A novel tissue model for angiogenesis: evaluation of inhibitors or promoters in tissue level

Abstract

A novel tissue model for angiogenesis (TMA) is established for effective evaluation of angiogenesis inhibitors or promoters in vitro. Lung tissues were cultured in fibrinogen "sandwich" structure which resembled the formation of neovessels in vivo. The cells and capillary-like structures grew from the lung tissues were identified as endothelial cells and neovessels. Both immunohistochemisty and western blot results indicated that autocrine VEGF bound to the KDR and induced KDR autophosphorylation that could induce the proliferation of endothelial cells and their migration as well as the formation of microvessels on the lung tissue edge. With addition of the TMA, the murine VEGF and cultured medium produced by A549 tumor cells apparently promoted the increase of neovessels. Sorafenib as a tumor angiogenesis inhibitor and Tongxinluo as an angiogenesis promoter were both used to evaluate the TMA performance and they exhibited a good effect on neovessels in the TMA. The model established imitated angiogenesis in vivo and could well serve as an effective method in evaluating the angiogenesis inhibitors or promoters, and could also be practical for screening small molecules that affect blood vessel formation.

Figure 1. Established tissue model for angiogenesis.

(a) The image of cells grew from the mouse lung tissue. Arrows were placed to indicate round shape, “cobblestone” shape and spindle shape cells. Bar = 100 μm. (b) The image of capillary-like structures grew from the lung tissues. Bar = 500 μm. (c) Peripheral cells were identified by transmission electron microscopy. The image showed that the electron micrograph of a weibel-palade body which showed typical longitudinal striations. Bar = 500 nm. (d) Effect of conditioned medium on the TMA. A549 cells cultured supernatant was added to the “sandwich” structure. Addition to the conditioned medium, the number of vessels increased quicker compared with vessels in RPMI-1640 medium group. Values are expressed as means ± SEM. (n = 4). (e) CD34 staining of control. (f) Immunofluorescence micrograph of CD34-stained endothelial cells. The montage image showed the merged fluorescent signal illustrating the human epithelial cells were stained with CD34 marker (green) whereas nuclei were stained with DAPI (blue). Bar = 100 μm. (g) vWF staining of control. (h) Capillary-like structures were stained with vWF-FITC. The representative image showed that the capillary-like structures were labeled with the vWF (green). Bar = 500 μm.

Figure 2. Mechanism of the angiogenesis in the TMA.

Immunohistochemical staining of CD34, vWF, KDR and p-KDR showed that positive cells labeled brown of fresh lung tissue (0 day) and cultured 9 days lung tissue from the TMA. Representative immunostaining pictures are presented (a₁–d₃). Magnification: 200×. (e) Change of VEGF secretion in culture system supernatant. The supernatant was collected every other day and used to quantitative the VEGF. The expression of VEGF in supernatant was evidently increased along with the time went on. Values are expressed as means ± SEM. (n = 2). *P < 0.05, **P < 0.01 versus the 5th day group. (f) Effect of VEGF on the TMA. VEGF was added to the culture system, vessels grew quicker than ones in control group. Values are expressed as means ± SEM. (n = 4). (g) Quantification of protein expression from 4 sections in each group. Data were expressed as mean values ± SEM. *P < 0.05, **P < 0.01 versus the 0 day group. (h) Western blot analysis of CD34, vWF, KDR and p-KDR expression in fresh lung tissue (0 day) and cultured lung tissues in the TMA (cultured 9 days). Gels were run under the same experimental conditions and GAPDH is shown as a control. The cropped gels images are shown in and the full-length blots/gels are presented in Supplementary Figure 1. (i) Quantification of CD34 and vWF protein expression. Data were expressed as mean values ± SEM. *P < 0.05, **P < 0.01 versus the 0 day group. (j) Quantification of p-KDR protein expression. Data were expressed as mean values ± SEM. *P < 0.05 versus the 0 day group.

Figure 3. Sorafenib inhibits angiogenesis and Tongxinluo capsule promotes angiogenesis in the TMA.

(a₁) The representative images of lung tissue blood vessels in the untreated group on the 9th day; a₂–a₄: lung tissue vessels in the sorafenib-treated group on the 9th day; (a₂) 20 nM, (a₃) 100 nM, (a₄) 500 nM. Vessels grew normally in control group; vessels in the sorafenib (20, 100, 500 nM)-treated group exhibited the slowly increase compared with the control group. Bar = 500 μm. (b₁) The representative images of lung tissue blood vessels in the untreated group on the 5th day; b₂–b₄: lung tissue vessels in the sorafenib-treated group on the 9th day; (b₂) 1 h serum, (b₃) 2 h serum, (b₄) 3 h serum. (c) Quantification of microvessels numbers in sorafenib treated group with the increased days. (n = 3) (d) Quantification of vessels length in sorafenib treated group along with the time prolonged. Data were expressed as mean values ± SEM. (n = 7). *P < 0.05, **P < 0.01 versus the control group. (e) Quantification of microvessels numbers in Tongxinluo capsule treated group with the increased days. (n = 3) (f) Quantification of vessels length in Tongxinluo capsule treated group along with the time prolonged. Data were expressed as mean values ± SEM. (n = 7). *P < 0.05, **P < 0.01 versus the control group.

Figure 4. Scheme of tissue model for angiogenesis (TMA).

(a) The cells and vessels sprout from the lung tissues in the cultured medium like as “sandwich” structure. (b) VEGF or Tongxinluo promotes angiogenesis of TMA. (c) Sorafenib inhibits angiogenesis of TMA.