Parsons-Wingerter P, Lwai B, Yang M C, Elliott K E, Milaninia A, Redlitz A, Clark J I, Sage E H
Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington 98195-7420, USA.
Microvasc Res. 1998 May;55(3):201-14. doi: 10.1006/mvre.1998.2073.
In a novel assay of angiogenesis in the quail chorioallantoic membrane (CAM), we measured vascular pattern and angiogenic rate after homogeneous exposure of the entire vascular tree to recognized modulators of vessel growth. In comparison to phosphate-buffered saline (PBS)-treated controls, the vascular stimulator, basic fibroblast growth factor (bFGF or FGF-2), increased the rate of angiogenesis by a maximum of 72%, whereas a recently discovered angiogenic inhibitor, angiostatin, decreased the rate of vascular growth by a maximum of 68%. The perturbants were applied in PBS to the CAM of 7-day-old embryos (E7) cultured in petri dishes, and the embryos were cultured further until fixation at E8 or E9. For morphometry of the quasi-two-dimensional CAM vasculature, digital images of arterial endpoints from the middle region of the CAM were acquired in grayscale at a magnification of 10x, binarized to black/white, and skeletonized. The pattern of vessel branching was assessed by measurement of the fractal dimension (Df), and vessel density (rhov), with the method of grid intersection. Correlations between these two statistical techniques were linear (r2 ranged from 0.967 to 0.985). For skeletonized images at E9, Df and rhov of bFGF-treated samples were 1.55 +/- 0.01 and 782 +/- 26/cm2, respectively (relative to 1.49 +/- 0.02 and 583 +/- 60/cm2 for controls), and of angiostatin-treated samples, 1.43 +/- 0.02 and 424 +/- 74/cm2 (relative to 1.50 +/- 0.02 and 616 +/- 59/cm2 for controls). To establish normalization values for rates of angiogenesis, we analyzed untreated CAMs of E6 to E12. From E7 to E10 in skeletonized images, Df increased linearly from 1.37 +/- 0.01 to 1.54 +/- 0.01 and rhov from 311 +/- 67 to 746 +/- 124/cm2 (in both cases, r2 = 1.000). Thus, the rates of normal angiogenic growth as measured by Df and rhov were 0.06/day and 138/cm2-day, respectively. From E10 to E12, Df and rhov declined slightly. Differences between the vasculature of untreated and PBS-treated CAMs were statistically insignificant. In conclusion, vascular branching pattern and density in the quail CAM were stimulated by bFGF and inhibited by angiostatin. We quantified these changes with statistical significance by Df and rhov, which are expressed relative to the rates of normal developmental angiogenesis measured for the two parameters in untreated quail embryos.
在一项针对鹌鹑绒毛尿囊膜（CAM）血管生成的新型检测方法中，我们在整个血管树均匀暴露于公认的血管生长调节剂后，测量了血管模式和血管生成速率。与磷酸盐缓冲盐水（PBS）处理的对照组相比，血管刺激剂碱性成纤维细胞生长因子（bFGF或FGF - 2）使血管生成速率最高增加了72%，而最近发现的血管生成抑制剂血管抑素使血管生长速率最高降低了68%。将这些干扰物用PBS施加到培养在培养皿中的7日龄胚胎（E7）的CAM上，然后将胚胎进一步培养至E8或E9固定。为了对近似二维的CAM脉管系统进行形态测量，从CAM中部区域获取动脉端点的数字图像，以10倍放大率获取灰度图像，二值化为黑白图像并进行骨架化处理。通过分形维数（Df）和血管密度（rhov）的测量，采用网格交叉法评估血管分支模式。这两种统计技术之间的相关性呈线性（r2范围为0.967至0.985）。对于E9时的骨架化图像，bFGF处理样本的Df和rhov分别为1.55±0.01和782±26/cm2（相对于对照组的1.49±0.02和583±60/cm2），血管抑素处理样本的Df和rhov分别为1.43±0.02和424±74/cm2（相对于对照组的1.50±0.02和616±59/cm2）。为了确定血管生成速率的标准化值，我们分析了E6至E12未处理的CAM。在骨架化图像中，从E7到E10，Df从1.37±0.01线性增加到1.54±0.01，rhov从311±67增加到746±124/cm2（在这两种情况下，r2 = 1.000）。因此，通过Df和rhov测量的正常血管生成生长速率分别为0.06/天和138/cm2 - 天。从E10到E12，Df和rhov略有下降。未处理和PBS处理CAM的脉管系统之间的差异无统计学意义。总之，鹌鹑CAM中的血管分支模式和密度受到bFGF的刺激和血管抑素的抑制。我们通过Df和rhov对这些变化进行了具有统计学意义的量化，它们相对于未处理鹌鹑胚胎中这两个参数测量的正常发育血管生成速率来表示。
