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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 knockdown (KD) impaired endothelial network formation, which can be rescued by exogenous transforming growth factor‐β1 (TGF‐β1). A, Network formation of control (CT) and LMO2 KD human umbilical vein endothelial cells (HUVECs). B, List of downregulated genes identified in LMO2 KD in comparison to CT HUVECs using angiogenesis polymerase chain reaction (PCR) array. C, Reverse transcription PCR (RT‐PCR) of TGF‐β1. D, ELISA for TGF‐β levels in culture medium of CT and LMO2 KD cells. E, RT‐PCR of TGF‐β1 in CT and LMO2 overexpression HUVECs. F, Network formation of CT and LMO2 KD HUVECs with or without supplementation of TGF‐β 10 ng/mL. G, Chromatin immunoprecipitation PCR analysis of LMO2 complex binding to TGF‐β1 promoter (–901 and –4030 region) in both CT and LMO2 KD cells. Data are presented as mean±SEM (n=3). *P<0.05 vs CT.
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jah31811-fig-0004: LMO2 knockdown (KD) impaired endothelial network formation, which can be rescued by exogenous transforming growth factor‐β1 (TGF‐β1). A, Network formation of control (CT) and LMO2 KD human umbilical vein endothelial cells (HUVECs). B, List of downregulated genes identified in LMO2 KD in comparison to CT HUVECs using angiogenesis polymerase chain reaction (PCR) array. C, Reverse transcription PCR (RT‐PCR) of TGF‐β1. D, ELISA for TGF‐β levels in culture medium of CT and LMO2 KD cells. E, RT‐PCR of TGF‐β1 in CT and LMO2 overexpression HUVECs. F, Network formation of CT and LMO2 KD HUVECs with or without supplementation of TGF‐β 10 ng/mL. G, Chromatin immunoprecipitation PCR analysis of LMO2 complex binding to TGF‐β1 promoter (–901 and –4030 region) in both CT and LMO2 KD cells. Data are presented as mean±SEM (n=3). *P<0.05 vs CT.

Mentions: The LMO2 KD impaired endothelial network formation in matrigel (Figure 4A). Further analysis of 84 angiogenesis‐related genes using the Qiagen Angiogenesis RT Profiler PCR Array revealed that there was a substantial reduction in the expression of core angiogenesis genes (33 of 84; Figure 4B) in the LMO2 KD cells. Among them, TGF‐β1 promoter region is predicted to have LMO2 complex binding sites, indicating that it could be a direct transcriptional target of LMO2. We confirmed by RT‐PCR (Figure 4C) and ELISA (Figure 4D) that TGF‐β1 was downregulated in LMO2 KD cells. To determine the contribution of TGF‐β downregulation to impaired angiogenic processes in LMO2 KD cells, we determined whether network formation could be rescued by TGF‐β. Indeed, supplementation of the HUVEC LMO2 KD with exogenous TGF‐β restored defective network formation (Figure 4F). ENCODE database prediction suggests that two regions on TGF‐β1 (Gene ID: 7040) promoter could potentially have LMO2 complex binding. One region, CCGCAGCTGCTGC, starts at the proximal –901 position and the other region, CAGATAGGGG, starts at the distal –4030 position. The ChIP assay revealed that LMO2 complex binds to both regions (Figure 4G), confirming that TGF‐β1 is a direct transcriptional target of LMO2. Interestingly, in LMO2 KO cells, the decreased binding of LMO2 complex was only observed in the proximal –901 binding site, suggesting that this binding site is more sensitive to LMO2 downregulation. Notably, OE of LMO2 did not further increase TGF‐β1 level, probably because LMO2 binding to DNA involves the participation of other components of the binding complex (Figure 4E). This explanation is consistent with the observation that LMO2 OE did not further promote endothelial network formation (Figure S3).


LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration
LMO2 knockdown (KD) impaired endothelial network formation, which can be rescued by exogenous transforming growth factor‐β1 (TGF‐β1). A, Network formation of control (CT) and LMO2 KD human umbilical vein endothelial cells (HUVECs). B, List of downregulated genes identified in LMO2 KD in comparison to CT HUVECs using angiogenesis polymerase chain reaction (PCR) array. C, Reverse transcription PCR (RT‐PCR) of TGF‐β1. D, ELISA for TGF‐β levels in culture medium of CT and LMO2 KD cells. E, RT‐PCR of TGF‐β1 in CT and LMO2 overexpression HUVECs. F, Network formation of CT and LMO2 KD HUVECs with or without supplementation of TGF‐β 10 ng/mL. G, Chromatin immunoprecipitation PCR analysis of LMO2 complex binding to TGF‐β1 promoter (–901 and –4030 region) in both CT and LMO2 KD cells. Data are presented as mean±SEM (n=3). *P<0.05 vs CT.
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jah31811-fig-0004: LMO2 knockdown (KD) impaired endothelial network formation, which can be rescued by exogenous transforming growth factor‐β1 (TGF‐β1). A, Network formation of control (CT) and LMO2 KD human umbilical vein endothelial cells (HUVECs). B, List of downregulated genes identified in LMO2 KD in comparison to CT HUVECs using angiogenesis polymerase chain reaction (PCR) array. C, Reverse transcription PCR (RT‐PCR) of TGF‐β1. D, ELISA for TGF‐β levels in culture medium of CT and LMO2 KD cells. E, RT‐PCR of TGF‐β1 in CT and LMO2 overexpression HUVECs. F, Network formation of CT and LMO2 KD HUVECs with or without supplementation of TGF‐β 10 ng/mL. G, Chromatin immunoprecipitation PCR analysis of LMO2 complex binding to TGF‐β1 promoter (–901 and –4030 region) in both CT and LMO2 KD cells. Data are presented as mean±SEM (n=3). *P<0.05 vs CT.
Mentions: The LMO2 KD impaired endothelial network formation in matrigel (Figure 4A). Further analysis of 84 angiogenesis‐related genes using the Qiagen Angiogenesis RT Profiler PCR Array revealed that there was a substantial reduction in the expression of core angiogenesis genes (33 of 84; Figure 4B) in the LMO2 KD cells. Among them, TGF‐β1 promoter region is predicted to have LMO2 complex binding sites, indicating that it could be a direct transcriptional target of LMO2. We confirmed by RT‐PCR (Figure 4C) and ELISA (Figure 4D) that TGF‐β1 was downregulated in LMO2 KD cells. To determine the contribution of TGF‐β downregulation to impaired angiogenic processes in LMO2 KD cells, we determined whether network formation could be rescued by TGF‐β. Indeed, supplementation of the HUVEC LMO2 KD with exogenous TGF‐β restored defective network formation (Figure 4F). ENCODE database prediction suggests that two regions on TGF‐β1 (Gene ID: 7040) promoter could potentially have LMO2 complex binding. One region, CCGCAGCTGCTGC, starts at the proximal –901 position and the other region, CAGATAGGGG, starts at the distal –4030 position. The ChIP assay revealed that LMO2 complex binds to both regions (Figure 4G), confirming that TGF‐β1 is a direct transcriptional target of LMO2. Interestingly, in LMO2 KO cells, the decreased binding of LMO2 complex was only observed in the proximal –901 binding site, suggesting that this binding site is more sensitive to LMO2 downregulation. Notably, OE of LMO2 did not further increase TGF‐β1 level, probably because LMO2 binding to DNA involves the participation of other components of the binding complex (Figure 4E). This explanation is consistent with the observation that LMO2 OE did not further promote endothelial network formation (Figure S3).

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