With the aim to study the effect of less-sulfated endothelial HS on tumor growth, Rip1-Tag2 (RT2) mice harbouring floxed Ndst-1 alleles (Ndst-1f/f) which are TekCre negative (TC-) and positive (TC+) were compared to each other in regard to total tumor volume. Analysis was performed on 12 and 15 week old mice. 
In general, 15 week old males show larger tumor volumes compared to females. Comparison of the two experimental groups yielded no significant differences in tumor volumes, neither for males nor females. However, in 12 week old males it can be speculated towards a trend to smaller tumor volumes in TC+ individuals. Each dot represents the total tumor volume of a single individual. 

In order to investigate if complexes are formed between heparin and HRG330-derived peptides, a gel-shift assay was conducted. HRG330 was shown to bind to heparin previously. Bovine serum albumin (BSA) was used as a negative control, since it is known not to bind to heparin. Protamine on the other hand, has a high affinity for heparin and was thus established as a positive control.
HRG330 and Peptide 8, which represents a shorter version of HRG330, were used. Upon incubation of 50 ng heparin and 200 μM HRG330 and Peptide 8, a shift in the heparin pattern is visible as compared to the heparin control. This suggests binding of both peptides to heparin. When incubated with 500 μM of protamine no heparin or bands are visible, indicating that heparin is completely bound and complexes do not enter the gel. No interaction between BSA and heparin is detectable, thus confirming that BSA is a suitable negative control.

To investigate the HRG-peptides for anti-angiogenic properties in vivo, the zebrafish sub-intestinal basket assay was conducted. In brief, zebrafish embryos were injected with HRG330 at 48 hours post fertilization (hpf) into the common cardinal vein. As a control phenol-red was injected. 24 hours later, the embryos were fixed and the overall length of entopic as well as ectopic vessels of the sub-intestinal basket were assessed.
Serbedzija et al. (2000) have shown in their study that upon VEGF injection basket and branching are significantly increased. Hence, if HRG330 is anti-angiogenic, we would expect smaller baskets. The number of animals per each group was ten. However, in any of the measured parameters no significant differences can be observed. Neither were significant differences in the overall length of embryos observed.