculino , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/farmacología , Forboles , Distribución Aleatoria , Estrés Mecánico , Sus scrofa/metabolismoRESUMEN
OBJECTIVE: This study aimed to compare arteriogenesis after femoral artery occlusion as influenced by exercise or arteriovenous shunt and follow changes in collateral transient receptor potential cation channel, subfamily V, member 4 (Trpv4). DESIGN: A prospective, controlled study wherein rats were subjected to femoral artery ligation (FAL), or FAL+arteriovenous shunt. Collateral Trpv4 was determined 0.5 and 6h post exercise. METHODS: Rats were subjected to exercise for 15 min, twice daily. The number and diameter of collaterals were assessed after 7 days. Collateral Trpv4 expression was quantified by reverse transcription-polymerase chain reaction. RESULTS: Collateral number and diameter per limb were significantly higher in the shunt group (number: 16.0+/-2.4 and diameter: 216.0+/-34 microm) compared to the ligature (number: 9.4+/-2 and diameter: 144+/-21 microm) and exercise groups (number: 9.9+/-2.5 and diameter: 151+/-15 microm). Trpv4 expression in collaterals harvested 0.5h post exercise was not significantly different from expression in shunted rats. It was significantly lower in collaterals harvested 6h post exercise (comparable to that in ligated rats). CONCLUSION: Collateral formation was greater in the shunt group than in the exercise group. Exercise-induced Trpv4 up-regulation, not significantly different from that achieved with shunt, returned to control values when evaluated 6h post exercise. More frequent exercise to chronically increase fluid shear stress, as with a shunt model, may be required for sufficient arteriogenesis to compensate for peripheral occlusion.
Asunto(s)
Arteriopatías Oclusivas/fisiopatología , Circulación Colateral , Músculo Esquelético/irrigación sanguínea , Condicionamiento Físico Animal , Canales Catiónicos TRPV/metabolismo , Animales , Arteriopatías Oclusivas/diagnóstico por imagen , Arteriopatías Oclusivas/genética , Arteriopatías Oclusivas/metabolismo , Arterias/metabolismo , Arterias/fisiopatología , Derivación Arteriovenosa Quirúrgica , Modelos Animales de Enfermedad , Arteria Femoral/cirugía , Vena Femoral/cirugía , Miembro Posterior , Masculino , Neovascularización Fisiológica , ARN Mensajero/metabolismo , Radiografía , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/genética , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Cardiovascular diseases account for more than half of total mortality before the age of 75 in industrialized countries. To develop therapies promoting the compensatory growth of blood vessels could be superior to palliative surgical interventions. Therefore, much effort has been put into investigating underlying mechanisms. Depending on the initial trigger, growth of blood vessels in adult organisms proceeds via two major processes, angiogenesis and arteriogenesis. While angiogenesis is induced by hypoxia and results in new capillaries, arteriogenesis is induced by physical forces, most importantly fluid shear stress. Consequently, chronically elevated fluid shear stress was found to be the strongest trigger under experimental conditions. Arteriogenesis describes the remodelling of pre-existing arterio-arteriolar anastomoses to completely developed and functional arteries. In both growth processes, enlargement of vascular wall structures was proposed to be covered by proliferation of existing wall cells. Recently, increasing evidence emerges, implicating a pivotal role for circulating cells, above all blood monocytes, in vascular growth processes. Since it has been shown that monocytes/ macrophage release a cocktail of chemokines, growth factors and proteases involved in vascular growth, their contribution seems to be of a paracrine fashion. A similar role is currently discussed for various populations of bone-marrow derived stem cells and endothelial progenitors. In contrast, the initial hypothesis that these cells -after undergoing a (trans-)differentiation- contribute by a structural integration into the growing vessel wall, is increasingly challenged.
Asunto(s)
Arterias/crecimiento & desarrollo , Circulación Colateral , Neovascularización Fisiológica , Células Madre/fisiología , Animales , Médula Ósea/fisiología , Diferenciación Celular , Perros , Endotelio/irrigación sanguínea , Fémur/irrigación sanguínea , Miembro Posterior/irrigación sanguínea , Masculino , PorcinosRESUMEN
BACKGROUND: The blood-brain barrier (BBB) forms a selective barrier between blood and brain and regulates the passage of most molecules. Pathological conditions such as ischemia lead to breakdown of the BBB. Vascular endothelial growth factor (VEGF) has been shown to be responsible for hypoxia-induced hyperpermeability of the BBB in vivo as well as in vitro. To eliminate factors which alter the permeability of the BBB in vivo, an in vitro model was used to test the effects of intravenous and volatile anesthetics on the permeability and on VEGF expression during normoxia and hypoxia. METHODS: The in vitro model of the BBB consisted of primary cultures of porcine brain microvascular endothelial cells (BMEC). The permeability was measured by the paracellular passage of [3H]inulin across the BMEC monolayer and the expression of VEGF was determined by northern blot analysis. RESULTS: All intravenous and volatile anesthetics tested (etomidate, ketamine, fentanyl, propofol, midazolam, sodium-gamma-hydroxybutyrate as well as halothane, enflurane, isoflurane, sevoflurane, desflurane) did not alter the permeability of the BBB or the expression of VEGF in vitro. Hypoxia (2 vol%) increased the permeability and the VEGF expression significantly which was not altered in the presence of the anesthetics. CONCLUSION: The in vitro model represents a suitable model of the BBB to investigate direct effects of anesthetics on functions of the BBB independent of hemodynamic factors.
Asunto(s)
Anestésicos/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Anestésicos por Inhalación/farmacología , Anestésicos Intravenosos/farmacología , Animales , Northern Blotting , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Hipoxia/metabolismo , Porcinos , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
BACKGROUND: Arteriogenesis refers to the development of collateral conductance arteries and is orchestrated by circulating monocytes, which invade growing collateral arteries and act as suppliers of cytokines and growth factors. CD44 glycoproteins are involved in leukocyte extravasation but also in the regulation of growth factor activation, stability, and signaling. Here, we explored the role of CD44 during arteriogenesis. METHODS AND RESULTS: CD44 expression increases strongly during collateral artery growth in a murine hind-limb model of arteriogenesis. This CD44 expression is of great functional importance, because arteriogenesis is severely impaired in CD44-/- mice (wild-type, 54.5+/-14.9% versus CD44-/-, 24.1+/-9.2%, P<0.001). The defective arteriogenesis is accompanied by reduced leukocyte trafficking to sites of collateral artery growth (wild-type, 29+/-12% versus CD44-/-, 18+/-7% CD11b-positive cells/square, P<0.01) and reduced expression of fibroblast growth factor-2 and platelet-derived growth factor-B protein. Finally, in patients with single-vessel coronary artery disease, the maximal expression of CD44 on activated monocytes is reduced in case of impaired collateral artery formation (poor collateralization, 1764+/-572 versus good collateralization, 2817+/-1029 AU, P<0.05). CONCLUSIONS: For the first time, the pivotal role of CD44 during arteriogenesis is shown. The expression of CD44 increases during arteriogenesis, and the deficiency of CD44 severely impedes arteriogenesis. Maximal CD44 expression on isolated monocytes is decreased in patients with a poor collateralization compared with patients with a good collateralization.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Circulación Colateral/fisiología , Receptores de Hialuranos/fisiología , Anciano , Animales , Circulación Colateral/genética , Endotelio Vascular/metabolismo , Femenino , Arteria Femoral , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Miembro Posterior/irrigación sanguínea , Humanos , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/genética , Isquemia/fisiopatología , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-sis/biosíntesis , Proteínas Proto-Oncogénicas c-sis/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Organismos Libres de Patógenos EspecíficosRESUMEN
To define the role of the plasminogen activators (PAs) urokinase PA (uPA) and tissue PA (tPA) as well as the uPA receptor (uPAR) in arteriogenesis, we investigated their impact in a rabbit and mouse model of adaptive collateral artery growth. Collateral artery growth was induced by occlusion of the femoral artery in rabbit and wild-type (WT) mice and in mice with targeted inactivation of uPA (uPA-/-), tPA (tPA-/-), or uPAR (uPAR-/-). Northern blot results revealed a significant up-regulation of uPA but not uPAR or tPA in the early phase of arteriogenesis in rabbit and WT mice. This up-regulation on RNA level was followed by an increased protein level and enzymatic activity. Impaired perfusion recovery upon femoral artery ligation was observed by laser Doppler analysis in vivo in uPA-deficient mice but not in uPAR or tPA deficiency compared with WT mice. Immunohistochemical studies revealed an association of leukocyte infiltration with arteriogenesis in WT mice that was strongly reduced in uPA-/- but not in uPAR- or tPA-deficient mice. We conclude that arteriogenesis is promoted by an uPA-mediated infiltration of leukocytes that is not dependent on uPAR.
Asunto(s)
Arterias/crecimiento & desarrollo , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Animales , Arterias/citología , Movimiento Celular , Arteria Femoral/cirugía , Miembro Posterior/irrigación sanguínea , Leucocitos/fisiología , Ligadura , Ratones , Ratones Noqueados , Modelos Cardiovasculares , ARN Mensajero/biosíntesis , Conejos , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Flujo Sanguíneo Regional , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Hypoxia has been identified as an important stimulus for gene expression during embryogenesis and in various pathological situations. Its influence under physiological conditions, however, has only been studied occasionally. We therefore investigated the effect of intermittent high altitude hypoxia on the mRNA expression of different cytokines and protooncogenes, but also of other genes described to be regulated by hypoxia, in the left ventricle (LV), the right ventricle (RV), atria and the lung of adult rats after simulation of hypoxia in a barochamber (5000 m, 4 hours to 10 days). Heme oxygenase-1 as well as transforming growth factor-beta1 showed an increased expression in all regions of the heart and the lung at different periods of hypoxia. For lactate dehydrogenase-A, we found a significant up-regulation in the RV and the lung, for lactate dehydrogenase-B up-regulation in the RV, but down-regulation in the LV and the atria. Vascular endothelial growth factor was up-regulated in the RV, the LV and the lung, but down-regulated in the atria. Its receptor Flk-1 mRNA was significantly increased in the atria and RV only. Expression of c-fos was found in the LV and RV only after 4 hours of hypoxia. The level of c-jun was significantly increased in the LV but decreased in the atria. Our data clearly demonstrate that intermittent hypoxia is a modulator of gene expression under physiological conditions. It differently regulates the expression of distinct genes not only in individual organs but even within one organ, i.e. in the heart.
Asunto(s)
Citocinas/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Ventrículos Cardíacos/enzimología , Hipoxia/enzimología , Hipoxia/genética , Pulmón/enzimología , Proteínas Proto-Oncogénicas/biosíntesis , Adaptación Fisiológica/genética , Altitud , Animales , Citocinas/genética , Perfilación de la Expresión Génica/métodos , Regulación Enzimológica de la Expresión Génica/genética , Corazón , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , L-Lactato Deshidrogenasa/biosíntesis , L-Lactato Deshidrogenasa/genética , Masculino , Miocardio/enzimología , Proteínas Proto-Oncogénicas/genética , Ratas , Ratas Wistar , Distribución TisularRESUMEN
Monocyte chemoattractant protein-1 (MCP-1) stimulates the formation of a collateral circulation on arterial occlusion. The present study served to determine whether these proarteriogenic properties of MCP-1 are preserved in hyperlipidemic apolipoprotein E-deficient (apoE-/-) mice and whether it affects the systemic development of atherosclerosis. A total of 78 apoE-/- mice were treated with local infusion of low-dose MCP-1 (1 microg/kg per week), high-dose MCP-1 (10 microg/kg per week), or PBS as a control after unilateral ligation of the femoral artery. Collateral hindlimb flow, measured with fluorescent microspheres, significantly increased on a 1-week high-dose MCP-1 treatment (PBS 22.6+/-7.2%, MCP-1 31.3+/-10.3%; P<0.05). These effects were still present 2 months after the treatment (PBS 44.3+/-4.6%, MCP-1 56.5+/-10.4%; P<0.001). The increase in collateral flow was accompanied by an increase in the number of perivascular monocytes/macrophages on MCP-1 treatment. However, systemic CD11b expression by monocytes also increased, as did monocyte adhesion at the aortic endothelium and neointimal formation (intima/media ratio, 0.097+/-0.011 [PBS] versus 0.257+/-0.022 [MCP-1]; P<0.0001). Moreover, Sudan IV staining revealed an increase in aortic atherosclerotic plaque surface (24.3+/-5.2% [PBS] versus 38.2+/-9.5% [MCP-1]; P<0.01). Finally, a significant decrease in the percentage of smooth muscle cells was found in plaques (15.0+/-5.2% [PBS] versus 5.8+/-2.3% [MCP-1]; P<0.001). In conclusion, local infusion of MCP-1 significantly increases collateral flow on femoral artery ligation in apoE-/- mice up to 2 months after the treatment. However, the local treatment did not preclude systemic effects on atherogenesis, leading to increased atherosclerotic plaque formation and changes in cellular content of plaques.
Asunto(s)
Arterias/efectos de los fármacos , Antígeno CD11b/biosíntesis , Quimiocina CCL2/farmacología , Circulación Colateral/efectos de los fármacos , Monocitos/metabolismo , Túnica Íntima/efectos de los fármacos , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Arterias/patología , Arteriosclerosis/patología , Quimiocina CCL2/efectos adversos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Arteria Femoral/efectos de los fármacos , Arteria Femoral/patología , Citometría de Flujo , Inmunohistoquímica , Lípidos/sangre , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/patología , Flujo Sanguíneo Regional/efectos de los fármacos , Túnica Íntima/patologíaRESUMEN
UNLABELLED: OBJECTIVES. We investigated the effect of short- and long-term swimming exercise, with or without insulin-like growth factor (IGF)-I administration, on the expression of myocardial IGFs and contractile proteins. METHODS: Sprague-Dawley male rats (n=36) were subjected to swimming exercise for 2 or 6 weeks. IGF-I (0.5mg/rat) was administered continuously for 1 week, using alzet osmotic pumps. Control groups remained sedentary. IGF-I, IGF-I receptor (IGF-IR), IGF-II, skeletal alpha-actin (sk-actin), and beta myosin heavy chain (beta MHC) mRNAs were measured using Northern blot analysis and RT-PCR. RESULTS: A significant 2-fold increase in myocardial IGF-I mRNA was found after 2 and 6 weeks of swimming in both IGF-I treated and untreated rats (p<0.001). IGF-IR mRNA was significantly (p<0.05) increased after 6 weeks of training only in the IGF-I treated animals. IGF-II mRNA remained unchanged at all time points. While beta MHC mRNA was significantly decreased (p=0.003) at 2 and 6 weeks, sk-actin mRNA remained unchanged. CONCLUSIONS: Short- and long-term swimming exercise training increase myocardial expression of IGF-I mRNA. Exogenous administration of IGF-I, during the first week of the exercise session, did not produce any effect on myocardial IGF-I but was associated with increased IGF-IR signal after the long-term exercise training. These data suggest a relationship between IGF-I expression and cardiac adaptation to exercise training.
Asunto(s)
Regulación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , Miocardio/metabolismo , Condicionamiento Físico Animal , Natación , Actinas/genética , Actinas/metabolismo , Animales , Northern Blotting , Cartilla de ADN/química , Corazón , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculos/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: The objective of our study was to quantify the arteriogenic potency of Monocyte Chemoattractant Protein-1 (MCP-1) under hyperlipidemic conditions. Additionally, we aimed to determine the effects of locally applied MCP-1 on systemic serum lipid levels as well as on atherosclerosis. METHODS: A total of sixty-four Watanabe rabbits was treated with either low dose MCP-1 (1 microg/kg/week), high dose MCP-1 (3.3 microg/kg/week) or PBS as a control substance. Substances were applied directly into the collateral circulation via an osmotic minipump with the catheter placed in the proximal stump of the ligated femoral artery. Either 1 week or 6 months after initiation of the treatment X-ray angiography was performed as well as measurements of collateral conductance using fluorescent microspheres. The extent of atherosclerosis was quantified in whole aortas using Sudan IV staining. RESULTS: One week after ligation of the femoral artery a significant increase in collateral conductance was observed in animals treated with high dose MCP-1 (control: 2.2+/-0.8 ml/min/100 mmHg vs. MCP-1 high dose: 8.9+/-2.0 ml/min/100 mmHg, P<0.05). Six months after femoral artery ligation no differences were found between the treated and the control group (PBS; 44.9+/-11.6 ml/min/100 mmHg, MCP-1; 47.8+/-11.5 ml/min/100 mmHg, P=NS). No influence was found on serum lipids or on the development of atherosclerosis in the present model. CONCLUSION: MCP-1 accelerates arteriogenesis upon femoral artery ligation under hyperlipidemic conditions. Six months after treatment these pro-arteriogenic effects of MCP-1 can no longer be observed. The present data do not show an effect of local MCP-1 treatment on serum lipids or on atherosclerosis. It should be noted however that a high standard deviation was observed for the data on atherosclerotic surface area, necessitating additional experiments in a different model of atherosclerosis.
Asunto(s)
Quimiocina CCL2/uso terapéutico , Circulación Colateral , Arteria Femoral , Hiperlipidemias/tratamiento farmacológico , Animales , Arteriosclerosis , Arteria Femoral/diagnóstico por imagen , Hiperlipidemias/sangre , Hiperlipidemias/diagnóstico por imagen , Ligadura , Lípidos/sangre , Modelos Animales , Conejos , RadiografíaRESUMEN
1927 Foundation of the DGHKF by Prof. A. Weber and Prof. B. Kisch in Bad Nauheim. The first executive board of the DGHKF consists of A. Weber, Bad Nauheim, H. E. Hering, Köln, J. Rihl, Prag, B. Kisch, Köln, and H. Eppinger, Freiburg. Registered office was William-G. Kerckhoff-Herzforschungsinstitut, Bad Nauheim, under direction of Prof. F. Groedel. 1933 Emigration of F. Groedel to the United States of America. 1933-1941 Prof. E. Koch becomes acting chairman but Groedel remains director and influences fate and finances from his New York domicile and practice. 1938 Emigration of B. Kisch to the United States of America. 1948 Foundation of the "American College of Cardiology" in the United States. (Executive board: Groedel and Kisch). 1948 Prof. H. Schäfer is the new executive secretary of the society (executive board: K. Matthes, Erlangen, F. Hildebrandt, Bad Nauheim/Giessen, C. Oelemann, Bad Nauheim, A. Weber, Bad Nauheim, E. Wollheim, Lund). 1952 Incorporation of the Kerckhoff-Institute into the Max-Planck-Society, Appointment of Prof. R. Thauer to the directorship of the Kerckhoff-Institute and election to the position of the executive secretary until 1976. 1972 Appointment of Prof. W. Schaper to the Kerckhoff-Institute, Bad Nauheim. 1976 Election of Prof. Wolfgang Schaper to the executive board of the society and assumption of the office of the executive secretary until 1989.
Asunto(s)
Cardiología/historia , Sociedades Médicas/historia , Alemania , Historia del Siglo XXRESUMEN
In vivo, hypoxia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Primary cultures of porcine brain derived microvascular endothelial cells were used as an in vitro BBB model to evaluate the mechanisms by which hypoxia regulates paracellular permeability. Paracellular passage across endothelial cell monolayers is regulated by specialized intercellular structures like the tight junctions (TJ). Zonula occludens-1 (ZO-1), a protein of the TJ, lines the cytoplasmic face of intact TJ. The continuity of the ZO-1 expression was disrupted during 24 h of hypoxia which correlated with a decrease of the protein level to 32 +/- 8% and with a twofold increase in the phosphorylation of ZO-1 in comparison to values determined at the start of the experiment. The localization and expression level of ZO-1 were maintained during hypoxia in the presence of a polyclonal antibody to vascular endothelial growth factor (VEGF) demonstrating that hypoxia-induced changes of the ZO-1 expression are mediated by VEGF. The effect of hypoxia on the ZO-1 distribution probably is not tissue- or cell-specific because similar changes of ZO-1 distribution were observed when the rat brain endothelial cell line RBE4 or the murine epithelial cell line CSG was used. Furthermore, ZO-1 changes correlated with small changes in actin distribution. These results suggest that hypoxia increases the paracellular flux across the cell monolayer via the release of VEGF, which in turn leads to the dislocalization, decreased expression, and enhanced phosphorylation of ZO-1. Science.
Asunto(s)
Encéfalo/irrigación sanguínea , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/metabolismo , Hipoxia , Linfocinas/metabolismo , Proteínas de la Membrana/biosíntesis , Microcirculación/metabolismo , Fosfoproteínas/biosíntesis , Actinas/biosíntesis , Animales , Barrera Hematoencefálica , Western Blotting , Encéfalo/metabolismo , Línea Celular , Células Cultivadas , Citoplasma/metabolismo , Endotelio Vascular/citología , Inmunohistoquímica , Ratones , Fosforilación , Pruebas de Precipitina , Ratas , Porcinos , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Proteína de la Zonula Occludens-1RESUMEN
We investigated the role of the colony stimulating factor for monocytes (GM-CSF) to test the hypothesis whether prolongation of the monocyte's life cycle will support arteriogenesis (rapid growth of preexisting collateral arteries). This appeared logical in view of our discovery that circulating monocytes play an important part in the positive remodeling of small preexisting arterioles into arteries to compensate for arterial occlusions (arteriogenesis) and especially following our findings that MCP-1 markedly increases the speed of arteriogenesis. The continuous infusion of GM-CSF for 7 days into the proximal stump of the acutely occluded femoral artery of rabbits by osmotic minipump produced indeed a marked arteriogenic response as demonstrated by an increase (2-fold) in number and size of collateral arteries on postmortem angiograms and by the increase of maximal blood flow during vasodilation measured in vivo by blood pump perfusion of the hindquarter (5-fold). When GM-CSF and MCP-1 were simultaneously infused the effects on arteriogenesis were additive on angiograms as well as on conductance. GM-CSF was also able to widen the time window of MCP-1 activity: MCP-1 treatment alone was ineffective when given after the third week following occlusion. When administered together with GM-CSF about 80% of normal maximal conductance of the artery that was replaced by collaterals were achieved, a result that was not reached before by any other experimental treatment. Experiments with cells isolated from treated animals showed that monocyte apoptosis was markedly reduced. In addition we hypothesize that GM-CSF may aid in releasing pluripotent monocyte (stem-) cells from the bone marrow into the circulation. In contrast to MCP-1, GM-CSF showed no activity on monocyte transmigration through- and also no influence on monocyte adhesion to cultured endothelial cells. In conclusion we have discovered a new function of the hemopoietic stem cell factor GM-CSF, which is also a powerful arteriogenic peptide that acts via prolongation of the life cycle of monocytes/macrophages.
Asunto(s)
Arteriopatías Oclusivas/tratamiento farmacológico , Quimiocina CCL2/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Lipoproteínas/efectos de los fármacos , Angiografía , Animales , Apoptosis , Circulación Colateral/efectos de los fármacos , Circulación Colateral/fisiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Arteria Femoral , Lipoproteínas/metabolismo , Monocitos/efectos de los fármacos , Monocitos/patología , Probabilidad , Conejos , Estadísticas no ParamétricasAsunto(s)
Inductores de la Angiogénesis/uso terapéutico , Arteriopatías Oclusivas/tratamiento farmacológico , Arteriopatías Oclusivas/fisiopatología , Arterias/efectos de los fármacos , Arterias/crecimiento & desarrollo , Circulación Colateral/efectos de los fármacos , Circulación Colateral/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Arterias/fisiopatología , HumanosRESUMEN
Vascular endothelial growth factor (VEGF) is known to play an important role in angiogenesis. Its place in collateral artery growth (arteriogenesis), however, is still debated. In the present study, we analyzed the expression of VEGF and its receptors (Flk-1 and Flt-1) in a rabbit model of collateral artery growth after femoral artery occlusion. Hypoxia presents the most important stimulus for VEGF expression. We therefore also investigated the expression level of distinct hypoxia-inducible genes (HIF-1alpha, LDH A) and determined metabolic intermediates indicative for ischemia (ATP, creatine phosphate, and their catabolites). We found that arteriogenesis was not associated with an increased expression of VEGF or the mentioned hypoxia-inducible genes. Furthermore, the high-energy phosphates and their catabolites were entirely within normal limits. Despite the absence of an increased expression of VEGF and its receptors, collateral vessels increased their diameter by a factor of 10. The speed of collateral development could be increased by infusion of the chemoattractant monocyte chemotactic protein-1 but not by infusion of a 30 times higher concentration of VEGF. From these data, we conclude that under nonischemic conditions, arteriogenesis is neither associated with nor inducible by increased levels of VEGF and that VEGF is not a natural agent to induce arteriogenesis in vivo.