Korean J Physiol Pharmacol.  2016 Sep;20(5):533-538. 10.4196/kjpp.2016.20.5.533.

Regulation of retinal angiogenesis by endothelial nitric oxide synthase signaling pathway

• Ha JM
• Jin SY
• Lee HS
• Shin HK
• Lee DH
• Song SH
• Kim CD
• Bae SS

• KMID: 2350510
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.533

Abstract

Angiogenesis plays an essential role in embryo development, tissue repair, inflammatory diseases, and tumor growth. In the present study, we showed that endothelial nitric oxide synthase (eNOS) regulates retinal angiogenesis. Mice that lack eNOS showed growth retardation, and retinal vessel development was significantly delayed. In addition, the number of tip cells and filopodia length were significantly reduced in mice lacking eNOS. Retinal endothelial cell proliferation was significantly blocked in mice lacking eNOS, and EMG-2-induced endothelial cell sprouting was significantly reduced in aortic vessels isolated from eNOS-deficient mice. Finally, pericyte recruitment to endothelial cells and vascular smooth muscle cell coverage to blood vessels were attenuated in mice lacking eNOS. Taken together, we suggest that the endothelial cell function and blood vessel maturation are regulated by eNOS during retinal angiogenesis.

Keyword

Angiogenesis; eNOS; Proliferation; Retina; Signal transduction

MeSH Terms

Animals
Blood Vessels
Embryonic Development
Endothelial Cells
Female
Mice
Muscle, Smooth, Vascular
Nitric Oxide Synthase Type III*
Pericytes
Pregnancy
Pseudopodia
Retina
Retinal Vessels
Retinaldehyde*
Signal Transduction
Nitric Oxide Synthase Type III
Retinaldehyde

Figure

• 

  Fig. 1 eNOS plays an essential role in retinal angiogenesis(A)A comparison of the body size between the wild type and eNOS knockout littermates was visualized by a digital camera at P6. (B) Lung, liver, spleen, heart, aorta, and retina were isolated from the wild type or eNOS-deficient mice, and the expression of eNOS was verified by a Western blot analysis with the indicated antibodies. (C and D) Retinas were isolated from the wild type and eNOS knockout mice at P6 and stained with IB4 (green). Angiogenesis was analyzed by measuring the angiogenic area and distance. Images were captured on confocal microscope at ×2.5 (zoom ×0.5) magnification. Angiogenic area and sprouting distance were quantified using Image J (National Institutes of Health, MD, USA) software. White arrowheads indicate optic nerve (ON). Data are presented as the means±SEM. Asterisks indicate statistical significance (p<0.05). Scale bars, 800 µm.

• 

  Fig. 2 eNOS regulates the activation of tip cells during retinal angiogenesis(A) Retinas from the wild type and eNOS-deficient mice at P6 were stained with IB4 (green). (B) Tip cell number and filopodia length were measured using Image J (National Institutes of Health, MD, USA) software. Images were visualized on confocal microscope at ×80 magnification. Data are presented as the means±SEM. Asterisks indicate statistical significance (p<0.05). Scale bars, 100 µm.

• 

  Fig. 3 eNOS is necessary for endothelial cell proliferation and angiogenic sprouting(A and B) Retinas isolated from the wild type and eNOS knockout mice at P6 were stained with IB4 (green) and pH3 (red). Images were visualized on confocal microscope at ×20 magnification. White arrowheads indicate pH3-positive cells. The number of pH3-positive cells was quantified using Image J (National Institutes of Health, MD, USA) software. Data are presented as the means±SEM. Asterisks indicate statistical significance (p<0.05). Scale bars, 100 µm. (C) Aortas were isolated from the wild type and eNOS knockout mice, and embedded in growth factor-reduced matrigel-coated plates in the presence of EGM-2. After 6 days, bright field images were captured under a microscope at ×5 magnification. Scale bars, 500 µm.

• 

  Fig. 4 eNOS regulates pericyte recruitment and VSMC coverage(A and B) Retinas from the wild type and eNOS knockout mice at P6 were stained with IB4 (blue) and either pericyte marker protein (NG2, red, panel A) or VSMC marker protein (SM22α, red, panel B). Images were visualized under a confocal microscope at ×40 magnification. Scale bars, 100 µm.

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