Table 2. In vivo and in vitro investigations of the effects of BCR-ABL TKIs on endothelial cell viability and major functions.
| Endpoints | Methods | Models | TKIs | Findings | Ref. |
|---|---|---|---|---|---|
| EC proliferation/survival | Cell counting; trypan blue staining | EA.hy 926 cell; HCAEC | Imatinib | = EC viability <10µM | 84 85 |
| Caspase assay; Annexin V staining; Hoechst staining; TUNEL assay | HMEC-1; HUVEC; Human pulmonary EC; Mouse EC | Imatinib | = EC apoptosis | 81 82 87 | |
| TUNEL assay; Annexin V staining | EA.hy 926 cell | Imatinib |
EC apoptosis
|
85 | |
| MTT cell proliferation assay; 3 H-thymidine incorporation; WST-1 assay; cell counting | HMEC-1; HUVEC; HCAEC | Imatinib | = EC proliferation | 39 81 82 84 | |
| Resazurin proliferation assay; PCNA expression | HUVEC; BAEC | Imatinib |
EC proliferation (≥1.2 µM)
|
83 | |
| Caspase assay; Hoechst staining; Annexin V staining; TUNEL assay | Human pulmonary EC | Dasatinib |
EC apoptosis
|
87 | |
| 3 H-thymidine incorporation; WST-1 assay; MTT assay | HUVEC; HCAEC; HMEC-1; HCtAEC | Nilotinib |
EC proliferation
|
39 82 89 | |
| Annexin V staining | HUVEC | Nilotinib | = EC apoptosis | 82 | |
| Caspase assay; Annexin V staining | HCAEC; HUVEC | Ponatinib |
EC apoptosis
|
82 90 | |
| 3 H-thymidine incorporation; WST-1 assay | HUVEC; HMEC-1; EPC | Ponatinib |
EC proliferation
|
82 90 | |
| Oxidative stress | Fluorescent ROS detection; Immunofluorescence (8-oxo-dG) | Human Pulmonary EC; Rat lung | Imatinib | = endothelial ROS | 87 |
| Fluorescent ROS detection; Immunofluorescence (8-oxo-dG) | Human Pulmonary EC; Rat lung | Dasatinib |
endothelial ROS
|
87 | |
| EC migration | Wound scratch assay; Microchemotaxis assay; Transwell migration assay | HMEC-1; HUVEC; EA.hy 926 cell; HCAEC | Imatinib | = EC migration | 81 82 84 85 |
| Wound scratch assay | HUVEC; HCAEC; HMEC-1 | Nilotinib |
EC migration
|
39 | |
| Transwell migration assay | HUVEC | Nilotinib | = EC migration | 82 | |
| Transwell migration assay | HUVEC | Ponatinib |
EC migration
|
82 | |
| Angiogenesis | Tube-formation assay | HMEC-1; HUVEC | Imatinib | = angiogenesis | 81 82 |
| Tube-formation assay | HUVEC; HCAEC; HMEC-1 | Nilotinib |
angiogenesis
|
39 | |
| Tube-formation assay | HUVEC | Nilotinib | = angiogenesis | 82 | |
| Tube-formation assay | HUVEC | Ponatinib |
angiogenesis
|
82 | |
| Permeability | Permeability to albumin | EA.hy 926 cell | Imatinib |
endothelial permeability (10 µM)
|
85 |
| Immunofluorescence (VE-cadherin) | EA.hy 926 cell; HPAEC | Imatinib |
membrane VE-cadherin (10 µM)
|
85 86 | |
| BAL protein levels | Mice (2-hit model of ALI) | Imatinib |
BAL protein levels
|
86 88 | |
| Permeability to FITC-Dextran; permeability to HRP | HMEC-1; HUVEC; Human lung microvascular EC | Imatinib | = endothelial permeability | 94 147 | |
| Immunostaining | HUVEC | Imatinib |
intercellular gaps
|
147 | |
| Evans blue/albumin extravasation | Mice | Imatinib |
Evans blue extravasation
|
147 | |
| Pulmonary microvascular permeability assay; permeability assay (FITC-Dextran) | Mice; HMEC-1; HPAEC | Dasatinib |
endothelial permeability
|
94 | |
| Permeability assay (FITC-Dextran) | HRMEC | Dasatinib |
VEGF-induced permeability
|
148 | |
| CAM expression | Confocal microscopy; ELISA; qRT-PCR; flow cytometry | HMEC-1; Pulmonary EC (rat lung); EA.hy926 | Imatinib | = ICAM-1, VCAM-1 and E-selectin expression = soluble ICAM-1, VCAM-1 and E-selectin |
81 87 149 |
| Immunoblotting (VCAM-1) | Human lung EC | Imatinib |
VCAM-1 expression
|
86 | |
| Confocal microscopy | Pulmonary EC (rat lung) | Dasatinib |
ICAM-1, VCAM-1 and E-selectin expression
|
87 | |
| ELISA | Rat | Dasatinib |
soluble ICAM-1, VCAM-1 and E-selectin
|
87 | |
| qRT-PCR; flow cytometry | EA.hy926 | Dasatinib | = ICAM-1, VCAM-1 and E-selectin expression | 149 | |
| Unknown | HUVEC | Nilotinib |
ICAM-1, VCAM-1 and E-selectin expression (≥1 µM)
|
39 | |
| qRT-PCR; flow cytometry | EA.hy926 | Nilotinib |
ICAM-1, VCAM-1 and E-selectin expression
|
149 | |
| Secretory | ELISA (IL-6; IL-8) | Stimulated HPAEC | Imatinib |
IL-8 and IL-6 (LPS induced)
|
86 |
| qRT-PCR ; ELISA (IL-1β; IL-6; TNF-α) | EA.hy926 cell ; HUVEC | Imatinib | = IL-1β, IL-6 and TNF-α expression and production | 149 | |
| qRT-PCR ; ELISA (IL-1β; IL-6; TNF-α) | EA.hy926 cell ; HUVEC | Dasatinib | = IL-1β, IL-6 and TNF-α expression and production | 149 | |
| qRT-PCR ; ELISA (IL-1β; IL-6; TNF-α) | EA.hy926 cell ; HUVEC | Nilotinib | = IL-6 and TNF-α expression and production
IL-1β expression and production
|
149 | |
| ELISA (t-PA; PAI-1; ET-1; vWF; total NO) | HCtAEC | Nilotinib |
t-PA
PAI-1, ET-1, vWF and total NO
|
89 | |
| Adhesion | Unknown | HUVEC | Ponatinib |
adhesion to plastic surface at 1 µM
|
90 |
Abbreviations: 8-oxo-dG, 8-hydroxy-2′-deoxyguanosine; ALI, acute lung injury; BAEC, bovine aortic endothelial cell; BAL, bronchoalveolar level ; EC, endothelial cell; ELISA, enzyme-linked immunosorbent assay; EPC, endothelial progenitor cell; ET-1, endothelin 1; FITC, fluorescein isothiocyanate; HCAEC, human coronary artery endothelial cell; HCtAEC, human carotid artery endothelial cell; HMEC-1, human microvascular endothelial cell; HPAEC, human pulmonary artery endothelial cell; HRMEC, human retinal microvascular endothelial cells; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; LPS, lipopolysaccharide; NO, nitric oxide; PAI-1, plasminogen activator inhibitor-1; ROS, reactive oxygen species; t-PA, tissue plasminogen activator; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VCAM-1, vascular cell adhesion molecule 1; VE-cadherin, vascular endothelial cadherin; vWF, Von Willebrand factor.