β3-Adrenergic receptors mediate choroidal endothelial cell invasion, proliferation, and cell elongation

doi:10.1016/j.exer.2004.08.015

Experimental Eye Research

Volume 80, Issue 1, January 2005, Pages 83-91

https://doi.org/10.1016/j.exer.2004.08.015 Get rights and content

Abstract

β₃-Adrenergic receptors have been reported to function primarily in adipose tissues to regulate thermogenesis. In this study, we determined if β-adrenergic receptors are present on human choroidal endothelial cells and examined their ability to promote invasion, proliferation, and/or cell elongation. Using western blotting techniques and assays of cell invasion, cell proliferation, and endothelial cell elongation, we were able to determine that human choroidal endothelial cells do possess all three subtypes of β-adrenergic receptors. Stimulation of the β₃-adrenergic receptor with BRL37344, a specific β₃-adrenergic receptor agonist, resulted in phosphorylation of Src, Akt, and ERK1/2. BRL37344 treatment also increased choroidal endothelial cell invasion by 103% above control values; the invasion response was inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), Akt inhibitor (Akt-I), and matrix metalloproteinase 2/9 inhibitor (MMP-I). Invasion was not affected by PD98059 (mek inhibitor) or KT5823 (protein kinase G inhibitor). BRL37344 produced a significant increase in the total elongation of choroidal endothelial cells formed on Matrigel over a 24 hr period. BRL37344 did significantly increase proliferation, although not to the same level as invasion. Stimulation of choroidal endothelial cells with dobutamine to activate β₁/β₂-adrenergic receptors did not affect invasion, proliferation, or endothelial cell elongation. In conclusion, β₃-adrenergic receptors may play a role in choroidal endothelial cell invasion and elongation, while playing a more limited function in regulation of cell proliferation.

Introduction

Age-related macular degeneration is the predominant cause of blindness in the elderly population (O'Shea, 1998). There are two different etiologies of macular degeneration, wet and dry. ‘Dry’ macular degeneration is characterised by hard exudate formation without neovascularisation. The ‘wet’ form of macular degeneration can be detected by the appearance of new blood vessel growth into the macular region of the eye. The exact causes of this disease are uncertain, yet it is clear that choroidal neovascularisation is a hallmark of the ‘wet’ form of the disease (Friedman, 1997). Aging can be associated with slowed or dysfunctional responses of the autonomic nerves, particularly sympathetic nerves (Schmidt et al., 1993). Little work has attempted to correlate dysfunctions in sympathetic innervation or neurotransmission with changes in vascular growth, as may occur in age-related macular degeneration.

While investigating sympathetic nerve regulation of ocular blood flow, we found that superior cervical ganglionectomy increased choroidal blood flow over 4-fold in the treated eye relative to a non-surgical control eye (Steinle et al., 2002a, Steinle et al., 2002b). Sympathectomy also produced significant increases in numbers of choroidal venules and arterioles (Steinle et al., 2002a, Steinle et al., 2002b). Additionally, we determined that the β-adrenergic receptor antagonist propranolol, a blocker of β₁ and β₂-adrenergic receptors, produced changes in the choroid similar to those after sympathectomy (Steinle and Smith, 2002).

β₁- and β₂-adrenergic receptors have been localised on a number of endothelial cells (Wang et al., 2000). β₃-adrenergic receptors, on the other hand, have been primarily localised to tissues involved in fat mobilisation and thermogenesis (Krief et al., 1993, Arch, 2001). Recently, we reported that retinal endothelial cells possess both β₃- and β₁-adrenergic receptors (Steinle et al., 2003). While retinal endothelial cells certainly may be involved in retinopathy, the endothelial cells lining the primary vascular bed of the eye, the choroid, are more likely to be involved in neovascularisation associated with age-related macular degeneration. However, because of a limited donor supply, work on human ocular endothelial cells has been lacking. The goal of this study was to determine which β-adrenergic receptor subtypes are present on human choroidal endothelial cells. Furthermore, we wanted to ascertain whether activation of these receptor subtypes could produce choroidal endothelial cell invasion, proliferation, and elongation, some common markers of angiogenesis.

Section snippets

Endothelial cell cultures

All usage of human tissues and cells in this study was in accordance with IRB approved protocols. Endothelial cell cultures were established using a protocol previously described (Silverman et al., 2001). Briefly, choroid tissue from anonymously donated human eyes (Lions Eye Bank, Portland, OR; donor age range 0–50 y/o in this study; no known history of ocular or cardiovascular disease) was digested in 0.2% type II collagenase (Sigma, St Louis, MO) in medium MCDB-131 for 20–30 min at 37°C.

β₃-Adrenergic receptor expression on choroidal endothelial cells

Choroidal endothelial cells express β₃-adrenergic receptor on the outer membrane (Fig. 1B). MM1 cells have limited β₃-adrenergic receptor expression (Fig. 1A).

β-Adrenergic receptor subtype protein expression in choroidal endothelial cells

Protein lysates from choroidal endothelial cells express β₃-adrenergic receptor (Fig. 2A). Additionally, both β₁ and β₂-adrenergic receptors are present in choroidal endothelial cell lysates, while retinal endothelial cell lysates contain only β₁-adrenergic receptor protein (Fig. 2B and C, respectively).

BRL37344 treatments causes phosphorylation of Akt, Src, and ERK1/2

Treatment of choroidal endothelial

Choroidal endothelial cells express all three subtypes of β-adrenergic receptors

We have previously found that propranolol, a β-adrenergic receptor antagonist, produces significant changes in the vascularity of the choroid (Steinle and Smith, 2002). Recently, we reported the retinal endothelial cells derived from blood vessels of the eye possess both β₃ and β₁-adrenergic receptors, with no noted expression of the β₂-subtype (Steinle et al., 2003). Unlike the blood vessels of the retina that are under strict autoregulation to maintain constant flow, the choroidal blood

Acknowledgements

Grant support. This work was supported by an NRSA from NHLBI (455991 to JJS); AHA SDG#0430344Z (JJS); R01 from NIH NHLBI (58062 to HJG); R01 from NIH NEI (EY06484 to JTR), NEI training grant #EY07123 and Casey Eye Institute Core Grant #EY10572, and a grant from Research to Prevent Blindness.

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β3-Adrenergic receptors mediate choroidal endothelial cell invasion, proliferation, and cell elongation

Abstract

Introduction

Section snippets

Endothelial cell cultures

β3-Adrenergic receptor expression on choroidal endothelial cells

β-Adrenergic receptor subtype protein expression in choroidal endothelial cells

BRL37344 treatments causes phosphorylation of Akt, Src, and ERK1/2

Choroidal endothelial cells express all three subtypes of β-adrenergic receptors

Acknowledgements

J. Immunol. Methods

Am. J. Ophthalmol.

J. Biol. Chem.

Exp. Eye Res.

J. Biol. Chem.

FEBS Lett.

The beta 3-adrenergic system and beta 3-adrenergic agonists

Rev. Endocr. Metab. Disord.

Reduced choroidal neovascular membrane formation in matrix metalloproteinase-2-deficient mice

Invest. Ophthalmol. Vis. Sci.

Tissue distribution of beta 3-adrenergic receptor mRNA in man

J. Clin. Invest.

β₃-Adrenergic receptors mediate choroidal endothelial cell invasion, proliferation, and cell elongation

β₃-Adrenergic receptor expression on choroidal endothelial cells