RESUMEN
Arterial Hypertension (AH) is characterized by reduced nitric oxide (NO) biosynthesis, activation of the Renin-Angiotensin-Aldosteron-System (RAAS), vasoconstriction, and microvascular rarefaction. The latter contributes to target organ damage, especially in left ventricular hypertrophy, and may partially be due to impaired angiogenesis. Angiogenesis, the formation of new microvessels and microvascular networks from existing ones, is a highly regulated process that arises in response to hypoxia and other stimuli and that relieves tissue ischemia. In AH, angiogenesis seems impaired. However, blood pressure alone does not affect angiogenesis, and microvascular rarefaction is present in normotensive persons with a family history for AH. Normal or increased NO in several processes and diseases enables or enhances angiogenesis (e.g. in portal hypertension) and reduced NO biosynthesis (for example, in a rat model of AH, in other disease models in vivo, and in endothelial NO Synthase knock out mice) impairs angiogenesis. Angiogenic growth factors such as Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF) induce NO and require NO to elicit an effect. Effector molecules and corresponding receptors of the RAAS either induce (Bradykinin, Angiotensin II) or perhaps inhibit angiogenesis. The pattern of Bradykinin- and Angiotensin II-receptor expression and the capacity to normalize NO biosynthesis may determine whether ACE-inhibitors, Angiotensin II-receptor antagonists and other substances affect angiogenesis. Reconstitution of a normally vascularized tissue by reversal of impaired angiogenesis with drugs such as ACE inhibitors and AT1 receptor antagonists may contribute to successful treatment of hypertension-associated target organ damage, e.g. left ventricular hypertrophy.
Asunto(s)
Vasos Sanguíneos/fisiopatología , Hipertensión/patología , Hipertensión/fisiopatología , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Animales , Humanos , Hipertensión/metabolismo , Microcirculación , Óxido Nítrico/biosíntesis , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Angiogenesis is crucial for many biological and pathological processes including the ovarian cycle and tumor growth. To identify molecules relevant for angiogenesis, we performed mRNA fingerprinting and subsequent Northern blot analysis using bovine cord-forming vs. monolayer-forming endothelial cells (EC) in vitro and staged bovine corpora lutea in vivo. We detected the receptor for activated C kinase 1 (RACK1), the specific receptor for activated protein kinase C beta (PKC beta), to be up-regulated in bovine cord-forming EC in vitro and in angiogenically active stages of bovine corpora lutea in vivo. Thereafter we established and determined the complete bovine RACK1 cDNA sequence. RACK1 was massively induced in subconfluent vs. contact-inhibited bovine EC, during angiogenesis in vitro, active phases of the murine ovarian cycle, human tumor angiogenesis, and in cancer cells in vivo as assessed by quantitative PCR and in situ hybridization. RACK1 transcripts were localized to proliferating EC in vitro and the endothelium of tumor neovascularizations in vivo by in situ hybridization. PKC beta plays an important role in angiogenesis and cancer growth. Our data suggest that downstream signaling of PKC beta in angiogenically active vs. inactive tissues and endothelium is affected by the availability of RACK1.
Asunto(s)
Carcinoma/genética , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Receptores de Superficie Celular/genética , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario/genética , Endotelio Vascular/metabolismo , Estro/fisiología , Femenino , Proteínas de Unión al GTP , Humanos , Datos de Secuencia Molecular , ARN Mensajero/aislamiento & purificación , ARN Neoplásico/aislamiento & purificación , Receptores de Cinasa C Activada , Homología de Secuencia de Ácido Nucleico , Regulación hacia ArribaRESUMEN
To investigate the potential role of tenascin-C (TN-C) on endothelial sprouting we used bovine aortic endothelial cells (BAECs) as an in vitro model of angiogenesis. We found that TN-C is specifically expressed by sprouting and cord-forming BAECs but not by nonsprouting BAECs. To test whether TN-C alone or in combination with basic fibroblast growth factor (bFGF) can enhance endothelial sprouting or cord formation, we used BAECs that normally do not sprout and, fittingly, do not express TN-C. In the presence of bFGF, exogenous TN-C but not fibronectin induced an elongated phenotype in nonsprouting BAECs. This phenotype was due to altered actin cytoskeleton organization. The fibrinogen globe of the TN-C molecule was the active domain promoting the elongated phenotype in response to bFGF. Furthermore, we found that the fibrinogen globe was responsible for reduced cell adhesion of BAECs on TN-C substrates. We conclude that bFGF-stimulated endothelial cells can be switched to a sprouting phenotype by the decreased adhesive strength of TN-C, mediated by the fibrinogen globe.
Asunto(s)
Endotelio Vascular/citología , Fibrinógeno/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Tenascina/metabolismo , Tenascina/farmacología , Empalme Alternativo , Animales , Aorta , Bovinos , Adhesión Celular/efectos de los fármacos , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Embrión de Pollo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Fibroblastos , Vidrio , Humanos , Peso Molecular , Neovascularización Fisiológica , Plásticos , Eliminación de Secuencia , Tenascina/química , Tenascina/genéticaRESUMEN
In this prospective, double-blind, monocenter drug trial, 48 primary care patients with mild to moderate essential hypertension were randomized to mibefradil, 50 mg, titrated to 100 mg, or enalapril, 20 mg, titrated to 2 x 20 mg. Ambulatory 24-h blood pressure measurements (ABPM) and echocardiography were performed at baseline and after 12 weeks' treatment. Complete data from 43 patients were analyzed. Mibefradil (n = 22; titration, 13 patients) reduced mean 24-h ABP from 159+/-14/102+/-7 mm Hg to 140+/-10/89+/-7 mm Hg after 12 weeks. Enalapril (n = 21; titration, six patients) reduced baseline ABP from 156+/-12/100+/-9 mm Hg to 140+/-17/89+/-10 mm Hg (12 weeks). Trough-to-peak ratios in DBP were 86% for mibefradil and 75% with enalapril. Left ventricular mass (LVM) decreased from 199+/-65 to 193+/-62 g [M-mode modified American Society of Echocardiography (ASE)] and from 184+/-65 to 173+/-50 g (truncated ellipsoid method) after 12 weeks in response to mibefradil (p > 0.2), and from 212+/-50 to 196+/-57 g and from 182+/-39 to 170+/-40 g (mean +/- SD, p < 0.02) with enalapril. Mibefradil matched enalapril in 24-h ABP control. Enalapril reduced LVM significantly after 12 weeks (p < 0.02). Mibefradil did not significantly reduce LVM after 12 weeks.
Asunto(s)
Antihipertensivos/uso terapéutico , Bencimidazoles/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Enalapril/uso terapéutico , Hipertensión/tratamiento farmacológico , Tetrahidronaftalenos/uso terapéutico , Antihipertensivos/farmacología , Bencimidazoles/farmacología , Método Doble Ciego , Enalapril/farmacología , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Mibefradil , Persona de Mediana Edad , Estudios Prospectivos , Tetrahidronaftalenos/farmacología , Vasodilatadores/farmacología , Vasodilatadores/uso terapéutico , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
To explore direct effects of platelet-derived growth factor (PDGF) on endothelial cells during angiogenesis in vitro, we have used cloned bovine aortic endothelial cells that spontaneously form cord structures. Recently we have shown that cells forming these endothelial cords express PDGF beta-receptors and that PDGF-BB can contribute to cellular proliferation and cord formation. In this study we investigated whether PDGF-induced cellular migration might also contribute to endothelial repair and angiogenesis in vitro. Ten individual endothelial cells in cords were tracked at an early stage of cord formation by video-timelapse microscopy. PDGF-BB (100 ng/ml) induced an increase in endothelial cell movement of 67 +/- 15% as compared with diluent control. Interestingly, PDGF-BB also increased movements of entire cord structures, followed at branching points, by 53 +/- 12% over diluent control. Taken together, these video-timelapse experiments suggested that the apparent movements of single endothelial cord cells might also be due to the motion of entire underlying cord structures in response to PDGF. To analyze the response of single endothelial cord cells we therefore examined whether PDGF-induced migration contributes to endothelial repair. Abrasions were applied with a razor blade to confluent monolayers of endothelial cells at an intermediate stage of cord formation. PDGF-BB concentration-dependently increased the distance to which cord-forming endothelial cells migrated into the abrasion. An increased number of elongated, i.e., probably migrating, endothelial cells was found in the abrasion in response to PDGF-BB. However, there was no effect of PDGF-BB on the total number of endothelial cells found in the abrasion. PDGF-AA affected neither the distance to which the cells migrated nor the number of elongated cells. Actin and tubulin stainings revealed that these cytoskeletal structures were not appreciably altered by PDGF-BB. Furthermore, urokinase-type plasminogen activator transcripts were not modulated in response to PDGF-BB. We conclude that in this model of angiogenesis in vitro PDGF-BB can elicit the movement of entire cord structures, possibly via u-PA-independent mechanisms. PDGF-BB also controls the migration of single cord-forming endothelial cells. Thus, PDGF-BB possibly contributes to endothelial repair and angiogenesis by direct effects on proliferation and composite movements of PDGF beta-receptor-expressing endothelial cells and cords.
Asunto(s)
Movimiento Celular/fisiología , Endotelio Vascular/fisiología , Neovascularización Fisiológica/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiología , Adulto , Animales , Northern Blotting , Bovinos , Técnicas de Cultivo de Célula , Análisis Factorial , Humanos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/agonistas , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
This review of angiogenesis aims to describe (a) stimuli that either elicit or antagonize angiogenesis, (b) the response of the vasculature to angiogenic or anti-angiogenic stimuli, i.e., processes required for the formation of new vessels, (c) aspects of angiogenesis relating to tissue remodeling and disease, and (d) the potential of angiogenic or antiangiogenic therapeutic measures. Angiogenesis, the formation of new vessels from existing microvessels, is important in embryogenesis, wound healing, diabetic retinopathy, tumor growth, and other diseases. Hypoxia and other as yet ill-defined stimuli drive tumor, inflammatory, and connective tissue cells to generate angiogenic molecules such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), and others. Natural and synthetic angiogenesis inhibitors such as angiostatin and thalidomide can repress angiogenesis. Angiogenic and antiangiogenic molecules control the formation of new vessels via different mechanisms. VEGF and FGF elicit their effects mainly via direct action on relevant endothelial cells. TGF-beta and PDGF can attract inflammatory or connective tissue cells which in turn control angiogenesis. Additionally, PDGF may act differently on specific phenotypes of endothelial cells that are engaged in angiogenesis or that are of microvascular origin. Thus phenotypic traits of endothelial cells committed to angiogenesis may determine their cellular responses to given stimuli. Processes necessary for new vessel formation and regulated by angiogenic/antiangiogenic molecules include the migration and proliferation of endothelial cells from the microvasculature, the controlled expression of proteolytic enzymes, the breakdown and reassembly of extracellular matrix, and the morphogenic process of endothelial tube formation. In animal models some angiogenesis-dependent diseases can be controlled via induction or inhibition of new vessel formation. Life-threatening infantile hemangiomas are a first established indication for antiangiogenic therapy in humans. Treatment of other diseases by modulation of angiogenesis are currently tested in clinical trials. Thus the manipulation of new vessel formation in angiogenesis-dependent conditions such as wound healing, inflammatory diseases, ischemic heart and peripheral vascular disease, myocardial infarction, diabetic retinopathy, and cancer is likely to create new therapeutic options.
Asunto(s)
Neovascularización Patológica , Neovascularización Fisiológica , Animales , Humanos , Neovascularización Patológica/terapiaRESUMEN
TNF-alpha stimulates DNA synthesis and proliferation of cultured human fibroblasts. Maximal DNA synthesis in response to TNF-alpha occurs approximately 28 h after addition of TNF-alpha to quiescent fibroblasts, a delay of about 12 to 14 h as compared with DNA synthesis elicited by platelet-derived growth factor (PDGF). TNF-alpha induces PDGF A chain gene expression with a maximum at 4 h. DNA synthesis is abrogated in response to TNF-alpha by a goat anti-PDGF IgG but not by nonimmune goat IgG, suggesting induction of an autocrine PDGF-AA loop by TNF-alpha. The response to PDGF-AA requires the presence of PDGF receptor alpha-receptors. TNF-alpha does not significantly affect PDGF alpha-receptor mRNA or protein expression, nor does it alter the proliferative response to externally added PDGF-AA. In contrast, TNF-alpha reduces the levels of PDGF beta-receptor mRNA, protein expression, and cell proliferation in response to PDGF-BB. Thus, DNA synthesis in response to TNF-alpha depends upon autocrinely induced PDGF-AA. At the same time, TNF-alpha may alter the response to PDGF-BB from exogenous sources.
Asunto(s)
Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptores del Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Northern Blotting , División Celular/efectos de los fármacos , Células Cultivadas , Replicación del ADN , Humanos , Sueros Inmunes/inmunología , Factor de Crecimiento Derivado de Plaquetas/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
To delineate potential angiogenic roles of platelet-derived growth factor (PDGF), we have investigated PDGF and its receptors on bovine aortic endothelial cells that exhibit spontaneous angiogenesis in vitro (angiogenic endothelial cells). Initiation of cord/tube formation by angiogenic endothelial cells required bovine or human serum. Neutralization of PDGF-BB in human serum with a monoclonal anti-PDGF-BB antibody reduced cord/tube formation by 37 +/- 10%, whereas neutralizing anti-PDGF-AA and an IgG isotype-matched control antibody had no effect. DNA synthesis in response to PDGF-BB increased as the cords and tubes developed; furthermore, PDGF-BB induced the incorporation of BrdU in the nuclei of cells associated with these structures. PDGF beta-receptor (PDGF-beta) mRNA increased concomitantly with cord/tube formation, and PDGFR-beta were specifically localized by immunocytochemistry to developing and mature cords and tubes. However, PDGFR-beta transcripts and protein were undetectable in nonangiogenic endothelial cells, and PDGF alpha-receptor mRNA was not expressed in either endothelial cell strain. In contrast to nonangiogenic endothelial cells, angiogenic endothelial cells did not express the PDGF B-chain, the required ligand for the PDGFR-beta. We conclude that (a) PDGF-BB can contribute to angiogenesis in vitro, (b) PDGFR-beta are specific for cord/tube-forming endothelial cells and mediate endothelial proliferation and cord/tube formation, and (c) in angiogenic and nonangiogenic endothelial cells, the expression of PDGFR-beta and PDGF B-chain is inversely correlated. We therefore suggest that paracrine PDGF might amplify angiogenesis via direct action on endothelially expressed PDGFR-beta.
Asunto(s)
Endotelio Vascular/citología , Neovascularización Patológica/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Becaplermina , Bovinos , División Celular , Células Cultivadas , Medios de Cultivo , Medio de Cultivo Libre de Suero , ADN/biosíntesis , Endotelio Vascular/metabolismo , Humanos , Neovascularización Patológica/etiología , Fenotipo , Proteínas Proto-Oncogénicas c-sis , Proteínas RecombinantesRESUMEN
Transforming growth factor-beta (TGF-beta) acts as a growth inhibitor, yet it can stimulate proliferation; 1-2 fg/cell of TGF-beta 1 elicits maximal proliferation of dense and sparse cultured smooth muscle cells (SMCs), whereas higher amounts are less stimulatory. This bimodal response is not limited to SMCs, as TGF-beta induces a similar response in human fibroblasts and chondrocytes. The amount of TGF-beta 1 per cell that induces maximal proliferation is identical for dense and sparse SMCs. At low concentrations of TGF-beta, there is a 10-12 hr delay in DNA synthesis compared with that elicited by PDGF. PDGF-AA is detected in the culture medium at 24 hr, and anti-PDGF IgG blocks DNA synthesis. At higher concentrations, TGF-beta 1 decreases transcripts and expression of PDGF receptor alpha subunits. Hence, TGF-beta induces proliferation of connective tissue cells at low concentrations by stimulating autocrine PDGF-AA secretion, which at higher concentrations of TGF-beta, is decreased by down-regulation of PDGF receptor alpha subunits and perhaps by direct growth inhibition.
Asunto(s)
Cartílago/citología , Replicación del ADN/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptores de Superficie Celular/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Cartílago/efectos de los fármacos , Cartílago/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Humanos , Recién Nacido , Sustancias Macromoleculares , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Receptores de Superficie Celular/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas , Transcripción Genética/efectos de los fármacosRESUMEN
Megakaryocyte colony formation, as identified by conventional techniques, was observed in precursor cell cultures from peripheral blood in 8 of 20 consecutive patients with diagnosis of myeloproliferative disease (4/11 patients with polycythemia vera, 3/5 with essential thrombocythemia, 1/2 with primary osteomyelofibrosis and 2 with a myeloproliferative syndrome not further assessable), but not in 50 healthy controls (p less than 0.0001). 7 cultures showed spontaneous erythroid colonies, but were negative for megakaryocyte colonies. Megakaryocyte colony formation was independent of added erythropoietin, plasma or human leukocyte-conditioned medium, but was dependent on the presence of accessory cells. The cells in megakaryocyte colonies had the characteristic morphology of megakaryocytes and stained positively with the IIIa/IIb monoclonal anti-platelet antibody. Thus, megakaryocyte colony formation by precursor cells from peripheral blood in the absence of exogenous stimulating factors seems to be a phenomenon specific for myeloproliferative disease. Differential diagnosis of thrombocythemia may be facilitated by demonstration of endogenous megakaryocyte colony formation, which does not occur in secondary disease.
Asunto(s)
Células Madre Hematopoyéticas/patología , Megacariocitos/patología , Trastornos Mieloproliferativos/sangre , Núcleo Celular/patología , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo , Citoplasma/patología , Diagnóstico Diferencial , Eritropoyetina/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Leucocitos/fisiología , Trastornos Mieloproliferativos/patología , Policitemia Vera/sangre , Policitemia Vera/patología , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/patología , Trombocitemia Esencial/sangre , Trombocitemia Esencial/diagnóstico , Trombocitemia Esencial/patologíaRESUMEN
This study was conceived to identify specific morphological characteristics associated with cytomegalovirus (CMV) infection of the kidney. 33 patients with manifest CMV infection at autopsy and 32 biopsies of kidney transplants with active or inactive CMV infection were studied. In 8 patients of the autopsy group a CMV infection of the kidney was detected (CMV cells in 3 cases, positive viral tissue culture in 4 cases, positive in-situ hybridization in one case), which was associated with severe dissemination into different organs. In situ hybridization was not superior to ordinary light microscopy in the detection of CMV cells. The biopsies were screened for the presence of glomerulopathy. No association of glomerulopathy with CMV could be found by light microscopy, whereas a significant correlation of glomerulopathy with vascular rejection was demonstrated. No differences as to the incidence of glomerulopathy were found, when non-transplant patients with manifest CMV infection at autopsy were compared with matched controls. With active infection electron microscopy revealed no osmiophilic deposits, but in immunofluorescence tiny IgG deposits were identified within the glomeruli (p less than 0.01). IgM or C3 deposition in the glomeruli was not specifically associated with either CMV infection or vascular rejection. Thus morphological identification of CMV infection of the kidney is difficult, necessitating the detection of CMV cells, positive viral tissue culture or positive in-situ hybridization. Glomerulopathy in light microscopy is associated with vascular rejection and is therefore termed transplant glomerulitis. Tiny IgG deposits may be indicative of CMV infection, although these are not always present.