Upregulation of placental growth factor by vascular endothelial growth factor via a post-transcriptional mechanism

FEBS 29269

FEBS Letters 579 (2005) 1227–1234

Upregulation of placental growth factor by vascular endothelial growth factor via a post-transcriptional mechanism Yong-Gang Yao, Hoseong S. Yang, Zhiming Cao, Jennifer Danielsson, Elia J. Duh* Department of Ophthalmology, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Jefferson 3-109, Baltimore, MD 21287, USA Received 18 November 2004; revised 2 January 2005; accepted 10 January 2005 Available online 21 January 2005 Edited by Ned Mantei

Abstract Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are key angiogenic stimulators during normal development and wound healing, as well as in a variety of pathological conditions. Recent studies have demonstrated a synergistic effect of VEGF and PlGF in pathological angiogenesis and suggest a role for PlGF in amplifying VEGF action in endothelial cells. We show here in the mouse model of oxygen-induced retinopathy that VEGF is significantly increased (P < 0.01) in the retina at both the mRNA and protein levels. In this mouse model, PlGF was significantly upregulated in the retina at the protein level (P < 0.01) without a corresponding change in mRNA levels. In cultured human retinal and umbilical vein endothelial cells, VEGF induced the production of PlGF protein by over 10-fold (P < 0.01) in a dose-dependent manner through a post-transcriptional mechanism. The increased PlGF expression upon VEGF treatment was significantly reduced by inhibition of the protein kinase C (PKC) and MEK signaling pathways, as well as by treatment with the calcium ionophore A23187. Taken together, our findings demonstrate that VEGF can amplify its effects on endothelial cells by inducing the production of PlGF via a post-transcriptional mechanism in a PKC-dependent manner, and provide a potential link between PKC inhibition and amelioration of vascular complications in the development of angiogenic diseases. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Vascular endothelial growth factor; Placental growth factor; Proliferative diabetic retinopathy; Angiogenesis; Endothelial cell; Protein kinase C

1. Introduction The role of growth factors in angiogenic disorders including retinal neovascularization has received great attention. Although many factors are likely to be important in the development of retinal neovascularization, it is clear that vascular endothelial growth factor (VEGF), in particular, plays a major role. Intraocular VEGF levels are significantly in*Corresponding author. Fax: +1 410 502 5244. E-mail address: [email protected] (E.J. Duh). Abbreviations: VEGF, vascular endothelial growth factor; PlGF, placental growth factor; HUVEC, human umbilical endothelial cell; HREC, human retinal endothelial cell

creased in patients with retinal neovascularization [1,2]. In animal models of ischemic retinopathy, VEGF expression correlates temporally and spatially with ocular neovascularization [3,4]. Increased expression of VEGF in the retina is sufficient to cause retinal and subretinal neovascularization [5], whereas inhibition of VEGF or VEGF signaling significantly reduces retinal neovascularization in animal models [6–10]. In addition to VEGF, however, other factors are likely to play a significant role in retinal neovascularization, and may therefore serve as additional targets for therapy. Placental growth factor (PlGF) has emerged as an important proangiogenic factor. PlGF is a member of the ‘‘VEGF family’’ of structurally related growth factors [11]. Unlike VEGF-A (which binds both VEGFR-1 and VEGFR-2), PlGF specifically binds VEGFR-1. Studies of PlGF-deficient mice have demonstrated that PlGF is required for pathologic angiogenesis, including retinal [12] and choroidal neovascularization [13]. Such studies have also demonstrated an important role for PlGF in plasma extravasation and collateral vessel growth during ischemia and wound healing [12], as well as inflammation [12,14]. PlGF has been found to stimulate angiogenesis and collateral growth in ischemic heart and limb with an efficiency comparable to that of VEGF [12,15,16]. PlGF and VEGF have been demonstrated to play a synergistic role in pathologic angiogenesis [12], and PlGF is thought to ‘‘amplify’’ VEGF-driven angiogenesis and activation of vascular endothelial cells [12,17]. PlGF could therefore represent an additional and perhaps safer target (given its binding specificity for VEGFR-1), for the treatment of neovascular disorders [18]. In this study, we sought to further explore the potential role of PlGF in retinal neovascularization by investigating the expression of PlGF both in vivo and in vitro. We found a significant increase in PlGF protein in mouse retinal tissue during neovascularization, which accompanied an increase in VEGF levels. We further studied the regulation of PlGF production in cultured endothelial cells and found that PlGF is upregulated by VEGF and phorbol-12-myristate-13-Acetate (PMA). While PMA increased PlGF mRNA, VEGF upregulation of PlGF was at the protein level and was dependent on the protein kinase C (PKC) signaling pathway, thus representing a potentially novel mechanism of regulation by VEGF. Our results suggest that VEGF can amplify its effects on endothelial cells by inducing endothelial cell production of PlGF.

0014-5793/$30.00 Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2005.01.017

1228

2. Materials and methods 2.1. Animal studies Oxygen-induced retinopathy was elicited in mice as previously described [19]. In brief, postnatal day 7 (P7) C57BL/6J mice were exposed to 75% O2, along with their nursing mother for 5 days. On P12, the mice were returned to room air. The mice were sacrificed at the indicated times. Retinas were extracted, snap-frozen in liquid nitrogen, and stored at
80 °C until further processing. For ELISA studies, total protein was harvested as previously described [20]. RNA was extracted from retinas using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturerÕs instructions. 2.2. Cell culture Human umbilical endothelial cells (HUVECs, Clonetics Corporation, San Diego, CA) and human retinal endothelial cells (HRECs, Cell Systems, Inc., Kirkland, WA) were cultured in EGM2-MV medium (Clonetics Corporation, San Diego, CA) and used between passages 4 and 10. HRECs were cultured on fibronectin-coated dishes or plates. Cells were grown to confluence in EGM2-MV medium in 12-well plates and washed with phosphate-buffered saline, switched to DulbeccoÕs modified EagleÕs medium (DMEM) containing 10% calf serum (CS), and treated with the following stimulators and/or signaling pathway inhibitors: human recombinant VEGF, TNF-a, and basic fibroblast growth factor (bFGF) were purchased from R&D Systems, Inc. (Minneapolis, MN); PD98059, U0126, GF109203X, Calphostin C, SB203580, LY294002, PP2 (4-amino-5-(4-chlorophenyl)-7-(tbutyl)pyrazolo[3,4-d]pyrimidine), cyclosporin A, and PMA were purchased from CalBiochem (San Diego, CA). LY333531, a specific PKCb isoform inhibitor, was from A.G. Scientific, Inc. (San Diego, CA). Culture medium was collected at the indicated time points and stored at
20 °C. Cells were harvested in Trizol and total RNA was isolated according to the manufacturerÕs instructions.

Y.-G. Yao et al. / FEBS Letters 579 (2005) 1227–1234 PCR for mouse VEGF was performed using primer pair mVEGF-F (5 0 -TTACTGCTGTACCTCCACC-3 0 )/mVEGF-R (5 0 -ACAGGACGGCTTGAAGATG-3 0 ) [24]. Real-time PCR for mouse PlGF was performed using primer pair mPGF-1198F (5 0 -TGAAGGCATGTAGAGGGGAC-3 0 )/mPGF-1370R (5 0 -CACTCTGCCTGTGTTCCAGA-3 0 ), which is located in exon 7 of mouse PlGF. Murine genes were normalized to mouse GAPDH, which was amplified using primer pair GAPDH-F (5 0 -AACGACCCCTTCATTGAC-3 0 )/GAPDH-R (5 0 TCCACGACATACTCAGCAC-3 0 ). 2.5. Statistical analyses All experiments were performed at least three times and results were expressed as means ± S.E. Statistical differences were assessed by the two-tailed t-test. A value of P < 0.05 was considered as statistically significant.

3. Results

2.3. ELISA assay PlGF levels in cell culture supernatants and lysates were measured by the Quantikine PlGF assay kit (R&D Systems) according to the manufacturerÕs instructions. Serial dilutions of recombinant human PlGF were included in all assays to serve as a standard. In our hands, this assay has a limit of sensitivity of 15.625 pg/ml. ELISA assay of recombinant human VEGF (100 ng/ml) using the Quantikine PlGF assay kit demonstrated no cross-reactivity, consistent with the manufacturerÕs specifications (R&D Systems). The VEGF and PlGF concentrations in mouse retina samples were detected using the mouse VEGF and PlGF-2 Quantikine ELISA kits, respectively (R&D Systems).

3.1. Increased PlGF protein, but not mRNA levels, in mouse oxygen-induced retinopathy model We investigated PlGF and VEGF protein and mRNA levels in the mouse model of oxygen-induced retinopathy [19]. In this experimental model, C57BL/6 mice are exposed to high oxygen from postnatal day 7 (P7) to P12, which results in extensive retinal capillary obliteration. On return of these mice to room air on P12, the inner retina becomes relatively hypoxic, which results in the formation of retinal neovascularization in 100% of animals by P17 [19]. Retinal VEGF mRNA expression has been found to be significantly increased in this model beginning at P12.5 [4]. In our experiment, there was a significant increase of retinal VEGF at both the mRNA and protein levels in P13 mice compared with P12 mice (P < 0.01), and the increased level of VEGF was maintained on P16 (Fig. 1). In addition, the mouse PlGF protein in the retina increased in a similar pattern to that of VEGF. However, real-time PCR for mouse PlGF revealed no increase of PlGF mRNA, and the mRNA level at P13 was actually slightly decreased compared with P12 (Fig. 1). Taken together, our results indicate that the increase of PlGF is regulated at the protein level.

2.4. Northern blot and quantitative real-time PCR For Northern blot analysis, samples of RNA (20 lg per lane) were size-fractionated on 1% agarose–formaldehyde gel, transferred onto a Gene Screen Hybridization transfer membrane (NEN Life Science), and UV crosslinked. After prehybridization for 2 h at 42 °C, the membrane was hybridized with 32P-labeled PlGF probe overnight. The probe was a 738-bp fragment (residing in the 3 0 -UTR of PlGF) purified from the NotI/EcoRI digested products of IMAGE Clone 2141182 and labeled using Prime It II Random Prime Labeling Kit (Stratagene). The blot was then washed three times, for 20 min at 42 °C, and exposed to Imaging Screen K-HD (Bio-Rad, Hercules, CA) for 24 h at
80 °C. Single-stranded cDNA was synthesized from 1 to 2 lg total RNA using an oligo(dT) 18mer as primer and the M-MLV Reverse Transcriptase (Promega Corp.) in a final reaction volume of 25 ll. Real-time PCR for human PlGF was performed with the QuantiTect SYBR Green PCR Kit (Qiagen Inc.) and LightCycler system (Roche Diagnostics Corporation) by using primer pair f581 (5 0 -GTTCAGCCCATCCTGTGTCT-3 0 )/r668 (5 0 -TTAGGAGCTGCATGGTGACA3 0 ), which targets exon 3 of PlGF and is present in all PlGF isoforms [21,22], according to human BLAT search (http://www.genome.ucsc.edu/cgi-bin/hgBlat). A thermal cycle at 95 °C for 15 min was used prior to cycling to activate the HotStarTaq DNA Polymerase, then followed by 30 cycles at 94 °C for 15 s, 55 °C for 20 s; and 72 °C for 10 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as described [23] and used as the reference for normalization. Real-time

3.2. VEGF increases PlGF protein production in cultured endothelial cells Endothelial cells are known to be an important source of PlGF [18,25]. We postulated that regulation of endothelial cell PlGF production might contribute to increased PlGF levels in mouse retina during neovascular conditions. Since VEGF is a major regulator of endothelial cells and we observed a correlation between PlGF and VEGF levels in mouse retina, we tested the kinetics of PlGF production in response to VEGF treatment in cultured HUVECs and HRECs. We collected the culture medium at designated time points and determined PlGF levels by ELISA. The concentration of PlGF in the culture supernatant showed a time-dependent change, with increase after 4 h VEGF treatment and peak increase after 24 h of VEGF stimulation (Fig. 2(a)). The regulation of PlGF production by VEGF was dose-dependent. VEGF at 2.5 ng/ml slightly increased PlGF compared with untreated controls. PlGF production was significantly upregulated by VEGF at 25 ng/ml, and upregulation of PlGF reached a plateau at a VEGF concentration of 100 ng/ml (Fig. 2(a)), with at least 10-fold increased PlGF production (Fig. 2(b) and (c)). We also

Y.-G. Yao et al. / FEBS Letters 579 (2005) 1227–1234

(a)

12

**

**

10

**

1800

**

4 2

1400 1200 1000 800

**

600 400 200

0 P12

P13

P14

0

P16

0.8

**

0.7

4h CN

(b)

0.6

4h VEGF

8h VEGF

0.5

**

0.4 0.3 0.2 0.1

24h VEGF

24h CN

HUVEC

2500

**

2000 PlGF (pg/ml)

PlGF (pg/ug)

2000 1600

8 6

(a)

PlGF (pg/ml)

mVEGF (pg/ug)

1229

**

1500

**

1000 500

0 P12

P13

P14

0

P16

0

2.5

25

(b) 4.5 4

VEGF

**

250

** (c)

PlGF

3500

3

3000

2.5

2500

PIGF (pg/ml)

Relative mRNA

3.5

100

VEGF (ng/ml)

2 1.5 1

†

0.5

HREC ** **

2000 1500 1000 500

0 P13

0

P15

Fig. 1. VEGF and PlGF levels in mouse model of oxygen-induced retinopathy. (a) VEGF and PlGF protein concentrations and (b) VEGF and PlGF mRNA expression in mouse retinas detected by ELISA assay and quantitative real-time PCR, respectively. \\P < 0.01 vs. controls (postnatal day 12, P12).  P < 0.05 vs. PlGF mRNA level in controls. Three mice were used for each time point.

observed VEGF-induced increase in PlGF levels in the cell lysate (data not shown). The upregulation of PlGF in endothelial cells was specific for VEGF, as PlGF levels in the culture supernatant were not significantly changed by treatment with bFGF (25 ng/ml) or TNF-a (25 ng/ml) for 24 h using the same culture conditions (Fig. 2(d)). 3.3. VEGF stimulation does not alter PlGF mRNA levels in cultured endothelial cells In order to determine whether VEGF upregulation of PlGF protein was accompanied by an increase in PlGF mRNA levels, HUVECs were treated with different concentrations of VEGF for 24 h. Cells were harvested at the indicated time points and total RNA was isolated. Similar to the findings in the mouse retina studies, real-time PCR for PlGF revealed that PlGF mRNA levels did not change significantly in either HUVEC or HREC treated with different doses of VEGF (Fig. 3(a) and (b)). This result was further confirmed by Northern blot

0

0.25

2.5

25

100

VEGF (ng/ml)

(d) 3000

** 2500 PlGF (pg/ml)

P12

2000 1500 1000 500 0 CN

VEGF

b-FGF

TNF-α

Fig. 2. VEGF induces PlGF production in cultured HUVECs and HRECs. (a) Time-course of PlGF protein in HUVEC supernatant in response to VEGF (100 ng/ml) stimulation. Dose-dependent regulation of PlGF production induced by 24 h VEGF treatment in the culture supernatant of (b) HUVEC and (c) HREC. (d) PlGF concentrations in HUVEC culture supernatant stimulated by bFGF (25 ng/ml) and TNF-a (25 ng/ml). Cells were grown to confluence in EGM-2MV in 12-well plates, then switched to 500 ll DMEM containing 10% CS and the indicated concentration of VEGF after washing with PBS. Culture supernatant was collected at indicated time points and PlGF levels measured by ELISA. The results shown are representative of four independent experiments, each performed in duplicate. \\P < 0.01 vs. controls.

1230

Y.-G. Yao et al. / FEBS Letters 579 (2005) 1227–1234

with VEGF treatment alone, but the difference was not statistically significant (Fig. 4(a)). The inhibition effect of each inhibitor at the designated concentration was tested by dephosphorylation of its respective signaling target (data not shown). Therefore, it appears that these signaling pathways are not playing a crucial role in the upregulation of PlGF by VEGF. Intriguingly, the PlGF concentration was significantly reduced by the Ca2+ ionophore, A23187 (1 lM) (Fig. 4(a)). PlGF concentrations in the culture supernatant were significantly reduced when the PKC (Calphostin C, 2.5 lM; GF109203X, 3 lM) and MEK (mitogen-activated protein kinase kinase: U0126, 10 lM; PD98059, 50 lM) signaling pathways were blocked (P < 0.05). The inhibition of the PKC signaling pathway showed a dose-dependent effect, whereas the inhibition of MEK was not sensitive to the inhibitor concentrations tested (Fig. 4(b) and (c)). Interestingly, inhibition of the PKCb isoform by LY333531 also reduced the PlGF production in a dose-dependent fashion (Fig. 4(d)). This suggests that the PKC signaling pathway plays an important role in the increased PlGF production induced by VEGF.

Fig. 3. PlGF mRNA expression is not altered by VEGF treatment. Quantitative real-time PCR for PlGF mRNA in HUVECs (a) and HRECs (b) in response to different concentrations of VEGF. PlGF mRNA values were normalized to GAPDH gene expression and were demonstrated as means ± S.E.M. (c) Northern blot analysis of PlGF mRNA levels in HUVECs with or without 24 h VEGF (100 ng/ml) treatment. Results are representative of two independent experiments, each performed in duplicate.

analysis for PlGF mRNA (Fig. 3(c)). Therefore, our results suggest that PlGF production in response to VEGF is via a post-transcriptional mechanism. 3.4. PKC signaling is involved in the VEGF induction of PlGF In order to characterize which signaling pathway(s) are involved in the upregulation of PlGF by VEGF treatment, we incubated confluent HUVEC and HREC in DMEM containing 10% CS together with VEGF (100 ng/ml) and signaling pathway inhibitors for 24 h. Inhibition of p38 MAP kinase (SB203580, 10 lM), phosphatidylinositol 3-kinase (PI 3-kinase: LY294002, 20 lM), src family of tyrosine kinases (PP2, 3 lM), and protein phosphatase activity of calcineurin (protein phosphatase 2B, PP2B; cyclosporin A, 1 lM), respectively, slightly affected PlGF production compared

3.5. PMA upregulates PlGF mRNA and protein levels Since the PKC signaling pathway was found to be important for PlGF production by endothelial cells, we speculated that PMA, a PKC activator even at nanomolar concentrations, could have a stimulatory effect on the production of PlGF protein. The incubation of HUVEC and HREC with VEGF (100 ng/ml) and PMA (100 ng/ml) had a synergistic effect on PlGF production, greater than 3-fold higher than VEGF (100 ng/ml) treatment alone (P < 0.01). PMA alone at concentrations of 10 and 100 ng/ml also significantly induced the production of PlGF in the culture supernatant, comparable to that of VEGF (100 ng/ ml) alone. In addition, PMA stimulation at a low concentration (10 ng/ml) resulted in a higher PlGF protein level compared with a high PMA concentration (100 ng/ml) (Fig. 5(a)). In parallel with its stimulating effects on PlGF protein levels, PMA upregulated RNA levels of PlGF by at least 4-fold (P < 0.01) (Fig. 5(b)). VEGF did not have a synergistic effect with PMA on PlGF mRNA levels (Fig. 5(b)), although VEGF did have a synergistic effect with PMA on PlGF protein levels (Fig. 5(a)). This result gives further support that VEGF upregulates PlGF protein at a post-transcriptional (protein) level, and that the PKC signaling pathway plays an active but complex role in PlGF expression.

4. Discussion VEGF and PlGF are key angiogenic stimulators for normal and pathological angiogenesis. By binding to its two main receptor tyrosine kinases, Flt1 (VEGFR1) and KDR (VEGFR2, serving as the major signaling receptor), VEGF induces angiogenesis and plays a critical role in embryonic vascular development, physiologic angiogenesis, and a variety of pathological conditions including tumor growth and blinding eye conditions such as diabetic retinopathy and age-related macular degeneration [26,27]. PlGF only binds to Flt1 and was initially regarded as a decoy factor serving to displace VEGF from Flt1 to KDR [28]. Recently, a series of studies showed that loss of PlGF, while not causing any vascular defects during normal development, impaired angiogenesis and

Y.-G. Yao et al. / FEBS Letters 579 (2005) 1227–1234

1231

(a) 8000

**

7000 6000 PlGF (pg/ml)

plasma extravasation in every pathological condition tested [12,14,29]. On the other hand, overexpression of PlGF in mice resulted in a substantial increase of dermal blood vessels, enhanced vascular permeability, and increased the inflammatory response [14,30]; it also contributed to the pathogenesis of pulmonary emphysema [31]. Moreover, PlGF stimulation has been shown to result in the phosphorylation of different tyro-

5000 4000 3000

**

**

**

(a)

3500

PlGF (pg/ml)

2000

3000 2500

1000 0

**

CN

1000

PMA 10ng/ml

PMA 100ng/ml

PMA 100ng/ml + VEGF

††

500

(b) 25 G F

Relative mRNA

VE +

C

yc lo s

po ri n

PP

A2 31 8

A

7

+

+ 2

+ 00 2

80

LY 29 4

20 35 SB

VE G F

F VE G

F VE G

VE G F +

C

N

VE G F

0

(b)

VEGF

2000 1500

2500

20

P > 0.05

15 10 P > 0.05

5 **

l

G VE

g/

0n

A

g/

m

10

l+

0n

ng 10

PM

A

10

PM

††

m

l /m

F G VE

A

††

PM

††

1000

N

†

F

0

† 1500

C

PlGF (pg/ml)

2000

500

Fig. 5. PMA up-regulates the production of PlGF and has a synergistic effect with VEGF treatment in HUVECs. \\P < 0.01 vs. controls. (b) Quantitative real-time PCR for PlGF mRNA in HUVECs treated with PMA and/or VEGF (100 ng/ml). The results shown are representative of 3 independent experiments, each performed in duplicate. \\P < 0.01 vs. untreated control samples.

9+ V

EG

F

F 05

05 50

10

10

uM

uM

P

P

D

D

98

98

01 U uM

U M 5u

9+ V

E +V 26

+V 26 01

01 U M 2u

EG

F G

F EG

EG +V 26

V

C

EG

N

F

F

0

(c) 2500 **

PlGF (pg/ml)

2000 1500 † †

1000

††

†† ††

500

(d)

3500

X+ VE G

F

F GF 10 92 03

03 X+ VE G

GF 10 92

3u M

2. 5u M

0. 6u M

Ca lph o

sti n

C+ VE GF

C+ VE GF

C+ VE G F

Ca lph os tin

0. 75 uM

0. 25 uM

Ca lph os tin

VE

CN

GF

0

b

**

3000 2500

†† ††

2000 1500

††

1000 500 EG F LY 33 35 31 +V

G F 10 uM

LY 33 35 31 +V E

2u M

20 0n M

LY 33 3

53 1+ V

VE

EG

G F

F

0 C N

PlGF (pg/ml)

sine residues as compared to VEGF stimulation [17]. PlGF stimulation of endothelial cells also results in a gene expression profile distinct from VEGF stimulation. PlGF regulates both intermolecular and intramolecular cross talk between Flt1 and Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1 [17]. All these studies suggest that PlGF plays an important role in pathologic angiogenesis and may be a potential therapeutic target. In this study, we first tested the levels of the pro-angiogenic molecule PlGF in the murine oxygen-induced retinopathy model of retinal neovascularization and found a similar pattern of increased VEGF and PlGF levels in mouse retina in reFig. 4. (a) Effects of signaling pathway inhibitors on the production of PlGF protein in response to VEGF treatment. HUVECs were grown confluent in 12-well plates, incubated with drugs (cyclosporin A, 1 lM; SB203580, 10 lM; LY294002, 20 lM; PP2, 3 lM; A23187, 1 lM) and VEGF (100 ng/ml) for 24 h. Culture supernatant was collected, and PlGF levels measured by ELISA. Results are representative of at least three independent experiments performed in duplicate. \\P < 0.01 vs. untreated control samples,   P < 0.01 vs. VEGF-treated samples. (b) Inhibition effects of MAP kinase (b) and PKC (c) signaling pathways on the production of PlGF protein in response to VEGF (100 ng/ml) treatment. (d) Inhibition of the PKC beta isoform by specific inhibitor, LY333531, shows a dose-dependent effect on the secreted PlGF level induced by VEGF (100 ng/ml). \\P < 0.01 vs. untreated control samples.  P < 0.05 or   P < 0.01 vs. VEGF-treated alone.

1232

sponse to oxygen-induced retinopathy. This result is consistent with observations that the vitreous PlGF and VEGF levels are significantly increased in proliferative diabetic retinopathy patients as compared to non-neovascular disease patients [1,2,32]. Interestingly, comparison of VEGF and PlGF mRNA in mouse retina revealed an increase in VEGF mRNA (which is consistent with a previous study [4]), whereas PlGF mRNA remained stable (Fig. 1). Our observations of increased PlGF in mouse retina in association with neovascularization led us to speculate whether or not the increased PlGF protein level was a consequence of the increased VEGF. Since endothelial cells are known to be an important source of PlGF [18,25], we postulated that regulation of endothelial cell PlGF production by VEGF might contribute to increased PlGF levels during the neovascular process; we investigated this hypothesis using cultured endothelial cells treated with VEGF. Indeed, the production of PlGF by both HUVEC and HREC was upregulated by VEGF in a dose-dependent fashion. Similar to our findings in mouse retina during retinal neovascularization (Fig. 1), the upregulation of PlGF protein was not accompanied by an increase in PlGF mRNA. This induction of PlGF protein in cultured endothelial cells was specific to VEGF and did not occur with TNF-a or bFGF treatment (Figs. 2 and 3). It should be mentioned that during preparation of our manuscript, a paper reported that PlGF is upregulated in bovine retinal microvascular endothelial cells at both the RNA and protein level (approximately 2-fold upregulation of PlGF RNA and protein) by 100 ng/ml VEGF compared with non-treated control [33]. In contrast, we observed a dramatic upregulation of PlGF protein by VEGF without a concomitant increase in PlGF mRNA. It is conceivable that the differences in our results are due in part to the cell types used (HUVEC and HREC) in our studies vs. bovine retinal endothelial cells in the study by Zhao et al. [33]. More important, the cell culture conditions are different and may be contributory, as we used serum-supplemented media for our experiments; we have found that serum starvation itself can upregulate PlGF mRNA (data not shown), thereby potentially blunting any observed effect of VEGF. In addition, we observed similar results of upregulation of PlGF protein but not RNA in the mouse retina during retinal neovascularization (Fig. 1), which supports the concept that upregulation of PlGF by VEGF via a post-transcriptional mechanism may be an important biological phenomenon in pathological angiogenic complications. Intriguingly, the induction of PlGF by VEGF was significantly affected by activation and inhibition of the PKC signaling pathway (Figs. 4 and 5), suggesting that PKC plays an active role in this process. It is noteworthy that activation of PKC, especially the b isoform, has been implicated for both the early and late-stage manifestations of diabetic retinopathy, and PKC inhibition has been found to slow or reverse the progression of diabetic retinopathy and diabetic macular edema [34–39]. A specific inhibitor of the PKCb isoform, LY333531, is currently under investigation in clinical trials for the amelioration of pathological changes associated with retinal permeability and angiogenesis [38–41]. Oral administration of LY333531 ameliorates the retinal mean circulation time [34], prevents diabetes-induced retinal vascular permeability [35], and attenuates leukocyte entrapment in the retinal microcirculation of diabetic rats [42]. Moreover, mice transgenic for the PKCb2 isoform develop a dramatic increase in retinal neovascularization in the oxygen-induced retinopathy model, whereas PKCb knockout mice demonstrate a significant de-

Y.-G. Yao et al. / FEBS Letters 579 (2005) 1227–1234

crease in retinal neovascularization [37]. The effects of activation or overexpression of PKC in diabetic pathological complications are similar to the effects observed with PlGF overexpression, such as increased vascularization, vessel permeability, as well as inflammatory responses associated with more pronounced vascular enlargement, edema, and inflammatory cell infiltration [12,14,29,30]. Moreover, inhibition of PKC has an ameliorating effect on pathologic complications, similar to that observed for PlGF deficiency in mice [12,14,29,30]. All these results suggest that inhibition of VEGF-induced increase in PlGF production by PKC inhibition may be one possible mechanism for the efficacy of LY333531 in diabetic retinopathy. Indeed, the production of PlGF in response to VEGF stimulation in endothelial cells was significantly inhibited by LY333531 (Fig. 4(d)). This gives supportive evidence that PKC is involved in the increased production of PlGF in proliferative diabetic retinopathy and other pathological angiogenesis complications. The angiogenic switch has been postulated to result from an imbalance between stimulators and inhibitors of angiogenesis in a given tissue bed [43]. VEGF is known to be a major angiogenic stimulator in diverse angiogenic conditions including retinal neovascularization [44]. Indeed, VEGF is known to increase the expression of several pro-angiogenic genes, including angiopoietin-2 [45,46], connective tissue growth factor [47,48], tissue factor [49], COX-2 [50], and IL-8 [51], by increasing RNA levels of these genes. This mode of VEGF gene regulation at the RNA level has been further demonstrated by microarray studies of endothelial cells stimulated with VEGF [52,53]. In this study, we demonstrate that VEGF upregulates the pro-angiogenic molecule, PlGF, at the protein level, which supports a versatile regulatory role for VEGF during angiogenesis. It is possible that VEGF may similarly upregulate other pro-angiogenic molecules at the post-transcriptional level. VEGF upregulation of PlGF serves as a potential mechanism for VEGF augmentation of its own effects on endothelial cells, since PlGF is known to play an important role in enhancing VEGF action [12,17], thereby contributing to the angiogenic switch. In summary, the significant increase in PlGF levels in human proliferative diabetic retinopathy and the mouse oxygen-induced retinopathy model raises the possibility of an active role for PlGF in ocular neovascular diseases, particularly in light of recent studies in PlGF transgenic and knockout mice demon-

VEGF

PKC PlGF

Pathological angiogenesis Fig. 6. Possible links among VEGF, PKC and PlGF in pathological angiogenic conditions.

Y.-G. Yao et al. / FEBS Letters 579 (2005) 1227–1234

strating an important role for PlGF in pathological angiogenic processes. With respect to retinal neovascularization, studies of PKC-b knockout mice have demonstrated a significant reduction in neovascularization, a result similar to PlGF knockout mice. Inhibition of the PKC signaling pathway in our study significantly affected the upregulation of PlGF by VEGF in cultured endothelial cells. These results provide a potential link between PKC inhibition and amelioration of vascular complications in the development of pathological angiogenic conditions (Fig. 6). Acknowledgments: We thank Yilin Yu and Manesh Dagli for technical assistance. This work was supported by funds from NIH (EY00398), the Juvenile Diabetes Research Foundation, and Michael B. Panitch. E.J.D. is a recipient of an RPB Career Development Award.

References [1] Adamis, A.P., Miller, J.W., Bernal, M.T., DÕAmico, D.J., Folkman, J., Yeo, T.K. and Yeo, K.T. (1994) Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am. J. Ophthalmol. 118, 445– 450. [2] Aiello, L.P., Avery, R.L., Arrigg, P.G., Keyt, B.A., Jampel, H.D., Shah, S.T., Pasquale, L.R., Thieme, H., Iwamoto, M.A. and Park, J.E., et al. (1994) Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331, 1480–1487. [3] Miller, J.W., Adamis, A.P., Shima, D.T., DÕAmore, P.A., Moulton, R.S., OÕReilly, M.S., Folkman, J., Dvorak, H.F., Brown, L.F. and Berse, B., et al. (1994) Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am. J. Pathol. 145, 574–584. [4] Pierce, E.A., Avery, R.L., Foley, E.D., Aiello, L.P. and Smith, L.E. (1995) Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc. Natl. Acad. Sci. USA 92, 905–909. [5] Tobe, T., Okamoto, N., Vinores, M.A., Derevjanik, N.L., Vinores, S.A., Zack, D.J. and Campochiaro, P.A. (1998) Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors. Invest. Ophthalmol. Vis. Sci. 39, 180–188. [6] Aiello, L.P., Pierce, E.A., Foley, E.D., Takagi, H., Chen, H., Riddle, L., Ferrara, N., King, G.L. and Smith, L.E. (1995) Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGFreceptor chimeric proteins. Proc. Natl. Acad. Sci. USA 92, 10457– 10461. [7] Adamis, A.P., Shima, D.T., Tolentino, M.J., Gragoudas, E.S., Ferrara, N., Folkman, J., DÕAmore, P.A. and Miller, J.W. (1996) Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch. Ophthalmol. 114, 66–71. [8] Robinson, G.S., Pierce, E.A., Rook, S.L., Foley, E., Webb, R. and Smith, L.E. (1996) Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc. Natl. Acad. Sci. USA 93, 4851–4856. [9] Seo, M.S., Kwak, N., Ozaki, H., Yamada, H., Okamoto, N., Yamada, E., Fabbro, D., Hofmann, F., Wood, J.M. and Campochiaro, P.A. (1999) Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor. Am. J. Pathol. 154, 1743–1753. [10] Ozaki, H., Seo, M.S., Ozaki, K., Yamada, H., Yamada, E., Okamoto, N., Hofmann, F., Wood, J.M. and Campochiaro, P.A. (2000) Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization. Am. J. Pathol. 156, 697–707. [11] De Falco, S., Gigante, B. and Persico, M.G. (2002) Structure and function of placental growth factor. Trends Cardiovasc. Med. 12, 241–246.

1233 [12] Carmeliet, P., Moons, L., Luttun, A., Vincenti, V., Compernolle, V., De Mol, M., Wu, Y., Bono, F., Devy, L., Beck, H., Scholz, D., Acker, T., DiPalma, T., Dewerchin, M., Noel, A., Stalmans, I., Barra, A., Blacher, S., Vandendriessche, T., Ponten, A., Eriksson, U., Plate, K.H., Foidart, J.M., Schaper, W., Charnock-Jones, D.S., Hicklin, D.J., Herbert, J.M., Collen, D. and Persico, M.G. (2001) Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat. Med. 7, 575–583. [13] Rakic, J.M., Lambert, V., Devy, L., Luttun, A., Carmeliet, P., Claes, C., Nguyen, L., Foidart, J.M., Noel, A. and Munaut, C. (2003) Placental growth factor, a member of the VEGF family, contributes to the development of choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 44, 3186–3193. [14] Oura, H., Bertoncini, J., Velasco, P., Brown, L.F., Carmeliet, P. and Detmar, M. (2003) A critical role of placental growth factor in the induction of inflammation and edema formation. Blood 101, 560–567. [15] Luttun, A., Tjwa, M., Moons, L., Wu, Y., Angelillo-Scherrer, A., Liao, F., Nagy, J.A., Hooper, A., Priller, J., De Klerck, B., Compernolle, V., Daci, E., Bohlen, P., Dewerchin, M., Herbert, J.M., Fava, R., Matthys, P., Carmeliet, G., Collen, D., Dvorak, H.F., Hicklin, D.J. and Carmeliet, P. (2002) Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat. Med. 8, 831–840. [16] Pipp, F., Heil, M., Issbrucker, K., Ziegelhoeffer, T., Martin, S., van den Heuvel, J., Weich, H., Fernandez, B., Golomb, G., Carmeliet, P., Schaper, W. and Clauss, M. (2003) VEGFR-1selective VEGF homologue PlGF is arteriogenic: evidence for a monocyte-mediated mechanism. Circ. Res. 92, 378–385. [17] Autiero, M., Waltenberger, J., Communi, D., Kranz, A., Moons, L., Lambrechts, D., Kroll, J., Plaisance, S., De Mol, M., Bono, F., Kliche, S., Fellbrich, G., Ballmer-Hofer, K., Maglione, D., MayrBeyrle, U., Dewerchin, M., Dombrowski, S., Stanimirovic, D., Van Hummelen, P., Dehio, C., Hicklin, D.J., Persico, G., Herbert, J.M., Shibuya, M., Collen, D., Conway, E.M. and Carmeliet, P. (2003) Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat. Med. 9, 936–943. [18] Luttun, A., Autiero, M., Tjwa, M. and Carmeliet, P. (2004) Genetic dissection of tumor angiogenesis: are PlGF and VEGFR1 novel anti-cancer targets. Biochim. Biophys. Acta 1654, 79–94. [19] Smith, L.E., Wesolowski, E., McLellan, A., Kostyk, S.K., DÕAmato, R., Sullivan, R. and DÕAmore, P.A. (1994) Oxygeninduced retinopathy in the mouse. Invest. Ophthalmol. Vis. Sci. 35, 101–111. [20] Gehlbach, P., Demetriades, A.M., Yamamoto, S., Deering, T., Duh, E.J., Yang, H.S., Cingolani, C., Lai, H., Wei, L. and Campochiaro, P.A. (2003) Periocular injection of an adenoviral vector encoding pigment epithelium-derived factor inhibits choroidal neovascularization. Gene Ther. 10, 637–646. [21] Cao, Y., Ji, W.R., Qi, P. and Rosin, A. (1997) Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing. Biochem. Biophys. Res. Commun. 235, 493–498. [22] Yang, W., Ahn, H., Hinrichs, M., Torry, R.J. and Torry, D.S. (2003) Evidence of a novel isoform of placenta growth factor (PlGF-4) expressed in human trophoblast and endothelial cells. J. Reprod. Immunol. 60, 53–60. [23] Yao, Y.G. and Duh, E.J. (2004) VEGF selectively induces Down syndrome critical region 1 gene expression in endothelial cells: a mechanism for feedback regulation of angiogenesis? Biochem. Biophys. Res. Commun. 321, 648–656. [24] Simpson, D.A., Feeney, S., Boyle, C. and Stitt, A.W. (2000) Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR. Mol. Vis. 6, 178–183. [25] Yonekura, H., Sakurai, S., Liu, X., Migita, H., Wang, H., Yamagishi, S., Nomura, M., Abedin, M.J., Unoki, H., Yamamoto, Y. and Yamamoto, H. (1999) Placenta growth factor and vascular endothelial growth factor B and C expression in microvascular endothelial cells and pericytes. Implication in autocrine and paracrine regulation of angiogenesis. J. Biol. Chem. 274, 35172– 35178. [26] Carmeliet, P. and Jain, R.K. (2000) Angiogenesis in cancer and other diseases. Nature 407, 249–257.

1234 [27] Ferrara, N. (2004) Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25, 581–611. [28] Park, J.E., Chen, H.H., Winer, J., Houck, K.A. and Ferrara, N. (1994) Placenta growth factor Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J. Biol. Chem. 269, 25646–25654. [29] Luttun, A., Brusselmans, K., Fukao, H., Tjwa, M., Ueshima, S., Herbert, J.M., Matsuo, O., Collen, D., Carmeliet, P. and Moons, L. (2002) Loss of placental growth factor protects mice against vascular permeability in pathological conditions. Biochem. Biophys. Res. Commun. 295, 428–434. [30] Odorisio, T., Schietroma, C., Zaccaria, M.L., Cianfarani, F., Tiveron, C., Tatangelo, L., Failla, C.M. and Zambruno, G. (2002) Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. J. Cell Sci. 115, 2559–2567. [31] Tsao, P.N., Su, Y.N., Li, H., Huang, P.H., Chien, C.T., Lai, Y.L., Lee, C.N., Chen, C.A., Cheng, W.F., Wei, S.C., Yu, C.J., Hsieh, F.J. and Hsu, S.M. (2004) Overexpression of placenta growth factor contributes to the pathogenesis of pulmonary emphysema. Am. J. Respir. Crit. Care Med. 169, 505–511. [32] Khaliq, A., Foreman, D., Ahmed, A., Weich, H., Gregor, Z., McLeod, D. and Boulton, M. (1998) Increased expression of placenta growth factor in proliferative diabetic retinopathy. Lab. Invest. 78, 109–116. [33] Zhao, B., Cai, J. and Boulton, M. (2004) Expression of placenta growth factor is regulated by both VEGF and hyperglycaemia via VEGFR-2. Microvasc. Res. 68, 239–246. [34] Ishii, H., Jirousek, M.R., Koya, D., Takagi, C., Xia, P., Clermont, A., Bursell, S.E., Kern, T.S., Ballas, L.M., Heath, W.F., Stramm, L.E., Feener, E.P. and King, G.L. (1996) Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 272, 728–731. [35] Aiello, L.P., Bursell, S.E., Clermont, A., Duh, E., Ishii, H., Takagi, C., Mori, F., Ciulla, T.A., Ways, K., Jirousek, M., Smith, L.E. and King, G.L. (1997) Vascular endothelial growth factorinduced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 46, 1473–1480. [36] Koya, D. and King, G.L. (1998) Protein kinase C activation and the development of diabetic complications. Diabetes 47, 859–866. [37] Suzuma, K., Takahara, N., Suzuma, I., Isshiki, K., Ueki, K., Leitges, M., Aiello, L.P. and King, G.L. (2002) Characterization of protein kinase C beta isoformÕs action on retinoblastoma protein phosphorylation, vascular endothelial growth factorinduced endothelial cell proliferation, and retinal neovascularization. Proc. Natl. Acad. Sci. USA 99, 721–726. [38] Aiello, L.P. (2002) The potential role of PKC beta in diabetic retinopathy and macular edema. Surv. Ophthalmol. 47 (Suppl 2), S263–S269. [39] Aiello, L.P., Cahill, M.T. and Cavallerano, J.D. (2004) Growth factors and protein kinase C inhibitors as novel therapies for the medical management diabetic retinopathy. Eye 18, 117–125. [40] Tuttle, K.R. and Anderson, P.W. (2003) A novel potential therapy for diabetic nephropathy and vascular complications: protein kinase C beta inhibition. Am. J. Kidney Dis. 42, 456–465.

Y.-G. Yao et al. / FEBS Letters 579 (2005) 1227–1234 [41] He, Z. and King, G.L. (2004) Protein kinase Cbeta isoform inhibitors: a new treatment for diabetic cardiovascular diseases. Circulation 110, 7–9. [42] Nonaka, A., Kiryu, J., Tsujikawa, A., Yamashiro, K., Miyamoto, K., Nishiwaki, H., Honda, Y. and Ogura, Y. (2000) PKC-beta inhibitor (LY333531) attenuates leukocyte entrapment in retinal microcirculation of diabetic rats. Invest. Ophthalmol. Vis. Sci. 41, 2702–2706. [43] Hanahan, D. and Folkman, J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353–364. [44] Aiello, L.P. (1997) Clinical implications of vascular growth factors in proliferative retinopathies. Curr. Opin. Ophthalmol. 8, 19–31. [45] Mandriota, S.J. and Pepper, M.S. (1998) Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia. Circ. Res. 83, 852–859. [46] Oh, H., Takagi, H., Suzuma, K., Otani, A., Matsumura, M. and Honda, Y. (1999) Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells. J. Biol. Chem. 274, 15732– 15739. [47] Suzuma, K., Naruse, K., Suzuma, I., Takahara, N., Ueki, K., Aiello, L.P. and King, G.L. (2000) Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase- akt-dependent pathways in retinal vascular cells. J. Biol. Chem. 275, 40725– 40731. [48] He, S., Jin, M.L., Worpel, V. and Hinton, D.R. (2003) A role for connective tissue growth factor in the pathogenesis of choroidal neovascularization. Arch. Ophthalmol. 121, 1283– 1288. [49] Armesilla, A.L., Lorenzo, E., Gomez del Arco, P., MartinezMartinez, S., Alfranca, A. and Redondo, J.M. (1999) Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression. Mol. Cell Biol. 19, 2032–2043. [50] Hernandez, G.L., Volpert, O.V., Iniguez, M.A., Lorenzo, E., Martinez-Martinez, S., Grau, R., Fresno, M. and Redondo, J.M. (2001) Selective inhibition of vascular endothelial growth factormediated angiogenesis by cyclosporin A: roles of the nuclear factor of activated T cells and cyclooxygenase 2. J. Exp. Med. 193, 607–620. [51] Lee, T.H., Avraham, H., Lee, S.H. and Avraham, S. (2002) Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells. J. Biol. Chem. 277, 10445– 10451. [52] Abe, M. and Sato, Y. (2001) cDNA microarray analysis of the gene expression profile of VEGF-activated human umbilical vein endothelial cells. Angiogenesis 4, 289–298. [53] Liu, D., Jia, H., Holmes, D.I., Stannard, A. and Zachary, I. (2003) Vascular endothelial growth factor-regulated gene expression in endothelial cells: KDR-mediated induction of Egr3 and the related nuclear receptors Nur77, Nurr1, and Nor1. Arterioscler. Thromb. Vasc. Biol. 23, 2002–2007.