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LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

View Article: PubMed Central - PubMed

ABSTRACT

Background: LIM domain only 2 (LMO2, human gene) is a key transcription factor that regulates hematopoiesis and vascular development. However, its role in adult endothelial function has been incompletely characterized.

Methods and results: In vitro loss‐ and gain‐of‐function studies on LMO2 were performed in human umbilical vein endothelial cells with lentiviral overexpression or short hairpin RNA knockdown (KD) of LMO2, respectively. LMO2 KD significantly impaired endothelial proliferation. LMO2 controls endothelial G1/S transition through transcriptional regulation of cyclin‐dependent kinase 2 and 4 as determined by reverse transcription polymerase chain reaction (PCR), western blot, and chromatin immunoprecipitation, and also influences the expression of Cyclin D1 and Cyclin A1. LMO2 KD also impaired angiogenesis by reducing transforming growth factor‐β (TGF‐β) expression, whereas supplementation of exogenous TGF‐β restored defective network formation in LMO2 KD human umbilical vein endothelial cells. In a zebrafish model of caudal fin regeneration, RT‐PCR revealed that the lmo2 (zebrafish gene) gene was upregulated at day 5 postresection. The KD of lmo2 by vivo‐morpholino injections in adult Tg(fli1:egfp)y1 zebrafish reduced 5‐bromo‐2′‐deoxyuridine incorporation in endothelial cells, impaired neoangiogenesis in the resected caudal fin, and substantially delayed fin regeneration.

Conclusions: The transcriptional factor LMO2 regulates endothelial proliferation and angiogenesis in vitro. Furthermore, LMO2 is required for angiogenesis and tissue healing in vivo. Thus, LMO2 is a critical determinant of vascular and tissue regeneration.

No MeSH data available.


Related in: MedlinePlus

lmo2 KD by vivo‐morpholino (Mo) in zebrafish reduced caudal fin regeneration and impaired vascular regeneration. Adult Tg(fli1:egfp)y1 zebrafish fish (2.5 months old) were injected with lmo2 or control (CT) vivo‐Mo, then the caudal fin was resected and fin regeneration was observed. A, Representative caudal fin images before resection and then at 0, 1, 2, and 5 days postresection (dpr) in fish injected with CT or lmo2 vivo‐Mo. B, Quantification of the regeneration. The whole area of the caudal fin at day 0 before resection and the new fin tissue area in each fish from the new distal fin edge to the amputation plane were quantified at 0, 1, 2, and 5 dpr. Caudal fin regeneration for each fin at each time point was defined as percentage of regeneration=100× (regenerated tissue area/original fin area amputated). C, Representative image showing decreased vasculature length in the regenerated fin in lmo2 KD fish compared with CT. The vertical white bar represents the length of the regenerated vasculature. D, Quantification of the regenerated vascular area in CT at 5 dpr. Data are presented as mean±SEM (n=10). *P<0.05 vs CT.
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jah31811-fig-0007: lmo2 KD by vivo‐morpholino (Mo) in zebrafish reduced caudal fin regeneration and impaired vascular regeneration. Adult Tg(fli1:egfp)y1 zebrafish fish (2.5 months old) were injected with lmo2 or control (CT) vivo‐Mo, then the caudal fin was resected and fin regeneration was observed. A, Representative caudal fin images before resection and then at 0, 1, 2, and 5 days postresection (dpr) in fish injected with CT or lmo2 vivo‐Mo. B, Quantification of the regeneration. The whole area of the caudal fin at day 0 before resection and the new fin tissue area in each fish from the new distal fin edge to the amputation plane were quantified at 0, 1, 2, and 5 dpr. Caudal fin regeneration for each fin at each time point was defined as percentage of regeneration=100× (regenerated tissue area/original fin area amputated). C, Representative image showing decreased vasculature length in the regenerated fin in lmo2 KD fish compared with CT. The vertical white bar represents the length of the regenerated vasculature. D, Quantification of the regenerated vascular area in CT at 5 dpr. Data are presented as mean±SEM (n=10). *P<0.05 vs CT.

Mentions: The caudal fin was resected at 4 days after the first vivo‐Mo injection and its regeneration was followed up to day 15 (Figure 7A). At day 1 and 2 postresection, the percentage of fin regeneration was similar in lmo2 KD and CT fish (Figure 7A and 7B). However, at 5 days postresection, the percentage of caudal fin regeneration was only 28% in LMO2 KD fish compared with 44% in CT fish (Figure 7A and 7B). Furthermore, neoangiogenesis was impaired as the regenerated vasculature area was significantly smaller (0.47 mm2) in lmo2 KD fish compared with CTs (0.74 mm2) at day 5 postresection (Figure 7C and 7D). Interestingly, the morphology of the neovessels in the lmo2 KD fish were aberrant with tangled vessel complexes (Figure 7C), in comparison to the standard vascular branching observed in the CT fish.


LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration
lmo2 KD by vivo‐morpholino (Mo) in zebrafish reduced caudal fin regeneration and impaired vascular regeneration. Adult Tg(fli1:egfp)y1 zebrafish fish (2.5 months old) were injected with lmo2 or control (CT) vivo‐Mo, then the caudal fin was resected and fin regeneration was observed. A, Representative caudal fin images before resection and then at 0, 1, 2, and 5 days postresection (dpr) in fish injected with CT or lmo2 vivo‐Mo. B, Quantification of the regeneration. The whole area of the caudal fin at day 0 before resection and the new fin tissue area in each fish from the new distal fin edge to the amputation plane were quantified at 0, 1, 2, and 5 dpr. Caudal fin regeneration for each fin at each time point was defined as percentage of regeneration=100× (regenerated tissue area/original fin area amputated). C, Representative image showing decreased vasculature length in the regenerated fin in lmo2 KD fish compared with CT. The vertical white bar represents the length of the regenerated vasculature. D, Quantification of the regenerated vascular area in CT at 5 dpr. Data are presented as mean±SEM (n=10). *P<0.05 vs CT.
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jah31811-fig-0007: lmo2 KD by vivo‐morpholino (Mo) in zebrafish reduced caudal fin regeneration and impaired vascular regeneration. Adult Tg(fli1:egfp)y1 zebrafish fish (2.5 months old) were injected with lmo2 or control (CT) vivo‐Mo, then the caudal fin was resected and fin regeneration was observed. A, Representative caudal fin images before resection and then at 0, 1, 2, and 5 days postresection (dpr) in fish injected with CT or lmo2 vivo‐Mo. B, Quantification of the regeneration. The whole area of the caudal fin at day 0 before resection and the new fin tissue area in each fish from the new distal fin edge to the amputation plane were quantified at 0, 1, 2, and 5 dpr. Caudal fin regeneration for each fin at each time point was defined as percentage of regeneration=100× (regenerated tissue area/original fin area amputated). C, Representative image showing decreased vasculature length in the regenerated fin in lmo2 KD fish compared with CT. The vertical white bar represents the length of the regenerated vasculature. D, Quantification of the regenerated vascular area in CT at 5 dpr. Data are presented as mean±SEM (n=10). *P<0.05 vs CT.
Mentions: The caudal fin was resected at 4 days after the first vivo‐Mo injection and its regeneration was followed up to day 15 (Figure 7A). At day 1 and 2 postresection, the percentage of fin regeneration was similar in lmo2 KD and CT fish (Figure 7A and 7B). However, at 5 days postresection, the percentage of caudal fin regeneration was only 28% in LMO2 KD fish compared with 44% in CT fish (Figure 7A and 7B). Furthermore, neoangiogenesis was impaired as the regenerated vasculature area was significantly smaller (0.47 mm2) in lmo2 KD fish compared with CTs (0.74 mm2) at day 5 postresection (Figure 7C and 7D). Interestingly, the morphology of the neovessels in the lmo2 KD fish were aberrant with tangled vessel complexes (Figure 7C), in comparison to the standard vascular branching observed in the CT fish.

View Article: PubMed Central - PubMed

ABSTRACT

Background: LIM domain only 2 (LMO2, human gene) is a key transcription factor that regulates hematopoiesis and vascular development. However, its role in adult endothelial function has been incompletely characterized.

Methods and results: In vitro loss&#8208; and gain&#8208;of&#8208;function studies on LMO2 were performed in human umbilical vein endothelial cells with lentiviral overexpression or short hairpin RNA knockdown (KD) of LMO2, respectively. LMO2 KD significantly impaired endothelial proliferation. LMO2 controls endothelial G1/S transition through transcriptional regulation of cyclin&#8208;dependent kinase 2 and 4 as determined by reverse transcription polymerase chain reaction (PCR), western blot, and chromatin immunoprecipitation, and also influences the expression of Cyclin D1 and Cyclin A1. LMO2 KD also impaired angiogenesis by reducing transforming growth factor&#8208;&beta; (TGF&#8208;&beta;) expression, whereas supplementation of exogenous TGF&#8208;&beta; restored defective network formation in LMO2 KD human umbilical vein endothelial cells. In a zebrafish model of caudal fin regeneration, RT&#8208;PCR revealed that the lmo2 (zebrafish gene) gene was upregulated at day 5 postresection. The KD of lmo2 by vivo&#8208;morpholino injections in adult Tg(fli1:egfp)y1 zebrafish reduced 5&#8208;bromo&#8208;2&prime;&#8208;deoxyuridine incorporation in endothelial cells, impaired neoangiogenesis in the resected caudal fin, and substantially delayed fin regeneration.

Conclusions: The transcriptional factor LMO2 regulates endothelial proliferation and angiogenesis in&nbsp;vitro. Furthermore, LMO2 is&nbsp;required for angiogenesis and tissue healing in&nbsp;vivo. Thus, LMO2 is a critical determinant of vascular and tissue regeneration.

No MeSH data available.


Related in: MedlinePlus