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Forskolin modifies retinal vascular development in Mrp4-knockout mice
Wataru Matsumiya1, Sentaro Kusuhara, Keiko Hayashibe
1Division of Ophthalmology, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
Purpose:
Multidrug resistance protein 4 (MRP4) effluxes a wide variety of endogenous compounds, including cyclic adenosine monophosphate (cAMP), and is exclusively expressed in vascular endothelial cells (ECs) of the retina. This study aimed to investigate the role of MRP4 in retinal vascular development.
Methods:
The retinal vascular phenotype of Mrp4(-/-) mice was examined by whole-mount immunohistochemistry at P3, P6, and P14. The retinas from P6 pups that received an intraperitoneal injection of either solvent control or forskolin, an inducer of intracellular cAMP formation, at P4 and P5 were analyzed in terms of their vascular formation (vascular length, vascular branching, vascular density, and the number of tip cells), cell proliferation and apoptosis, and vessel stability.
Results:
The Mrp4(-/-) mice exhibited no overt abnormalities in the development of the retinal vasculature, but retinal vascular development in the Mrp4(-/-) mice was suppressed in response to forskolin administration. There was a significant decrease in the vascular length, vascular branching, and vascular density, and inhibited tip cell formation at the vascular front. The forskolin-treated Mrp4(-/-) mice showed an increased number of Ki67-positive and cleaved caspase 3-positive ECs, a significant decrease in the amount of pericyte coverage, and a reduced number of empty sleeves. In pups exposed to hyperoxia (75% oxygen) from P7 to P12, the Mrp4(-/-) mice showed a significant increase in the unvascularized retinal area.
Conclusions:
Mrp4(-/-) mice exhibited suppressed retinal vascular development in response to forskolin treatment. Thus, Mrp4 might have protective roles in retinal vascular development by regulating the intracellular cAMP level.
Insights
Multidrug resistance protein 4 (MRP4) normally supports retinal vascular development. Loss of MRP4 function suppressed vascular growth when cAMP levels were artificially increased, suggesting a protective role.
Area of Science:
- Ophthalmology
- Vascular Biology
- Molecular Biology
Background:
- Multidrug resistance protein 4 (MRP4) is expressed in retinal vascular endothelial cells (ECs) and effluxes cyclic adenosine monophosphate (cAMP).
- MRP4's role in retinal vascular development is not fully understood.
Purpose of the Study:
- To investigate the function of MRP4 in the development of retinal vasculature.
- To determine if MRP4 influences retinal vascular growth by regulating intracellular cAMP levels.
Main Methods:
- Retinal vascularization in Mrp4(-/-) mice was assessed using whole-mount immunohistochemistry.
- Mice were treated with forskolin (a cAMP inducer) or exposed to hyperoxia to evaluate vascular formation, cell proliferation, apoptosis, and pericyte coverage.
Main Results:
- Mrp4(-/-) mice showed suppressed retinal vascular development, reduced vascularization, and inhibited tip cell formation when treated with forskolin.
- Forskolin-induced treatment in Mrp4(-/-) mice led to increased EC proliferation and apoptosis, decreased pericyte coverage, and more "empty sleeves."
- Mrp4(-/-) mice exhibited increased avascular areas under hyperoxic conditions.
Conclusions:
- MRP4 plays a protective role in retinal vascular development.
- Regulating intracellular cAMP levels by MRP4 is crucial for normal retinal vascular growth and stability.
