We demonstrated previously that VEGF-induced proliferation of HPVEC is mediated by VEGF-R2-induced NFATc1 translocation into the nucleus (Johnson et al., [2003]). Therefore, we tested whether AAC 789 and PTK 787 could block VEGF-induced NFATc1 nuclear translocation in HPVEC. Pretreatment with either inhibitor, for one hour prior to VEGF stimulation, effectively blocked NFATc1 nuclear translocation (Fig. 1). These results showed that the receptor tyrosine kinase inhibitors disrupt VEGF-mediated NFATc1 nuclear translocation, and therefore would be useful for probing events downstream of VEGF-Rs. For comparison, FK506 was tested in parallel and, as expected, blocked VEGF-induced nuclear translocation of NFATc1 (Fig. 1). (FK506 is an immunosuppresant drug that inhibits the phosphatase activity of calcineurin, which in turn blocks de-phosphorylation and nuclear translocation of NFATs.) The tyrosine kinase domain between human and zebrafish VEGF-R2/KDR is highly conserved (Habeck et al., [2002]). In addition, PTK 787 has been shown to block vascular development in zebrafish embryos (Chan et al., [2002]; Lee et al., [2002]) and VEGF-induced autophoshorylation of zebrafish VEGF-R2/flk-1(Chan et al., [2002]). Hence, we used PTK 787, and additionally AAC 789, to test our hypothesis that signaling through VEGF-Rs is required for valve development.
PTK 787 or AAC 789 Cause Toggling of Blood Within the Hearts of Zebrafish Embryos
To avoid pleiotropic effects on vascular development, zebrafish were treated with 5 M AAC 789 or 5 M PTK 787 beginning at 17 hpf (15 somite stage) when heart development is already underway. The inhibitors were removed after 4, 12, or 24 hr, and embryos were allowed to continue their growth until 48 hpf, a time when valve formation is normally complete (Walsh and Stainier, [2001]). Treatment with either inhibitor, individually, for the three time periods resulted in toggling of blood between the atrial and ventricular chambers of the heart. To visualize this defect in real time, video microscopy was performed on live embryos [see Supplementary Movie S1, control embryo (note directional flow of blood through the heart and robust circulation); Supplementary Movie S2, embryo treated for 4 hr with AAC 789; Supplementary Movie S3, embryo treated for 4 hr with PTK 787, which are available online at www.interscience.wiley.com/jpages/1058-8388/suppmat]. The toggling of blood seen in the VEGF-R-tyrosine kinase inhibitor-treated embryos represents ineffective and non-directional blood flow through the heart chambers. The phenotype of the AAC 789- and PTK 787-treated embryos was strongly reminiscent of the jekyll mutation in which the cardiac valve fails to develop (Walsh and Stainier, [2001]). Not surprisingly, since NFATc1 is required for valve development in mice, FK506 (2 M) also induced toggling of blood in the hearts of zebrafish embryos (Supplementary Movie S4, embryo treated for 4 hr with FK506). Delaying the addition of PTK787 until 22 hpf resulted in normal embryos without evident toggling in the heart (data not shown) suggesting that VEGF-R signaling is most critical for valve development at the 15-18 somite stage, corresponding to 17-19 hpf. A dose-response experiment in which PTK 787 was tested at 1, 2, 5, 7.5, and 10 M revealed that PTK 787 caused toggling at 1 and 2 M, with nearly complete penetrance at 5 M. Hence, 5 M was used for all subsequent experiments, including those performed with AAC 789.
VEGF-R2/KDR is required for vasculogenesis and hematopoiesis (Eichmann et al., [1997]; Shalaby et al., [1997]). Thus, prolonged inhibition of VEGF-R signaling could inhibit hematopoiesis in zebrafish embryos, and thereby affect blood viscosity and shear forces in the developing vasculature. Intracardiac fluid forces have been shown to play an important epigenetic role in heart valve formation in zebrafish (Hove et al., [2003]). To test whether exposure to PTK 787 decreased blood cell number in the circulation, and thus potentially altered hemodynamic forces in the developing heart, embryos were stained with o-dianisidine to detect hemoglobinized blood (Fig. 3). Normal circulation throughout the heart and the ducts of Cuvier on the yolk sac in a control embryo at 48 hpf is shown in Figure 3a. At 17 hpf, embryos were treated with PTK-787 or FK506 for 4, 12, and 24 hr, washed thoroughly, and then allowed to develop until 48 hpf. Blood circulation and levels of hemoglobinized blood appeared normal except in embryos treated for 24 hr with PTK 787. Hence, gross alterations in the blood viscosity are unlikely to have contributed to the cardiac valve defect seen in embryos treated for 4 hr PTK 787 or with FK506.
Рис.3 | Effects of PTK 787 and FK506 on hemoglobinized blood levels and circulation. Embryos treated with PTK 787 and FK506 as described in Figure 2 were stained with o-dianisidine for circulating hemoglobinized red blood cells (Iuchi and Yamamoto, [1983]) in the yolk sac and heart (30). a: Control embryos at 48 hpf. b: PTK787 treatment for 4 hr had no effect on oxygenated blood levels and circulation. Twelve-hour (c) and 24-hr (d) treatment with PTK 787 resulted in decreased blood in yolk-sac and heart, especially in embryos treated for 24 hr (d). FK506 did not affect oxygenated blood levels or circulation after 4-hr, 12-hr, or 24-hr exposure (panels e, f, g, respectively).
PTK 787 and AAC 789 Alter Expression Patterns of notch 1b and bmp-4 in the AV Boundary Region
To investigate whether the toggling of blood in the heart is associated with defects in cell differentiation in the endocardial cushions, we examined expression of early markers of valve differentiation. The expression patterns of notch 1b (Westin and Lardelli, [1997]) and bmp-4 (Nikaido et al., [1997]) in the AV valve, and the endothelial marker, VEGFR2/flk-1, were examined by whole mount in situ hybridization. Notch 1b and bmp-4 were initially expressed throughout the anterio-posterior extent of the heart (data not shown), but then became restricted to the valve region (Fig. 4a and i), as reported previously (Westin and Lardelli, [1997]; Walsh and Stainier, [2001]). Notch 1b at 48 hpf was clearly restricted to the AV boundary of the endocardium in normal mock-treated embryos (Fig. 4a). In contrast, AAC789, PTK787, and FK506 induced dispersed and ectopic expression of Notch1b at the constriction and ventricular endocardium as well as weak atrial expression (Fig. 4b-d). FK506 caused a similar loss of cell-restricted expression of notch 1b in the valve region (Fig. 4d).
Рис.4 | Disruption of VEGF or calcineurin signaling results in altered expression of notch 1b and bmp4 in cardiac valves. In situ hybridization analysis of the endocardial marker notch 1b (a-d), the myocardial marker bmp-4 (i-l), and the endothelial marker flk-1 (q) in the AV valve region. a: notch 1b was restricted to the AV region of the endocardium in control embryos (see arrow). b: notch 1b was ectopically expressed beyond the AV boundary into the ventricle and weakly in the atrium of embryos treated with AAC 789. c,d: Similar ectopic expression of notch 1b in embryos treated with PTK 787 and FK506, respectively. e-h: Schematic figures for the expression patterns seen in a-d. i: bmp-4 expression was prominent in the in myocardial region of AV valve at 48 hpf in control embryos (see arrow). j: AAC 789 treatment for 4 hr resulted in strong ectopic expression of bmp-4, especially in the ventricle region. k,l: PTK787 and FK 506 induced similar mis-localized expression patterns. m-p: Schematic figures for expression patterns of bmp-4 seen i-l. q: Expression of flk-1 in the endocardium of control embryos at 48 hpf.
Bmp-4 expression was restricted to the myocardium of the AV boundary at 48 hpf zebrafish embryos (Fig. 4i), as reported previously (Walsh and Stainier, [2001]). AAC 789- and PTK 787-treated embryos showed dispersed expression of bmp-4 in the ventricle and to a lesser extent in the atrium (Fig. 4j and k). FK506-treated embryos displayed a similar diffuse pattern of bmp-4 expression (Fig. 4l). Expression of VEGF-R2/flk-1, the target of AAC 789 and PTK 787, is seen weakly throughout the endocardium of the developing heart at 48 hpf (Fig. 4q), consistent with previous reports (Thompson et al., [1998]). The defects in endocardial and myocardial patterning, as indicated by ectopic expression of both notch 1b and bmp-4, may disrupt cell-cell communication within the developing valve region and thereby disrupt cell fate decisions during valve development.
AAC 789 and PTK 787 Cause a Morphological Defect in the AV Boundary Region
We used three approaches to gain insight into the morphological defect caused by the small molecule inhibitors of VEGF-R signaling. The first approach was to use the signal generated by the notch1b in situ hybridization probe to highlight the AV boundary region in histological sections (Fig. 5A,C). In control embryos, notch 1b was detected in a constricted region at the base of the ventricle in a region consistent with the AV boundary (Fig. 5A). The small size of the embryos at 48 hpf made it difficult to find sections that included the atrium. In contrast, the notch 1b signal was dispersed throughout the ventricular region of embryos treated with AAC789 for 4 hr beginning at the 15 somite stage (Fig. 5C). This dispersed localization is consistent with results in Figure 4 and with dysregulated notch 1b expression observed in other published cases of morphological defects in zebrafish heart valve development (Walsh and Stainier, [2001]).
Рис.5 | Defects in the AV boundary region of AAC 789-treated embryos. Control embryos (A, B) and embryos treated with 5 M AAC 789 for 4 hr beginning 17 hpf (C, D) were analyzed by two methods. In situ hybridization for notch 1b was performed on whole mount embryos, which were then embedded in plastic resin, sectioned, and stained with eosin (A,C). Microangiography using FITC-Dextran was performed on embryos at 56 hpf (B, D). Fluorescence images were captured within 30 min of injection. a, atrium; v, ventricle. E-J: H&E stained histological sections of embryos untreated (E,H) or treated with 5 M PTK 787 for 4 hr beginning at 17 hpf (F, I) or with 2 M FK (G, J). Sagittal (E-G) or transverse (H-J) sections through embryos at 68 hpf are shown.
We also used micro-angiography to gain information about the morphological defect. Embryos were treated without or with AAC789 for 4 hr beginning at the 15 somite stage, washed to remove the inhibitor, and allowed to develop until approximately 50 hpf. The hearts of anesthetized embryos were micro-injected with FITC-dextran, and then observed and the images captured on a fluorescence microscope within 30 min. The FITC-dextran filled the heart and circulatory path of the embryo: this provided a striking view of the boundary between the atrial and ventricular chambers of the heart in control embryos (Fig. 5B). In contrast, the demarcation between the two heart chambers was absent in embryos treated with AAC-789 for 4 hr beginning at the 15 somite stage.
We also examined the endocardial cushion by H&E staining paraffin sections from control and treated embryos (Fig. 5E-J). Figure 5E-G shows sagittal sections and Figure 5H-J shows transverse sections through embryos sectioned at 68 hpf. The endocardial/myocardial cellular borders, as well as blood cells within the heart chamber, were seen in all embryos. In control embryos (Fig. 5E and H), cells with a distinct morphology were seen in the mid-region of the heart chamber perhaps indicating these are valvular cells. In PTK787-treated (Fig. 5F and I) and FK506- treated (Fig. 5G and J) embryos, there was a notable lack of cells in this region of the heart corresponding to the AV boundary; this was most apparent in the sagittal sections (see arrows). These histological findings are consistent with a lack of valvular development seen by microangiography (Fig. 5D). In summary, the results from these three types of morphological analyses revealed defective valve development in the AV boundary, consistent with the functional toggling defect seen in live embryos.
DISCUSSION
Химическое разрушение передачи сигналов VEGF-R у эмбрионов рыбок данио фенокопирует генетическое разрушение UDP-glucose dehydrogenase (Walsh and Stainier, [2001]). В обоих случаях, toggling крови, т.е. обратный заброс крови внутри сердца виден у живых эмбрионов, а потеря клеточно-ограниченной экспрессии notch-1b и bmp-4 выявляется с помощью in situ гибридизации. Walsh and Stainier полагают, что UDP-glucose dehyrogenase является важным предшественником для биосинтез агиалуроновой кислоты, т.к. сходные jekyll-подобные дефекты наблюдаются у мышей, дефицитных по hyaluronan synthase-2 (Camenisch et al., [2000]). Многие исследования показали, что VEGF модулирует биосинтез внеклеточного матрикса и в свою очередь ECM влияет на поведение клеток во время развития (Ortega et al., [1998]). Могут ли передача сигналов VEGF-R и функция биосинтеза гиалуроновой кислоты быть интегрированы во время развития клапанов, остается неизвестным.
TGF-β семейство, как было установлено, играет важные роли в морфогенезе эндокардиальных подушек (Harvey and Rosenthal, [1999]). TGFβ3 экспрессируется в эндокарде и вносит вклад в ремоделирование сердечных клапанов после EMT (Camenisch et al., [2002]). Недавно было показано, что TGFβ1 индуцирует EMT в клонированных популяциях из эндотелиальных клеток клапанов (Paranya et al., [2001]). TGFβ1 ? как было показано, увеличивает экспрессию endoglin (Sanchez-Elsner et al., [2002]), вспомогательного TGFβ рецептора, который важен для развития клапанов сердца (Arthur et al., [2000]). Усиление активности endoglin с помощью TGFβ1 сопровождается гипоксией, которая является вышестоящим регулятором VEGF, подтверждая тем самым взаимодействие между VEGF и TGFβ1 в генезе клапанов. Исходя из полученных здесь результатов и опубликованных данных, мы полагаем, что VEGF и TGFβ1 могут действовать вместе благодаря кооперативным взаимодействиям во время развития сердечных клапанов.
Мыши, дефицитные по VEGF-R2/flk-1 погибают in utero из-за отсутствия эндотелиальных и кровяных клеток (Shalaby et al., [1995]). Недавний анализ мутантов flk-1 рыбок данио показал, что flk-1 не является существенным для васкулогенеза до 36 hpf (Habeck et al., [2002]); однако, отсутствует ангиогенез межсегментных сосудов, центральных артерий и вен, которые снабжают кровью мозг, кишечник и грудные плавники. Дефекты в образовании клапанов сердца не отмечены. Средней выраженности фенотипы мутантных flk-1 рыбок данио указывают на присутствие второго VEGF рецептора у рыбок данио, который частично компенсирует потерю перекрывающейся изоформы (Habeck et al., [2002]). Следовательно, использование молекулярных ингибиторов, таких как AAC 789 и PTK787, подчеркивает уникальные преимущества устранения передачи сигналов через множественные, перекрывающиеся VEGF рецепторы. Такие ингибиторы, фактически, позволяют легко осуществить эксперимент по условному нокауту и тем самым выявить новые и специфические функции исследуемых белков. В самом деле, использование малых проникающих в клетки молекул, чтобы нарушить функцию специфических белков у рыбок данио, становится многообещающей областью химической геномики (Pichler et al., [2003]).
Итак, базируясь на полученных результатах, а также на предыдущих исследованиях (Dor et al., [2001]; Johnson et al., [2003]), мы полагаем, что в результате индукции ядерной транслокации NFATc1 у рыбок данио VEGF-R(s) регулируют гены, участвующие в пролиферации, миграции и дифференцировке эндокардиальных клеток в областях, формирующих клапаны сердца, в точно регулируемом пространственно-временном паттерне. Эти предположения согласуются с недавними находками, описанными Chang и др. (Chang et al., [2004]). В их исследовании генеза клапанов у мышей было показано, что передача сигналов NFAT в миокарде на ст. E9 супрессирует экспрессию VEGF, создавая условия, которые инициируют EMT. На ст. E11, передача сигналов NFAT в эндокарде вносит вклад в элонгацию створок клапанов и в дальнейшее ремоделирование, которое скорее всего использует пролиферацию, миграцию эндотелия и вообще дополнительный EMT. Этот сценарий согласуется с позитивной ролью VEGF в EMT, обнаруживаемой на ст. E10.5 (Hallaq et al., [2004]). В этом элегантном исследовании передача сигналов VEGFa в AV эксплантах от эмбрионов ст. E10.5, как было показано, способствует морфологическим изменениям в эндокардиальных клетках, активирует клеточную миграцию в коллагеновый гель и экспрессию гладкомышечного alpha-actin, маркера для клеток, подвергающихся EMT. Мы полагаем, что на этой последней стадии EMT, VEGF-обусловленная пролиферация эндотелиальных клеток необходима для пополнения эндотелиального монослоя развивающихся створок клапанов, т.к. до этого эндотелиальные клетки мигрировали в кардиальный гель, чтобы стать мезенхимными клетками. Без достаточной передачи сигналов VEGF переход EMT может остановиться из-за недостаточного количества эндотелиальных клеток. Следовательно, высокие уровни VEGF, особенно в начале EMT, ингибируют развитие клапанов, но слишком низкая передача сигналов VEGF, особенно на поздних сроках, когда начинаются пролиферация и миграция клеток, будет ограничивать развитие клапанов. Наши результаты представляют первое прямое доказательство in vivo , что передача сигналов VEGF-R необходима для развития кардиальных клапанов. Дальнейшие исследования необходимы для идентификации генетического разнообразия VEGF-R и NFAT изоформ у рыбок данио и для корреляции их гомологов с соотв. развитием у млекопитающих.
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