RESUMO
Mechanisms regulating coronary vascularization are not well understood. To test hypotheses regarding the influence of key growth factors and their interactions, we studied vascular tube formation (vasculogenesis) in collagen gels onto which quail embryonic ventricles were placed and incubated in the presence of growth factors or inhibitors. Vasculogenesis in this model is dependent on tyrosine kinase receptors, since tube formation was totally blocked by genestein. Tube formation was attenuated when anti-bFGF or anti-VEGF neutralizing antibodies were added to the medium and nearly completely inhibited when the both were added. The attenuation associated with anti-VEGF was due primarily to a decrease in assembly of endothelial cells, while that associated with bFGF was primarily due to a reduction in endothelial cells. Soluble tie-2, the receptor for angiopoietins, also had an inhibitory effect and, when added with either anti-bFGF or anti-VEGF, markedly attenuated tube formation. At optimal doses, tube formation was enhanced 6.5-fold by bFGF and 2.5-fold by VEGF over the controls. Each of these growth factors was dependent upon the other for optimal induction of tube formation, since neutralizing antibodies to one markedly reduced the potency of the other. VEGF potency was also markedly reduced when soluble tie-2 was added to the medium. Tube formation was virtually totally blocked by exogenous TGF-beta at doses > 1 ng/ml, while neutralizing TGF-beta antibodies enhanced tube formation 2-fold in the 30 ng-30 microg range. These data provide the first documentation of multiple growth factor regulation of coronary tube formation.
Assuntos
Vasos Coronários/embriologia , Fatores de Crescimento Endotelial/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Vasos Coronários/fisiologia , Fatores de Crescimento Endotelial/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Linfocinas/farmacologia , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Codorniz , Receptores Proteína Tirosina Quinases/farmacologia , Receptor TIE-2 , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary growth that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar growth, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles (<15 microm) was markedly reduced, whereas the percentage of large arterioles (26 to 50 microm) more than doubled. Thus, inhibition of both growth factors negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary growth, (2) bFGF facilitates arteriolar growth, and (3) the two growth factors interact to establish the normal hierarchy of the arteriolar tree.
Assuntos
Arteríolas/fisiologia , Vasos Coronários/fisiologia , Fatores de Crescimento Endotelial/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Linfocinas/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/farmacologia , Arteríolas/citologia , Arteríolas/efeitos dos fármacos , Western Blotting , Capilares/efeitos dos fármacos , Capilares/fisiologia , Capilares/ultraestrutura , Contagem de Células , Circulação Coronária/efeitos dos fármacos , Circulação Coronária/fisiologia , Vasos Coronários/citologia , Vasos Coronários/efeitos dos fármacos , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/farmacologia , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/farmacologia , Imuno-Histoquímica , Linfocinas/antagonistas & inibidores , Linfocinas/farmacologia , Microcirculação/fisiologia , Microcirculação/ultraestrutura , Microscopia Eletrônica , Miocárdio/citologia , Miocárdio/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
It has been documented that hypoxia enhances coronary vasculogenesis and angiogenesis in cultured embryonic quail hearts via the upregulation of vascular endothelial growth factor (VEGF). In this study, we compared the functions of two VEGF splice variants. Ventricles from 6-day-old embryonic quail hearts were cultured on three-dimensional collagen gels. Recombinant human VEGF(121) or VEGF(165) were added to the culture medium for 48 h, and vascular growth was visualized by immunostaining with a quail-specific endothelial cell (EC) marker, QH1. VEGF(165) enhanced vascular growth in a dose-dependent manner: 5 ng/ml of VEGF(165) slightly increased the number of ECs, 10 ng/ml of VEGF(165) increased the incorporation of ECs into tubular structures, and at 20 ng/ml of VEGF(165) wider tubes were formed. This pattern plateaued at the 50 ng/ml dose. In contrast, VEGF(121) did not enhance either the number of ECs or tube formation at these or higher dosages. Combined effects of hypoxia and exogenous VEGF(165) were then compared. Tube formation from the heart explants treated with both hypoxia and 50 ng/ml of VEGF(165) had a morphology intermediate to those treated with hypoxia or VEGF(165) alone. Immunocytochemistry study revealed EC lumenization under all culture conditions. However, the addition of VEGF(165) stimulated the coalescence of ECs to form larger vessels. We conclude the following: 1) VEGF(121) and VEGF(165) induced by hypoxia have different functions on coronary vascular growth, 2) unknown factors induced by hypoxia can modify the effect of VEGF(165), and 3) EC lumenization observed in the heart explant culture closely mimics in vivo coronary vasculogenesis.
Assuntos
Circulação Coronária/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Linfocinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Embrião de Galinha , Endotélio Vascular/ultraestrutura , Coração/embriologia , Hipóxia/fisiopatologia , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Oxigênio/farmacologia , Codorniz , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
To test the hypotheses that cyclic stretch of 1) cardiac myocytes produces factors that trigger angiogenic events in coronary microvascular endothelial cells (CMEC) and 2) CMEC enhances the expression of growth factors, cardiac myocytes and CMEC were subjected to cyclic stretch in a Flexercell Strain Unit. Vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor mRNA and protein levels increased approximately twofold in myocytes after 1 h of stretch. CMEC DNA synthesis increased approximately twofold when conditioned medium from stretched myocytes or VEGF protein was added, and addition of VEGF neutralizing antibody blocked the increase. CMEC migration and tube formation increased with the addition of conditioned media but were markedly attenuated by VEGF neutralizing antibody. Myocyte transforming growth factor-beta [corrected] (TGF-beta) increased 2.5-fold after 1 h of stretch, and the addition of TGF-beta neutralizing antibodies inhibited the stretch-induced upregulation of VEGF. Stretch of CMEC increased VEGF mRNA in these cells (determined by Northern blot and RT-PCR) and increased the levels of VEGF protein (determined by ELISA analysis) in the conditioned media. Therefore, cyclic stretch of cardiac myocytes and CMEC appears to be an important primary stimulus for coronary angiogenesis through both paracrine and autocrine VEGF pathways. These data indicate that 1) CMEC DNA synthesis, migration, and tube formation are increased in response to VEGF secreted from stretched cardiac myocytes; 2) VEGF in CMEC subjected to stretch is upregulated and secreted; and 3) TGF-beta signaling may regulate VEGF expression in cardiac myocytes.
Assuntos
Vasos Coronários/fisiologia , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/citologia , Linfocinas/metabolismo , Neovascularização Fisiológica/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Anticorpos/farmacologia , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/fisiologia , Northern Blotting , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Vasos Coronários/citologia , Meios de Cultivo Condicionados/farmacologia , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/imunologia , Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica/fisiologia , Linfocinas/genética , Linfocinas/imunologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Testes de Neutralização , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Angiogenesis, the formation of vessels from pre-existing vessels, is of critical importance not only during normal growth, but also in pathological situations. In the latter, some diseases are enhanced by excessive vascular growth (e.g., tumors), whereas in others inadequate vascular growth contributes to morbidity and mortality (e. g., ischemic heart disease). Our current state of knowledge makes it clear that the cascade of angiogenic events depends on complex processes that include cell-cell interactions, various intracellular signaling pathways, and the appropriate extracellular microenvironment. The literature regarding angiogenesis has increased exponentially during the last decade. Progress in this area is largely a consequence of advances in our understanding of angiogenic growth factor and cytokine function, in part due to the determination of their complete amino acid sequences and cloning of their genes. Other factors also play key roles in angiogenesis, including the extracellular matrix, adhesion molecules and their inhibitors, and metabolic and mechanical factors. The potential for developing therapeutic protocols has been enhanced by data from both in vitro and in vivo studies and has provided the rationale for clinic trials. Angiogenic therapy strategies include inhibition of aberrant angiogenesis, as seen in tumors or diabetes, as well as stimulation of angiogenesis in conditions of ischemia, such as ischemic heart or peripheral vascular disease. Anat Rec (New Anat) 261:126-135, 2000.
Assuntos
Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Indutores da Angiogênese/farmacologia , Indutores da Angiogênese/uso terapêutico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Humanos , Isquemia Miocárdica/tratamento farmacológico , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacosRESUMO
Hypoxia is known to stimulate vascular growth by up-regulating vascular endothelial growth factor (VEGF), but little is known about the function of hypoxia in the development of the coronary vasculature, and the relationship between hypoxia and VEGF in this event. To test the effects of hypoxia and VEGF on coronary vasculogenesis/angiogenesis in the developing heart, ventricles from 6-day-old quail embryos were cultured on three-dimensional collagen gels. After 2 days of growth in normal medium and 1 day of starvation in low serum medium (0.5% fetal bovine serum), the heart explants were further cultured under various oxygen levels for another 24, 48, and 72 hr. Angioblasts and endothelial cells, which migrated out from the heart explants, were identified by QH1 antibody using immunofluorescence and confocal microscopy. In the normoxic culture environment, the endothelial cells began to proliferate and migrate out from the heart explants after 3 days of growth; they formed tubes mainly after another 72 hr. In contrast, this vascular growth was accelerated under hypoxic conditions, as evidenced by increased tube formation with significant differences observed at 48 hr. On the other hand, hyperoxia delayed this process. Reverse transcription-polymerase chain reaction results indicated that VEGF (including VEGF(122), VEGF(166), and VEGF(190)) was up-regulated in the heart explants under hypoxia and down-regulated under hyperoxia. VEGF neutralizing antibody added to the culture medium partially blocked this vascular growth. We conclude from this study that hypoxia can stimulate or up-regulate coronary vasculogenesis/angiogenesis and that VEGF signaling plays a major role in this event. Dev Dyn 1999;216:28-36.
Assuntos
Vasos Coronários/embriologia , Coração/embriologia , Hipóxia/fisiopatologia , Neovascularização Fisiológica , Animais , Sequência de Bases , Bovinos , Coturnix , Primers do DNA/genética , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/fisiologia , Expressão Gênica , Hipóxia/genética , Linfocinas/antagonistas & inibidores , Linfocinas/genética , Linfocinas/fisiologia , Testes de Neutralização , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: We tested the hypothesis that the degree of coronary microvessel formation in the embryonic heart is regulated by the magnitude of myocardial growth. METHODS: The outflow tract of Hamburger-Hamilton stage 21 chicken hearts (prior to the onset of coronary vasculogenesis) was constricted in ovo with a loop of 10-0-nylon suture, and the hearts were studied at stages 29 and 36. RESULTS: At stage 29 ventricular mass was 64% greater in the pressure-overloaded than in the hearts of sham-operated controls, but vascular volume density and numerical density, determined by electron microscopic morphometry, were identical. As demonstrated by histological morphometric evaluation, the compact region of the left ventricle at stage 29 was 43% thicker than the shams. However, by stage 36 heart mass, thickness of the compact region, and overall wall thickness (demonstrated by scanning electron microscopy) were significantly less than in the sham group of this stage, but vascular volume density was virtually identical in the two groups. Formation of the two main coronary arteries was clearly impeded in the banded hearts, i.e., the coronaries were stunted in their development or failed to completely form coronary ostia. CONCLUSIONS: Vascular growth is proportional to myocardial growth in the embryonic, overloaded heart, but the persistence of the pressure overload results in a failure of or severe limitations in coronary artery development. These data support the hypothesis that vascular growth during this period of development is regulated, at least in part, by the rate and magnitude of myocardial growth.
Assuntos
Embrião de Galinha/fisiologia , Circulação Coronária/fisiologia , Coração/embriologia , Neovascularização Fisiológica , Animais , Vasos Coronários/embriologia , Ventrículos do Coração/embriologia , Microcirculação , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Pressão VentricularRESUMO
A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily. Heart rate decreased by 32% for 20 to 40 minutes after injection and was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Arterial pressure and cardiac output were unchanged. Left ventricular capillary length density (mm/mm(3)) increased gradually with alinidine administration; a 15% increase after 2 weeks and a 40% increase after 3 weeks of alinidine treatment were documented. Left ventricular weight, body weight, and their ratio were not significantly altered by alinidine treatment. After 1 week of treatment, before an increase in capillary length density, VEGF mRNA increased >2-fold and then declined to control levels after 3 weeks of treatment. VEGF protein was higher in alinidine-treated rats than in controls after 2 weeks and increased further after 3 weeks of treatment. Injection of VEGF-neutralizing antibodies over a 2-week period completely blocked alinidine-stimulated angiogenesis. In contrast, bFGF mRNA was not altered by alinidine treatment. These data suggest that VEGF plays a key role in the angiogenic response that occurs with chronic bradycardia. The mechanism underlying this VEGF-associated angiogenesis may be an increase in stretch due to enhanced diastolic filling.
Assuntos
Bradicardia/fisiopatologia , Fatores de Crescimento Endotelial/genética , Linfocinas/genética , Neovascularização Fisiológica/fisiologia , Doença Aguda , Animais , Antiarrítmicos/farmacologia , Anticorpos/farmacologia , Northern Blotting , Western Blotting , Peso Corporal , Bradicardia/induzido quimicamente , Capilares/química , Capilares/fisiologia , Clonidina/análogos & derivados , Clonidina/farmacologia , Vasos Coronários/química , Vasos Coronários/fisiologia , Fatores de Crescimento Endotelial/análise , Fatores de Crescimento Endotelial/imunologia , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/imunologia , Expressão Gênica/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Linfocinas/análise , Linfocinas/imunologia , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Função Ventricular EsquerdaRESUMO
Vascular endothelial growth factor (VEGF) plays an important role in early embryonic vasculogenesis. To establish its temporal expression and localization in the heart during development, we studied rat hearts from the first embryonic day (E) of myocardial vascular tube formation through the early postnatal period. Ventricular VEGF immunoreactivity was noted in the epicardium and the thin underlying myocardium in E10 ventricles. During the earliest stages of vascularization (E13-E16) immunoreactivity was highest in the compact myocardium nearest the epicardium, and subsequently (E18 and thereafter) became more evenly distributed transmurally. By birth (E22) immunoreactivity was most intense around microvessels. Similarly, VEGF mRNA localization, demonstrated by in situ hybridization, was initially highest near the epicardium and then became more evenly distributed transmurally by late gestation. Within the interventricular septum, the highest expression occurred in the middle of the wall where it correlated with the greatest vascularization. Northern blot analysis showed that from E12 through the first 10 days of postnatal life, VEGF was two to three times higher than in the adult. Western blot analysis showed that VEGF tended to be higher in the atria than the ventricles, and negligible in the outflow tract. Our data indicate that VEGF localization and expression 1) correspond to the pattern of vascularization in the embryonic/fetal heart, and 2) remain high during the early postnatal period when capillary proliferation is high. Because VEGF is stimulated by hypoxia, its preferential mRNA expression near the epicardium, that is, farthest from the ventricular lumen and the O2 source, fits with the hypothesis that a hypoxic gradient is a driving force in the transmural vascularization process.
Assuntos
Sistema Cardiovascular/embriologia , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Neovascularização Fisiológica , Fatores Etários , Animais , Sistema Cardiovascular/anatomia & histologia , Fatores de Crescimento Endotelial/fisiologia , Linfocinas/fisiologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Distribuição Tecidual , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Myocardial hypertrophy is one of the basic mechanisms by which the heart compensates for hemodynamic overload. The mechanisms by which hemodynamic overload is transduced by the cardiac muscle cell and translated into cardiac hypertrophy are not completely understood. Candidates include activation of the renin-angiotensin system (RAS) and angiotensin II receptor (AT1) stimulation. In this study, we tested the hypothesis that load, independent of the RAS, is sufficient to stimulate cardiac growth. Four groups of cats were studied: 14 normal controls, 20 pulmonary artery-banded (PAB) cats, 7 PAB cats in whom the AT1 was concomitantly and continuously blocked with losartan, and 8 PAB cats in whom the angiotensin-converting enzyme (ACE) was concomitantly and continuously blocked with captopril. Losartan cats had at least a one-log order increase in the ED50 of the blood pressure response to angiotensin II infusion. Right ventricular (RV) hypertrophy was assessed using the RV mass-to-body weight ratio and ventricular cardiocyte size. RV hemodynamic overload was assessed by measuring RV systolic and diastolic pressures. Neither the extent of RV pressure overload nor RV hypertrophy that resulted from PAB was affected by AT1 blockade with losartan or ACE inhibition with captopril. RV systolic pressure was increased from 21 +/- 3 mmHg in normals to 68 +/- 4 mmHg in PAB, 65 +/- 5 mmHg in PAB plus losartan and 62 +/- 3 mmHg in PAB plus captopril. RV-to-body weight ratio increased from 0.52 +/- 0.04 g/kg in normals to 1.11 +/- 0.06 g/kg in PAB, 1.06 +/- 0.06 g/kg in PAB plus losartan and 1.06 +/- 0.06 g/kg in PAB plus captopril. Thus 1) pharmacological modulation of the RAS with losartan and captopril did not change the extent of the hemodynamic overload or the hypertrophic response induced by PAB; 2) neither RAS activation nor angiotensin II receptor stimulation is an obligatory and necessary component of the signaling pathway that acts as an intermediary coupling load to the hypertrophic response; and 3) load, independent of the RAS, is capable of stimulating cardiac growth.
Assuntos
Hipertensão/complicações , Hipertrofia Ventricular Direita/etiologia , Sistema Renina-Angiotensina/fisiologia , Angiotensina II/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Gatos , Constrição Patológica , Elasticidade/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Hipertensão/fisiopatologia , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/fisiopatologia , Losartan/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Artéria Pulmonar/fisiopatologiaRESUMO
We tested the hypothesis that early vascularization of the embryonic heart is enhanced after bolus injections of vascular, endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) into the vitelline vein before the onset of myocardial vasculogenesis (3.5 days, stage 21). Electron and light microscopy were utilized to obtain morphometric data. At stages 29 and 31, myocardial vessel volume or numerical density were higher in embryos injected with 50 ng bFGF than in the saline-injected controls. A VEGF injection increased vascular volume density at stage 29 and both volume and numerical, density at stage 31, bFGF, but not VEGF, was associated with an enhancement of the sinusoidal system (spongy layer of the ventricle) at stage 29. This effect disappeared by stage 31. In conclusion, 1) enhancement of bFGF or VEGF before myocardial vascularization increases vascular growth, but the initial effect of bFGF is greater; 2) the effects of these growth factors on vascular volume and numerical density are temporally dependent; and 3) bFGF, in addition to its effects on the coronary vasculature, influences ventricular modeling by apparently acting on myocytes as well as endothelial cells.
Assuntos
Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiologia , Fatores de Crescimento Endotelial/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Coração/embriologia , Linfocinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Embrião de Galinha , Vasos Coronários/embriologia , Coração/fisiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
In view of the evidence that thyroid hormone administration has angiogenic effects on the hypertrophic myocardium, we tested the hypothesis that the capillary supply in the hypertrophic myocardium surviving infarction would be improved by administration of the thyroid hormone analog, diiodothyroproprionic acid (DITPA). We administered DITPA (MI-DITPA) or saline (MI-saline), s.c., to rats for 10 days following experimental infarction of the left ventricle (LV). Morphometric methods were used to assess capillarity and myocyte cross-sectional area in three regions of the left ventricle: (1) border (next to the scar of infarction); (2) adjacent (next to the border); and (3) remote (interventricular septum). Infarct size ranged from 20-85% of the LV free-wall, and both groups had similar mean infarct size. Capillary length density (LV) was significantly higher in the remote region of the treated group than in the MI-saline rats. LV in the border region, which experienced the most marked increase in cardiocyte cross-sectional area, was not significantly lower than in the other regions, indicating a more marked angiogenic response. In hearts with large infarcts (> or = 40%) LV in the border region was higher in the DITPA group than in the non-treated rats. In the MI-DITPA group, cardiocyte size in the border region was positively correlated with that of the other regions, which contrasts with the negative correlations noted for the MI-saline rats. These data suggest that DITPA therapy (1) may improve maximal perfusion potential of the hypertrophied myocardium surviving a myocardial infarction, and (2) is selectively effective in the border region of hearts with large infarcts.
Assuntos
Di-Iodotironinas/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Propionatos/uso terapêutico , Disfunção Ventricular/tratamento farmacológico , Análise de Variância , Animais , Masculino , Ratos , Ratos Sprague-Dawley , Estimulação QuímicaRESUMO
BACKGROUND: Thyroid hormone-induced cardiac hypertrophy is a model of enhanced physiological growth and angiogenesis. This study addressed the growth and geometry of the capillary bed in relation to the development of cardiac hypertrophy. METHODS: Thyroxine was administered daily (0.2 mg/kg, s.c.) for 5 or 10 days to Fischer 344 rats. After obtaining ventricular function and hemodynamic data, the hearts were perfuse fixed, and specimens from the left ventricle (LV) were subjected to image analysis to determine indices of capillary growth. RESULTS: After 5 days of treatment, prior to cardiac enlargement, capillary length density was significantly greater in the epimyocardium of the thyroxine rats than in the controls (saline injected). Most of the increase could be attributed to an increase in capillary numerical density, but some enhancement in capillary profile axial ratio suggests that enhanced tortuosity or formation of oblique channels also occurred. After 10 days of treatment, all capillary parameters (length, volume, and surface densities) were similar to the controls despite a 30% enlargement of the LV. We estimate that total LV capillary length increased by 14% during the first 5 days and by 9% during the next 5 days of treatment. CONCLUSIONS: These findings indicate that capillary angiogenesis precedes the development of ventricular enlargement due to thyroxine administration. Therefore, angiogenesis in this model is not stimulated by the presence of hypertrophy.
Assuntos
Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Coração/efeitos dos fármacos , Coração/crescimento & desenvolvimento , Tiroxina/farmacologia , Animais , Capilares/fisiologia , Cardiomegalia/induzido quimicamente , Coração/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Masculino , Neovascularização Fisiológica/fisiologia , Ratos , Ratos Endogâmicos F344RESUMO
Although a substantial coronary angiogenesis occurs after thyroid hormone treatment, its regulation and relationship to cardiac hypertrophy are not understood. This study was designed to determine (1) the onset of capillary proliferation, (2) the sites of capillary proliferation, and (3) whether basic fibroblast growth factor (bFGF) upregulation occurs in response to thyroxine administration. Male Sprague-Dawley rats were injected daily with L-thyroxine (T4, 0.2 mg/kg s.c.). Bromodeoxyuridine labeling of capillary endothelial cells increased during the first 24 hours of treatment and peaked after 2 days of treatment. Northern blot analysis revealed a slight increase in bFGF mRNA during this period, followed by a doubling of expression by 48 hours, at which time bFGF protein was also increased. In situ hybridization, used to localize bFGF mRNA, showed an increase in transcripts within 24 hours after T4. This enhancement was uniform in the epimyocardium and endomyocardium. Histochemical analysis (double staining for alkaline phosphatase and dipeptidyl peptidase) of frozen sections, used to discriminate capillary profiles as arteriolar and venular, respectively, showed that growth occurred in the latter, since the percentage of capillary profiles positive for dipeptidyl peptidase was higher than the control value after 4 days of T4 administration. These data indicate that in the thyroxine model of cardiac hypertrophy (1) capillary DNA synthesis occurs after a single injection of thyroxine, (2) capillary growth coincides with an upregulation in bFGF mRNA and increase in bFGF protein, and (3) proliferation occurs in the venular capillaries.
Assuntos
Vasos Coronários/fisiologia , Fator 2 de Crescimento de Fibroblastos/genética , Neovascularização Fisiológica/efeitos dos fármacos , Tiroxina/farmacologia , Animais , Capilares/citologia , Capilares/fisiologia , Vasos Coronários/química , Fator 2 de Crescimento de Fibroblastos/análise , Expressão Gênica/efeitos dos fármacos , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hibridização In Situ , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The main goal of this study was to determine the temporal and spatial relationship of several components of the extracellular matrix (ECM) to coronary vascularization during prenatal and early postnatal development. Rat microvessels were visualized by immunolabeling for platelet endothelial cell adhesion molecule (PECAM-1), and by exposure to the lectin from Griffonia simplicifolia I. Coronary vasculogenesis, which first occurs in gestation day 13 (E13) hearts, was preceded by the deposition of fibronectin. The onset of laminin immunoreactivity in basement membranes coincided with tube formation and was followed by the appearance of collagen IV. Discontinuous collagen IV staining of basement membranes typified early tube formation but progressed to completely encircle capillaries. Sparse staining of collagen I and III was observed in prenatal hearts, but increased after birth. Staining for both molecules was limited mainly to the adventitia of vessels larger than capillaries, and as a component of septa and the epicardium. To determine the effects of loading conditions on key ECM molecules relating to neovascularization, avascular E12 rat hearts were grafted to the anterior eye chamber of adult hosts. In these hearts, which are hemodynamically unloaded, the appearance and distribution of ECM components were similar to hearts developing in utero. It was concluded that during heart development: (1) fibronectin may provide a primary scaffolding for the migration of primordial endothelial cells/angioblasts; (2) tube formation coincides with lamin deposition and is closely followed by the appearance of collagen IV; (3) collagens I and III are not related to tube formation in the prenatal heart; and (4) the relationship of the ECM to vessel formation is not notably altered in the absence of a ventricular load. Furthermore the early onset of PECAM-1 immunoreactivity suggests that it is a useful endothelial marker and may play a role in tube formation.
Assuntos
Vasos Coronários/fisiologia , Matriz Extracelular/fisiologia , Coração/crescimento & desenvolvimento , Neovascularização Fisiológica/fisiologia , Animais , Endotélio Vascular/química , Proteínas da Matriz Extracelular/análise , Coração/embriologia , Coração/fisiologia , Hemodinâmica , Lectinas/farmacologia , Microcirculação , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Our overall aims were to elucidate the temporal and spatial sequence of coronary vascularization during development in the rat, and to determine whether basic fibroblast growth factor expression corresponds to any phase of the vascularization process. METHODS: Immunohistochemical, histochemical, morphometric and in situ hybridization analyses were performed on prenatal and postnatal hearts of various ages. RESULTS: Coronary vascularization, which begins at embryonic day 13 (E13) with blood island-like structures in the epicardium, progresses from this layer toward the endocardium as indicated by a transmural gradient of vascular volume throughout the ventricles. Vascular smooth muscle first appears in E17 hearts at the time a capillary-like plexus coalesces and penetrates the aorta to form the main coronary arteries. These vessels maintain an anastomatic morphology and must undergo subsequent remodeling in order to assume adult branching characteristics. The early postnatal period is characterized by development of the arterial tree and the enzymatic differentiation of the arteriolar and venular ends of the capillary bed. Although bFGF is expressed both prenatally and postnatally, the highest mRNA expression was noted during the early period of vascularization (E14 and E15), and the early neonatal period (1-6 days) which corresponds to a period of substantial microvascular growth. CONCLUSIONS: Coronary vascularization follows a temporal sequence which includes transmural expansion of the capillary bed, arteriolar formation subsequent to vascular penetration of the aorta, and postnatal growth, differentiation, and remodeling. Since high levels of bFGF expression are correlated with key time points in coronary vascular growth, bFGF may play an important role in this process.
Assuntos
Vasos Coronários/embriologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Fosfatase Alcalina/análise , Animais , Capilares/embriologia , Capilares/enzimologia , Vasos Coronários/química , Dipeptidil Peptidases e Tripeptidil Peptidases/análise , Endocárdio/embriologia , Fator 2 de Crescimento de Fibroblastos/análise , Imuno-Histoquímica , Hibridização In Situ , Pericárdio/embriologia , Ratos , Ratos Sprague-DawleyRESUMO
Myocardial vascularization is initiated after endothelial cell precursors from the region of the liver or septum transversarium migrate to the newly formed epicardium. Blood island-like structures appear and form vascular channels along the epicardium and into the myocardium. Prior to this time myocardial cells receive nutrition directly from the ventricular lumen, a process which is facilitated by the highly trabecular arrangement of the ventricles. A venous system is formed prior to any evidence of arteries or arterioles. The formation of a vascular plexus in the region of the outflow tract is followed by penetration of these microvessels into the wall of the aorta. These ingrowing vessels merge, acquire a muscular coat and form the left and then the right coronary vessels. These events occur during a short period of time, e.g., 2 weeks in humans, 4 days in rats. Maturation of the arterial tree occurs primarily after birth. Our knowledge of the regulation of coronary vascularization during development is very limited. Accordingly, future studies need to focus on the role of growth factors, chemotactic factors, extracellular matrix molecules, and mechanical events.
Assuntos
Vasos Coronários/embriologia , Endotélio Vascular/embriologia , Animais , Artérias , Capilares , Humanos , VeiasRESUMO
We have shown that sympathetic denervation increases subendocardial capillarity during left ventricular hypertrophy. To determine the direct effects of sympathetic innervation on development of the coronary microvasculature in the absence of hemodynamic load, we grafted avascular fetal rat atrial or ventricular tissue into the anterior eye chamber of host rats which had undergone unilateral superior cervical gangliectomies. Innervation to the contralateral eye chamber remained intact. The grafts were harvested 14 or 35 days later, and volume densities of blood vessels, myocytes, and extracellular matrix were determined using standard point-counting techniques on low-power electron micrographs. Graft perfusion and metabolism were assessed simultaneously with thallium-201 and 2-[14C]deoxy-D-glucose uptake, respectively. Innervation did not significantly alter the vascular volume densities or cellular composition of atrial or ventricular tissue compared with noninnervated tissue after either 14 or 35 days in oculo. Similarly, innervation did not significantly alter graft perfusion or metabolism. We conclude that sympathetic innervation does not directly influence the growth of the microvasculature or myocardial metabolism in hemodynamically unloaded, developing heart tissue.
Assuntos
Olho , Coração Fetal/transplante , Sistema de Condução Cardíaco/fisiologia , Transplante de Coração , Neovascularização Fisiológica/fisiologia , Transplante Heterotópico , Animais , Desoxiglucose/farmacocinética , Coração Fetal/inervação , Coração Fetal/metabolismo , Átrios do Coração , Sistema de Condução Cardíaco/embriologia , Ventrículos do Coração , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/fisiologiaRESUMO
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate endothelial cell proliferation, migration, and vascular tube formation. We tested the hypotheses that these growth factors stimulate (1) cell migration and (2) assembly into cord-like structures in embryonic rat heart explants cultured on collagen gels. Atrial and ventricular explants from rat embryos at 12 (E12, avascular) and 14 (E14, early vascularization stage) days of gestation were cultured on a collagen substrate. Western blot analysis of the explants indicated that endogenous VEGF was present in both atria and ventricles during incubation. Addition of bFGF to E12 explants markedly increased cell migration, whereas VEGF had no significant effect. In E14 explants neither growth factor influenced cell migration. Cotreatment with VEGF and bFGF did not have a synergistic effect on the migration distance of cells from either E12 or E14 embryonic hearts. However, VEGF stimulated the appearance of cord-like structures in E14, but not E12, explants. Transmission electron microscopy analysis showed that these cord-like structures consist of elongated cells, some of which aggregate into clusters, or form tube-like structures, similar to capillaries. Serial sections of monolayers revealed that tube formation occurs beneath the surface of collagen gel. We conclude that in this model system VEGF and bFGF play distinct roles, at specific time points, in coronary vascular tube formation in the developing heart.