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Influence of levamisole and other angiogenesis inhibitors on angiogenesis and endothelial cell morphology in vitro.

Friis T, Engel AM, Bendiksen CD, Larsen LS, Houen G - Cancers (Basel) (2013)

Bottom Line: Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size.This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling.Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark. gh@ssi.dk.

ABSTRACT
Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.

No MeSH data available.


Related in: MedlinePlus

VEGF-dependent capillary-like tube formation of HMVECs propagated for three days in coculture with fibroblasts in (A) HMVEC5 medium, (B) HMVEC5 medium without bFGF, (C) HMVEC5 medium + 20 µg/mL goat anti-bFGF, (D) HMVEC5 medium without bFGF and VEGF, (E) HMVEC5 medium without VEGF, (F) HMVEC5 medium + 40 µg/mL goat anti-VEGF.
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cancers-05-00762-f004: VEGF-dependent capillary-like tube formation of HMVECs propagated for three days in coculture with fibroblasts in (A) HMVEC5 medium, (B) HMVEC5 medium without bFGF, (C) HMVEC5 medium + 20 µg/mL goat anti-bFGF, (D) HMVEC5 medium without bFGF and VEGF, (E) HMVEC5 medium without VEGF, (F) HMVEC5 medium + 40 µg/mL goat anti-VEGF.

Mentions: In this assay, an elaborate network of differentiated ECs is formed when HUVECs are grown on a layer of living NHDFs in the presence of VEGF (Figure 3A). However, in the absence of VEGF, clusters of non-differentiated ECs are formed (Figure 3D–E). The same morphological features are observed when replacing HUVECs with human microvascular ECs (HMVECs) in a coculture system, where NHDFs and HMVECs are cocultured for 4 days in HMVEC5 medium (an EGF-depleted TFSM2 medium with 5% fetal calf serum (FCS) and 100 ng/mL VEGF instead of 2% FCS and 10 ng/mL VEGF as used for HUVECs (Figure 4A,D–E). Recently, Sarkanen et al. improved the sensitivity of this assay by increasing the coculture period from three to six days [38] and other variations have been described which allow real time microscopy of ECs [39].


Influence of levamisole and other angiogenesis inhibitors on angiogenesis and endothelial cell morphology in vitro.

Friis T, Engel AM, Bendiksen CD, Larsen LS, Houen G - Cancers (Basel) (2013)

VEGF-dependent capillary-like tube formation of HMVECs propagated for three days in coculture with fibroblasts in (A) HMVEC5 medium, (B) HMVEC5 medium without bFGF, (C) HMVEC5 medium + 20 µg/mL goat anti-bFGF, (D) HMVEC5 medium without bFGF and VEGF, (E) HMVEC5 medium without VEGF, (F) HMVEC5 medium + 40 µg/mL goat anti-VEGF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795364&req=5

cancers-05-00762-f004: VEGF-dependent capillary-like tube formation of HMVECs propagated for three days in coculture with fibroblasts in (A) HMVEC5 medium, (B) HMVEC5 medium without bFGF, (C) HMVEC5 medium + 20 µg/mL goat anti-bFGF, (D) HMVEC5 medium without bFGF and VEGF, (E) HMVEC5 medium without VEGF, (F) HMVEC5 medium + 40 µg/mL goat anti-VEGF.
Mentions: In this assay, an elaborate network of differentiated ECs is formed when HUVECs are grown on a layer of living NHDFs in the presence of VEGF (Figure 3A). However, in the absence of VEGF, clusters of non-differentiated ECs are formed (Figure 3D–E). The same morphological features are observed when replacing HUVECs with human microvascular ECs (HMVECs) in a coculture system, where NHDFs and HMVECs are cocultured for 4 days in HMVEC5 medium (an EGF-depleted TFSM2 medium with 5% fetal calf serum (FCS) and 100 ng/mL VEGF instead of 2% FCS and 10 ng/mL VEGF as used for HUVECs (Figure 4A,D–E). Recently, Sarkanen et al. improved the sensitivity of this assay by increasing the coculture period from three to six days [38] and other variations have been described which allow real time microscopy of ECs [39].

Bottom Line: Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size.This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling.Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark. gh@ssi.dk.

ABSTRACT
Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.

No MeSH data available.


Related in: MedlinePlus