Effects of Angiogenic Growth Factor Combinations on Retinal Endothelial Cells☆

doi:10.1006/exer.2001.1161

Experimental Eye Research

Volume 74, Issue 4, April 2002, Pages 523-535

https://doi.org/10.1006/exer.2001.1161 Get rights and content

Abstract

The aim of this paper was to determine if growth factors, known to be upregulated in proliferative diabetic retinopathy, exerted combined effects on retinal endothelial cells. The authors explored the individual and collective actions of insulin-like growth factor I (IGF-I), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor-2 (FGF-2) and placenta growth factor (PlGF) on several parameters that reflect the angiogenic potential of endothelial cells. The effect of growth factors on cell migration and survival/proliferation was examined using primary cultures of bovine retinal endothelial cells (BREC). The authors also determined the growth factor action on capillary-like tube formation on a reconstituted basement membrane matrix and on the newly described phenomenon of secondary sprouting, in which endothelial cell colonies spontaneously survive, proliferate, migrate and invade the matrix after the original capillary-like tubes have collapsed. Sprouting cells were positive for von Willebrand factor and could aggregate into larger tubes with lumens. Incubation with VEGF+IGF-I or PlGF+FGF-2 enhanced tube stability by 40–50%, more than each growth factor alone or other combinations (5–20%). The concurrent addition of four growth factors did not improve the response seen with growth factor pairs. Surprisingly, PDGF-BB induced tube collapse. IGF-I and FGF-2 mildly enhanced BREC proliferation/survival (5–15%). However, VEGF+IGF-I or PlGF+FGF-2 increased BREC proliferation/survival by 25% under low serum conditions, whereas combinations of all four growth factors exerted a clearly synergistic effect (250% increase). PDGF-BB or FGF-2 stimulated secondary sprouting and were the only factors capable of exerting this effect alone. Even though VEGF, IGF-I or PlGF were not effective, if administered in pairs, they demonstrated increased responses. PDGF-BB was also able to enhance the effect of FGF-2+IGF-I+VEGF on BREC secondary sprouting, but not of any of them individually. No other growth factor tested was able to significantly improve the action of combinations of three other growth factors. VEGF increased cell migration in a wounded monolayer assay two-fold and PDGF-BB, 2.5 times, but other individual growth factors were ineffective. PlGF+FGF-2 enhanced cell migration more than each factor alone. VEGF+IGF-I+PlGF+FGF-2, however, increased cell migration four-fold. In summary, this study indicates that growth factors, overexpressed in diabetic retinopathy eyes, enhance the angiogenic characteristics of cultured cells (tube formation, proliferation, secondary sprouting and migration). Their effects, however, can be greatly augmented by other growth factors that alone exert little or no action. Therefore, diabetic retinal neovascularization may result from the additive or synergistic action of several growth factors.

References (67)

A.P. Adamis et al.
Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy
Am. J. Ophthalmol.
(1994)
L.P. Aiello et al.
Diabetic eye disease
Endocrinol. Metab. Clin. North Am.
(1996)
Y. Cao et al.
Heterodimers of placenta growth factor/vascular endothelial growth factor. Endothelial activity, tumor cell expression, and high affinity binding to Flk-1/KDR
J. Biol. Chem.
(1996)
J. DiSalvo et al.
Purification and characterization of a naturally occurring vascular endothelial growth factor–placenta growth factor heterodimer
J.Biol. Chem.
(1995)
R.O. Dull et al.
Kinetics of placenta growth factor/vascular endothelial growth factor synergy in endothelial hydraulic conductivity and proliferation
Microvasc. Res.
(2001)
I. Grierson et al.
Hepatocyte growth factor/scatter factor in the eye
Prog. Retin. Eye Res.
(2000)
H. Kurz et al.
Automated evaluation of angiogenic effects mediated by VEGF and PlGF homo- and heterodimers
Microvasc. Res.
(1998)
S. Liekens et al.
Angiogenesis: regulators and clinical applications
Biochem. Pharmacol.
(2001)
H. Ozaki et al.
Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization
Am. J. Pathol.
(2000)
M.S. Pepper et al.
Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro
Biochem. Biophys. Res. Commun.
(1992)

R.S. Piotrowicz et al.

Inhibition of cell migration by 24-kDa fibroblast growth factor-2 is dependent upon the estrogen receptor

J. Biol. Chem.

(2001)

P.A. Randazzo et al.

Characterization of the growth of murine tibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

Exp. Cell Res.

(1990)

M.J. Tolentino et al.

Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate

Ophthalmology

(1996)

S. Vukicevic et al.

Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components

Exp. Cell Res.

(1992)

L.P. Aiello

Vascular endothelial growth factor and the eye: biochemical mechanisms of action and implications for novel therapies

Ophthalmic Res.

(1997)

L.P. Aiello

Vascular endothelial growth factor. 20th-century mechanisms, 21st-century therapies

Invest. Ophthalmol. Vis. Sci.

(1997)

L.P. Aiello et al.

Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders

N. Engl. J. Med.

(1994)

L.P. Aiello et al.

Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective β-isoform-selective inhibitor

Diabetes

(1997)

L.P. Aiello et al.

Diabetic retinopathy

Diabetes Care

(1998)

L.P. Aiello et al.

Molecular mechanisms of growth factor action in diabetic retinopathy

Curr. Opin. Endocrinol. Diabetes

(1999)

L.P. Aiello et al.

Role of vascular endothelial growth factor in diabetic vascular complications

Kidney Int.

(2000)

S. Baatout

Endothelial differentiation using Matrigel (review)

Anticancer Res.

(1997)

R. Benelli et al.

In vitro models of angiogenesis: the use of Matrigel

Int. J. Biol. Markers

(1999)

R. Birkenhager et al.

Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor

Biochem. J.

(1996)

M. Boulton et al.

Intravitreal growth factors in proliferative diabetic retinopathy: correlation with neovascular activity and glycaemic management

Br. J. Ophthalmol.

(1997)

R. Burgos et al.

Vitreous levels of IGF-I, IGF binding protein 1, and IGF binding protein 3 in proliferative diabetic retinopathy: a case-control study

Diabetes Care

(2000)

S.C. Butterwith et al.

Regulation of adipocyte precursor DNA synthesis by acidic and basic fibroblast growth factors: interaction with heparin and other growth factors

J. Endocrinol.

(1993)

P.A. Campochiaro

Retinal and choroidal neovascularization

J. Cell Physiol.

(2000)

A. Canton et al.

Hepatocyte growth factor in vitreous and serum from patients with proliferative diabetic retinopathy

Br. J. Ophthalmol.

(2000)

Y. Cao et al.

In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor

J. Clin. Invest.

(1996)

P. Carmeliet et al.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions

Nat. Med.

(2001)

S.R. Edmondson et al.

Interactions between growth hormone, insulin-like growth factor I, and basic fibroblast growth factor in melanocyte growth

J. Clin. Endocrinol. Metab.

(1999)

C. Favard et al.

Vascular endothelial growth factor and retinal neovascularisation: a new therapeutic approach for diabetic retinopathy

Diabetes Metab.

(1996)

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^☆: Address correspondence to: Raquel Castellon, Cedars-Sinai Medical Center, Ophthalmology Research Laboratories, 8700 Berverly Boulevard, Davis-2025, Los Angeles, CA 90048, U.S.A. E-mail: [email protected]

^f2: ^☆Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, FL, May 2001.

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Regular ArticleEffects of Angiogenic Growth Factor Combinations on Retinal Endothelial Cells☆☆

Abstract

Am. J. Ophthalmol.

Endocrinol. Metab. Clin. North Am.

J. Biol. Chem.

J.Biol. Chem.

Microvasc. Res.

Prog. Retin. Eye Res.

Microvasc. Res.

Biochem. Pharmacol.

Am. J. Pathol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Exp. Cell Res.

Ophthalmology

Exp. Cell Res.

Vascular endothelial growth factor and the eye: biochemical mechanisms of action and implications for novel therapies

Ophthalmic Res.

Vascular endothelial growth factor. 20th-century mechanisms, 21st-century therapies

Invest. Ophthalmol. Vis. Sci.

Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders

N. Engl. J. Med.

Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective β-isoform-selective inhibitor

Diabetes

Diabetic retinopathy

Diabetes Care

Molecular mechanisms of growth factor action in diabetic retinopathy

Curr. Opin. Endocrinol. Diabetes

Role of vascular endothelial growth factor in diabetic vascular complications

Kidney Int.

Endothelial differentiation using Matrigel (review)

Anticancer Res.

In vitro models of angiogenesis: the use of Matrigel

Int. J. Biol. Markers

Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor

Biochem. J.

Intravitreal growth factors in proliferative diabetic retinopathy: correlation with neovascular activity and glycaemic management

Br. J. Ophthalmol.

Vitreous levels of IGF-I, IGF binding protein 1, and IGF binding protein 3 in proliferative diabetic retinopathy: a case-control study

Diabetes Care

Regulation of adipocyte precursor DNA synthesis by acidic and basic fibroblast growth factors: interaction with heparin and other growth factors

J. Endocrinol.

Retinal and choroidal neovascularization

J. Cell Physiol.

Hepatocyte growth factor in vitreous and serum from patients with proliferative diabetic retinopathy

Br. J. Ophthalmol.

In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor

J. Clin. Invest.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions

Nat. Med.

Interactions between growth hormone, insulin-like growth factor I, and basic fibroblast growth factor in melanocyte growth

J. Clin. Endocrinol. Metab.

Vascular endothelial growth factor and retinal neovascularisation: a new therapeutic approach for diabetic retinopathy

Diabetes Metab.

Regular Article
Effects of Angiogenic Growth Factor Combinations on Retinal Endothelial Cells^☆☆