Figure 1.
CX dose-dependently promotes angiogenic sprouting and vessel growth in vitro and in vivo.
(A) Dose-response curve for CX in endothelial sprouting assay using HUVEC in type I collagen gels (EC50 value: 8.8 µM). Cumulative sprout length (CSL) was calculated from the ten longest sprouts from ten HUVEC spheres. (B-E) Mouse retinas analyzed at day 5 after systemic treatment of mice, between postnatal day 3-4, with vehicle, B or CX at 5 mg/kg, C, 10 mg/kg, D and 20 mg/kg, E. Endothelial cells were stained using Isolectin B4, white. There was a clear dose-dependent increase in the vascular density after CX dosing. (F) There was a 68% increase in the number of filopodia protrusions per vessel length when comparing CX-treated (10 mg/kg) retinas to vehicle (CX; 9.8 versus vehicle; 5.8 filopodia protrusions/100 µm, P-value = 0.0041). (G-H) High-magnification images of vehicle and CX-treated (10 mg/kg) retinas showing endothelial tip cells (white asterisks) with extensive filopodia protrusions, isolectin B4-positive staining in black. Error bars represent standard deviation. The scale bars in B-E are 100 µm and in G-H 25 µm. ** = P<0.01.
Figure 2.
CX treatment is anti-tumorigenic and promotes angiogenic sprouting in mouse renal cell carcinoma model.
At day 0 twelve mice were orthotopically inoculated with RENCA cells under the kidney capsule. (A) Mean animal weight plotted in relation to their initial weight. There was a weight reduction in the control group, 1.0 g (a loss of 5.5% of initial weight), P = 0.049 between day 1 and 20. There was no significant weight reduction in the CX- treated cohort. At day 21 the study was terminated. (B) Tumor volume was reduced in the CX- treated group by 53% of control, P = 0.0055. (C) Tumor wet weight was reduced in the CX- treated group by 38% of controls, P = 0.041. (D) Microvascular density (MVD) was measured as the number of CD31 positive structures per area, and was increased by 29% of control after CX treatment, P = 0.0080. (E) The number of filopodia-like protrusions from the vessels profiles were increased by 130% in CX 10 mg/kg treated tumors compared to vehicle treated tumors (CX 10 mg/kg; 0.84 versus vehicle; 0.37 endothelial protrusions per vessel profile, P = 0.017). (F) Vessel perimeter was calculated as the length surrounding the CD31+ blood vessel areas and there was a significant increase in the mean vessel perimeter in the CX-treated tumor as compared to control, 107 µm versus 85 µm, P<0.001. (G) The mural cell coverage was quantified as the fraction of CD31+ vessels with associated ASMA+ cells, and was 0.16 in the vehicle group and 0.06 in the CX-treated group, P = 0.048. (H) The number of occluded vessels per area was increased 1.93-fold in CX- treated tumors as compared to controls, P = 0.010. (I and J) Representative tumor fields from control and CX-treated tumors stained with CD31. Black structures are CD31 positive vessels in I-M. (K–L) A higher frequency of endothelial filopodia-like protrusions were observed after CX treatment compared to control, black arrowheads point at filopodia-like protrusions. (M) Occluded vessels were identified as large vessel structures completely filled with CD31-positive structures (arrowheads), clearly distinguished from small, and presumably, tangentially sectioned vessels. (N) Control tumors stained for CD31, white structures. (O) CD31-stained CX-treated tumors form long continuous vessels structures that seem to arise from fusion of several previously distinct vessels, e.g. center of image. (P) Example of CD31 (green) and ASMA (red) stained tumor section. Error bars represent standard deviation. Compound X: CX. Scale bars are 100 µm, except K-L which are 25 µm. Significance level were indicated * = <0.05, ** = P<0.01. *** = P<0.001.
Figure 3.
CX treatment leads to formation of abnormal angiogenesis and vessel occlusion in model for VEGF-A driven angiogenesis.
(A–H) Cross section of rabbit skeletal muscle semimembranous muscle, hematoxylin staining in blue and endothelial CD31/PECAM1 or Podocalyxin staining in brown, as indicated. (A) Thin-walled capillaries are evenly interspersed in the AdLacZ-treated (control) skeletal muscle. (B) 2 mg/kg CX treatment did not alter the vascular morphology of the skeletal muscle capillaries. (C) Administration of VEGF-A165-isoform by adenoviral vector promotes an angiogenic response resulting in dilation and proliferation of the existing vessels, day six after gene transfer. (D–E) Addition of 1 or 2 mg/kg CX (D and E, respectively) dose-dependently abrogates the normal VEGF-A response and leads to the formation of PECAM1-positive structures occluding the vessel lumen (arrowheads, F is a magnification of E). (G–H) The endothelial marker, Podocalyxin, labeled the endothelial cells specifically in AdVEGF-A and CX-treated treated skeletal muscles, brown. (H) Podocalyxin revealed the same cellular structures occluding the vascular lumens after CX treatment as the CD31 staining. Scale bars are 25 µm.
Figure 4.
CX treatment leads to decreased perfusion and increased leakage after VEGF-A induced angiogenesis.
Contrast pulse sequence (CPS) ultrasound was used to measure vascular perfusion in the semimembranous muscle. The CPS-ratio between the VEGF-A adenovirus gene transferred leg and the non-treated leg was compared. (A) VEGF-A induced a 10.3-fold increase in perfusion. There was a significant dose-dependent reduction in the CPS ratio between VEGF-A transferred leg and the control leg when CX was systemically administered. The CPS ratio was 5.5 and 3.6 after administration of 1 and 2 mg/kg CX, respectively, ANOVA P = 0.0040. (B) Evans blue was injected into the blood stream prior to extraction of the muscle tissue. The signal between the VEGF-A treated muscle versus the non-treated leg was compared. There was a 35-fold increase in Evans blue leakage after VEGF-A gene transfer, which was further increased by administration of CX. Doses of CX at 1 mg/kg or 2 mg/kg resulted in a 40- or 76-fold increase in Evans blue leakage, respectively, ANOVA P = 0.041. (C) Biopsies from the semimembranous muscle from either AdVEGF-A165 treated or intact leg with the systemic treatment as indicated in the figure. The blue dye comes from extravasated Evans blue that is trapped in the tissue. Compound X: CX. Error bars represent standard deviation. Significance level were indicated * = P<0.05, ** = P<0.01. *** = P<0.001.
Figure 5.
CX treatment alters liver morphology.
(A–C) Liver samples from rabbits were stained with CD31/PECAM1 (brown) and counter-stained with hematoxylin (blue). (A) AdVEGF-A + vehicle, (B) AdVEGF-A + 1 mg/kg CX and (C) AdVEGF-A + 2 mg/kg CX. Microphotographs show representative liver portal triads. (B–C) Note the dose dependent dilation of the liver sinusoids (arrowheads in C) as well as the increased staining intensity of CD31 in CX-treated livers. The bile ducts seemed occluded by intraluminal epithelial cells (asterisks in B and C). A: Artery, V: Vein, B: bile duct. Scale bars are 100 µm.
Table 1.
Clinical chemistry parameters of VEGF-A and CX-treated rabbits.
Table 2.
Quantification of occluded capillaries in rabbit skeletal muscle upon VEGF-A and CX treatment.