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Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration.

Chan LY, Craik DJ, Daly NL - Biosci. Rep. (2015)

Bottom Line: Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum.The present study has demonstrated a novel approach to stabilize the active region of TSP-1.The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane 4072, Queensland, Australia.

No MeSH data available.


Related in: MedlinePlus

A schematic overview of the results from the present studyNewly designed cyclic TSP-1 analogues (SFTI-TSP-1 and MCo-TSP-1) were able to suppress HMVEC migration. Interestingly, these peptides had better inhibition of HMVEC migration (highlighted with a square box) compared with HUVECs, probably due to the overexpression of CD36 receptors in HMVECs but not in HUVECs. It is also possible that these peptides have the ability to inhibit VEGF as well since VEGF was added during the migration assay. Weaker inhibition was also observed in HUVEC migration although CD36 was not expressed in these cells. This might be due to the possibility of TSP-1 being able to inhibit β1-integrin receptors expressed in HUVECs. This was also observed in Short et al. [35]. Overall, cyclic TSP-1 analogues have better stability and potency (pathway shown in solid line) than the linear heptapeptide TSP-1 (pathway shown in dashed line).
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Figure 6: A schematic overview of the results from the present studyNewly designed cyclic TSP-1 analogues (SFTI-TSP-1 and MCo-TSP-1) were able to suppress HMVEC migration. Interestingly, these peptides had better inhibition of HMVEC migration (highlighted with a square box) compared with HUVECs, probably due to the overexpression of CD36 receptors in HMVECs but not in HUVECs. It is also possible that these peptides have the ability to inhibit VEGF as well since VEGF was added during the migration assay. Weaker inhibition was also observed in HUVEC migration although CD36 was not expressed in these cells. This might be due to the possibility of TSP-1 being able to inhibit β1-integrin receptors expressed in HUVECs. This was also observed in Short et al. [35]. Overall, cyclic TSP-1 analogues have better stability and potency (pathway shown in solid line) than the linear heptapeptide TSP-1 (pathway shown in dashed line).

Mentions: Figure 6 gives a schematic overview of the effects of the grafted cyclic peptides on cell migration. The linear and cyclic TSP-1 mimetics and native frameworks were examined in a human endothelial cell migration assay using two cell types, i.e., HUVECs and HMVECs. TSP-1 derived peptides target HMVECs through the CD36 receptor [12,56] and have an inhibitory effect on HUVEC migration via targeting the β-integrin receptor instead of CD36 [35]. We showed that cyclic TSP-1 mimetics have higher potency towards HMVECs compared with HUVECs. This difference in potency is likely a result of the difference in expression of CD36 receptors in HMVECs compared with large vessel cells such as HUVECs. Previous studies using flow cytometric analysis have shown the presence of CD36 on the surface of HMVECs, in contrast with HUVECs where the antibodies failed to detect the presence of CD36 [57,58]. This difference in potency is also consistent with previous studies on thrombospondin-1 type-1 repeats (TSR) by Short et al. [35], who reported 35% inhibition could be achieved on HMVECs at 5 nM but 16 nM was required for 60% inhibition on HUVECs. The TSP-1 heptapeptide grafted into the MCoTI-II framework resulted in better inhibition of cell migration than the SFTI-1 framework grafts, possibly because the larger framework provides better opportunity for the delivery of the TSP-1 fragment to its receptor than SFTI-1 [59]. Additionally, the site of the incorporation of the TSP-1 peptide in loop 6 of MCoTI-II offers greater flexibility compared with SFTI-1 as the SFTI-1 framework is small, with a β-hairpin constrained structure [26]. Although SFTI-TSP-1 was observed to be less potent than MCo-TSP-1, both peptides showed better stability and potency than their linear counterpart. On the other hand, the cell penetration properties of cyclic frameworks could also play a role in the different route of entry into cells, as D'Souza et al. [60] have recently shown that MCoTI-II is more efficiently internalized into cells than SFTI-1. This fits the results of cell migration study, where MCo-TSP-1 has better potency than SFTI-TSP-1. Although these peptides do not have as high potency as ABT-510 (inhibition of HMVEC migration: IC50=0.89 nM [14]), they are still active at low micromolar (SFTI-TSP-1) or nanomolar (MCo-TSP-1) concentrations. These data suggest TSP-1 mimetics act against HMVECs via a CD36-dependent pathway.


Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration.

Chan LY, Craik DJ, Daly NL - Biosci. Rep. (2015)

A schematic overview of the results from the present studyNewly designed cyclic TSP-1 analogues (SFTI-TSP-1 and MCo-TSP-1) were able to suppress HMVEC migration. Interestingly, these peptides had better inhibition of HMVEC migration (highlighted with a square box) compared with HUVECs, probably due to the overexpression of CD36 receptors in HMVECs but not in HUVECs. It is also possible that these peptides have the ability to inhibit VEGF as well since VEGF was added during the migration assay. Weaker inhibition was also observed in HUVEC migration although CD36 was not expressed in these cells. This might be due to the possibility of TSP-1 being able to inhibit β1-integrin receptors expressed in HUVECs. This was also observed in Short et al. [35]. Overall, cyclic TSP-1 analogues have better stability and potency (pathway shown in solid line) than the linear heptapeptide TSP-1 (pathway shown in dashed line).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660582&req=5

Figure 6: A schematic overview of the results from the present studyNewly designed cyclic TSP-1 analogues (SFTI-TSP-1 and MCo-TSP-1) were able to suppress HMVEC migration. Interestingly, these peptides had better inhibition of HMVEC migration (highlighted with a square box) compared with HUVECs, probably due to the overexpression of CD36 receptors in HMVECs but not in HUVECs. It is also possible that these peptides have the ability to inhibit VEGF as well since VEGF was added during the migration assay. Weaker inhibition was also observed in HUVEC migration although CD36 was not expressed in these cells. This might be due to the possibility of TSP-1 being able to inhibit β1-integrin receptors expressed in HUVECs. This was also observed in Short et al. [35]. Overall, cyclic TSP-1 analogues have better stability and potency (pathway shown in solid line) than the linear heptapeptide TSP-1 (pathway shown in dashed line).
Mentions: Figure 6 gives a schematic overview of the effects of the grafted cyclic peptides on cell migration. The linear and cyclic TSP-1 mimetics and native frameworks were examined in a human endothelial cell migration assay using two cell types, i.e., HUVECs and HMVECs. TSP-1 derived peptides target HMVECs through the CD36 receptor [12,56] and have an inhibitory effect on HUVEC migration via targeting the β-integrin receptor instead of CD36 [35]. We showed that cyclic TSP-1 mimetics have higher potency towards HMVECs compared with HUVECs. This difference in potency is likely a result of the difference in expression of CD36 receptors in HMVECs compared with large vessel cells such as HUVECs. Previous studies using flow cytometric analysis have shown the presence of CD36 on the surface of HMVECs, in contrast with HUVECs where the antibodies failed to detect the presence of CD36 [57,58]. This difference in potency is also consistent with previous studies on thrombospondin-1 type-1 repeats (TSR) by Short et al. [35], who reported 35% inhibition could be achieved on HMVECs at 5 nM but 16 nM was required for 60% inhibition on HUVECs. The TSP-1 heptapeptide grafted into the MCoTI-II framework resulted in better inhibition of cell migration than the SFTI-1 framework grafts, possibly because the larger framework provides better opportunity for the delivery of the TSP-1 fragment to its receptor than SFTI-1 [59]. Additionally, the site of the incorporation of the TSP-1 peptide in loop 6 of MCoTI-II offers greater flexibility compared with SFTI-1 as the SFTI-1 framework is small, with a β-hairpin constrained structure [26]. Although SFTI-TSP-1 was observed to be less potent than MCo-TSP-1, both peptides showed better stability and potency than their linear counterpart. On the other hand, the cell penetration properties of cyclic frameworks could also play a role in the different route of entry into cells, as D'Souza et al. [60] have recently shown that MCoTI-II is more efficiently internalized into cells than SFTI-1. This fits the results of cell migration study, where MCo-TSP-1 has better potency than SFTI-TSP-1. Although these peptides do not have as high potency as ABT-510 (inhibition of HMVEC migration: IC50=0.89 nM [14]), they are still active at low micromolar (SFTI-TSP-1) or nanomolar (MCo-TSP-1) concentrations. These data suggest TSP-1 mimetics act against HMVECs via a CD36-dependent pathway.

Bottom Line: Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum.The present study has demonstrated a novel approach to stabilize the active region of TSP-1.The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane 4072, Queensland, Australia.

No MeSH data available.


Related in: MedlinePlus