Vascular endothelial growth factor in diabetes induced early retinal abnormalities

Vascular endothelial growth factor in diabetes induced early retinal abnormalities

Diabetes Research and Clinical Practice 65 (2004) 197–208 Vascular endothelial growth factor in diabetes induced early retinal abnormalities Mark Cuk...

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Diabetes Research and Clinical Practice 65 (2004) 197–208

Vascular endothelial growth factor in diabetes induced early retinal abnormalities Mark Cukiernik a , Denise Hileeto a , Terry Evans a , Suranjana Mukherjee a , Donal Downey b , Subrata Chakrabarti a,∗ b

a Department of Pathology, University of Western Ontario, London, Ont., Canada N6A 5C1 Department of Nuclear Medicine and Diagnostic Radiology, University of Western Ontario, London, Ont., Canada N6A 5C1

Received in revised form 17 November 2003; accepted 2 February 2004

Abstract Increased vascular permeability and blood flow alterations are characteristic features of diabetic retinal microangiopathy. The present study investigated vascular endothelial growth factor (VEGF) and its interactions with endothelin (ET) 1 and 3, endothelial, and inducible nitric oxide synthase (eNOS, iNOS) in mediating diabetes induced retinal vascular dysfunction. Male Sprague Dawley rats with streptozotocin (STZ) induced diabetes, with or without VEGF receptor signal inhibitor SU5416 treatment (high or low dose) were investigated after 4 weeks of follow-up. Colour Doppler ultrasound of the ophthalmic/central retinal artery, retinal tissue analysis with competitive RT-PCR and microvascular permeability were studied. Diabetes caused increased microvascular permeability along with increased VEGF mRNA expression. Increased vascular permeability was prevented by SU5416 treatment. Diabetic animals showed higher resistivity index (RI), indicative of vasoconstriction with increased ET-1 and ET-3 mRNA expression, whereas eNOS and iNOS mRNA expressions were un-affected. SU5416 treatment corrected increased RI via increased iNOS in spite of increased ET-1, ET-3 and VEGF mRNA expression. Cell culture (HUVEC) studies indicate that in part, an SU5416 induced iNOS upregulation may be mediated though a MAP kinase signalling pathway. The present data suggest VEGF is important in mediating both vasoconstriction and permeability in the retina in early diabetes. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Diabetes; Blood flow; VEGF; SU5416; Permeability

1. Introduction The major target of diabetic retinopathy is the retinal microvasculature. The integrity of capillary endothelial cells is crucial to maintain homeosta∗ Corresponding author. Tel.: +1-519-661-2030; fax: +1-519-661-2930. E-mail address: [email protected] (S. Chakrabarti).

sis of the surrounding retinal tissue [1]. Endothelial cells produce and are responsive to the autocrine and paracrine activities of several vasoactive molecules like vascular endothelial growth factor (VEGF), endothelin (ET) and nitric oxide (NO) [2]. Diabetes increases the expression of VEGF secondary to protein kinase C (PKC) activation [3]. The VEGF protein family is comprised of several members including VEGF A, B, C, D, E and placental growth

0168-8227/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2004.02.002

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factor [4]. VEGF mediates its activities through interactions with receptor tyrosine kinase proteins; VEGFR1 (Flt-1), VEGFR2 (KDR), or VEGFR3 (Flt-4). A recently discovered co-receptor, neuropilin has been demonstrated to associate with VEGFR2 [5,6]. VEGF is an important factor promoting angiogenesis during proliferative diabetic retinopathy [7]. However, since its discovery, VEGF has demonstrated the ability to increase vascular tissue permeability in non-diabetic conditions [8]. Reduced microvascular blood flow and increased vascular permeability are two early characteristic abnormalities of diabetic microangiopathy [9]. There are reports outlining a possible pathogenetic role of VEGF activation in early diabetes, where VEGF may play a role in the breakdown of the blood retinal barrier [10,11]. Recently, it has been demonstrated that VEGF neutralising antibody treatment is capable of preventing the diabetes induced increased permeability [12]. VEGF also reacts extensively with other vasoactive factors. We have reported how an up-regulation of ET-1 and ET-3 mRNA levels in response to short term diabetes, contributed to reduced blood flow in the retina [13]. We have further demonstrated in human umbilical vein endothelial cells both ET-1 and VEGF may be responsible for the production of glucose induced, increased endothelial permeability [14]. Furthermore, a co-stimulatory relationship between glucose induced ET and VEGF may exist [15]. In addition, VEGF effector pathways involve increased nitric oxide synthase (NOS) mRNA expression and NO production [16]. NO has been demonstrated to cause a down-regulation of VEGF [17,18]. Interestingly, an up-regulation of ET can lead to a down-regulation of NO [19,20]. Hence, an intricate relationship may exist among these factors. In order to delineate the pathogenetic mechanisms in early diabetic microangiopathy, the present study investigated the role of VEGF and its interactions with other vasoactive factors in the pathogenesis of increased microvascular permeability in the retina of the streptozotocin (STZ) diabetic rat. We investigated VEGF alterations in mediating retinal blood flow changes in short term diabetes. Finally, we have examined the role of the MAP kinase signalling pathway in inducible nitric oxide synthase (iNOS) upregulation.

2. Materials and methods 2.1. Animals All animals were cared for under the conditions and rules designated by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research with approval by the University of Western Ontario Animal Care and Ethics Committee. Male Sprague Dawley rats of approximately 200 g received a single intravenous injection of streptozotocin (65 mg/kg in citrate buffer, pH 6.5). Control animals received an equivalent injection of citrate buffer. After confirmation of diabetes (blood glucose > 20 mmol/l on 2 consecutive days), animals were randomised to one of three treatment groups: poorly controlled diabetics, poorly controlled diabetics treated with high dose (30 mg/kg sub cutaneous daily) SU5416 (SUGEN Inc. San Francisco CA, USA courtesy of Drs. A. Howlett, N. Patel and G. McMahon) or poorly controlled diabetics with low dose (20 mg/kg subcutaneous every second day) SU5416. Control animals received an equal volume injection of vehicle. SU5416 is a VEGFR2 signal inhibitor [21,22]. Animals were followed up for a 4 week treatment period with rat chow and water ad libitum. Animals were monitored for body weight changes, glucosuria and ketouria. All diabetic rats were implanted with slow release insulin implants to prevent ketosis (approximately 2 U/day) (LinShin, Scarborough, ON, Canada). Prior to sacrifice, colour Doppler ultrasound analysis of the right eye was performed using previously described techniques [13]. Briefly, following anaesthesia with ketamine and xylazine the central retinal vasculature was located with an 8L5, 80 MHz colour Doppler ultrasound probe (Acuson Mountainview, CA, USA). Doppler waveforms were examined and colour images were obtained in real time with Doppler spectral analysis using the arterial tracings. At least three measurements were recorded for each animal and the mean was calculated. The resistivity index (RI) of the central retinal artery was calculated by subtracting the diastolic velocity (DV) from the peak systolic velocity (SV) and dividing by the systolic velocity[(SV−DV)/SV]. An increased central retinal artery RI value is indicative of vasoconstriction at the capillary or pre-capillary level within the retina [13]. Furthermore, colour Doppler and echocardiographic studies on the heart were also

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performed. RI values for the mitral valve and pulmonary artery were obtained. Tissue harvesting meant the right retina was snap frozen in liquid nitrogen while the left retina was preserved in 10% buffered formalin solution. Blood was collected for analysis of glucose levels (LifeScan, Burnaby, BC, Canada) glycated haemoglobin and plasma levels of SU5416. 2.2. RNA isolation Using TRIZOLTM reagent (Invitrogen Inc. Burlington, ON, Canada), RNA was extracted with chloroform. Centrifugation separated the solution into aqueous and organic phases, where RNA was recovered from the aqueous phase by precipitation with isopropyl alcohol. After suspension in DEPC-treated water, RNA was quantified by measuring absorbance at 260 and 280 nm. RNA samples were stored at −70 ◦ C. 2.3. First strand cDNA synthesis Using the Superscript-II system (Invitrogen Inc.) first strand cDNA was made. Five micrograms of RNA and oligo (dT) primers (Invitrogen Inc.) were denatured for 10 min at 65 ◦ C, and the reaction was terminated by placing the samples on ice. Addition of MMLV-reverse transcriptase and dNTP at 42 ◦ C for 50 min and a termination step of 15 min at 70 ◦ C, produced a final 20 ␮l volume of RT product, which was stored at −20 ◦ C.

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2.4. Competitive RT-PCR Due to the small amount of retinal tissues available to perform multiple gene analysis, competitive RT-PCR was used to quantify the expression of the specific mRNAs. Competitive PCR amplifies a known amount of sample (the competitor) to an unknown amount (the target) under the same reaction conditions. The method is superior to semi-quantitative PCR which is based on quantification of a house keeping gene. Competitors were created using the TaKaRa DNA Construction Kit (Panvera, Madison WI USA). Briefly, competitors were constructed using the primers found in Table 1. Sequences at both ends of the competitor were complementary to primers for amplifying the target DNA. Competitors were of comparable size to target DNA fragments assuring similar amplification dynamics of both target and competitor DNA fragments. 20 pmol/l of sense and antisense primers along with 25 ␮l of PCR water were added to the premix solution. Samples were amplified by 30 cycles consisting of 30 s steps at the temperatures: 94, 60 and 72 ◦ C. Samples were filtered with the Suprec-02 cartridge and diluted with PCR water to a final volume of 50 ␮l. Copies per microliter were calculated according to manufacturer’s instructions. Each competitor was diluted and preliminary experiments were performed to establish the optimal dilution by measuring the product of the area and average intensity between competitor and target was a 1:1 ratio.

Table 1 Rat primer sequences used for competitive RT-PCR Primer name

Competitor size (bp)

Target size (bp)

Sequence (5 →3 )

ET-1

484

500

(+) gct cct gct cct cct tga tgg tac ggt cat cat ctg aca c (−) ctc gct cta tgt aag tca tgg gcg tga gta tta cga cga agg tg

ET-3

294

383

(+) gca ctt gct tca ctt ata agg gta cgg tca tca tct gac ac (−) aca gaa gca aga agc atc agt tga gag ttt ctg cgg cag tta a

eNOS

290

207

(+) gca aga ccg att aca cga cag tac ggt cat cat ctg aca c (−) gtc ctc agg agg tct tgc aca gag ttt ctg cgg cag tta a

iNOS

301

220

(+) atg gaa cag tat aag cga aac acc gta cgg tca tca tct gac ac (−) gtt tct ggt cga tgt cat gag caa agg aga gtt tct gcg gca gtt aa

VEGF 188/164/120

340

635/563/431

(+) ctg ctg tct tgg gtg cat tgg gta cgg tca tca tct gac ac (−) cac cgc ctt ggc ttg tca cat cgc cat cct ggg aag act cc

The complete sequence represents the primer sequence used to construct the competitor fragments, while the underlined sequence represents the primers used to amplify the target gene.

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Amplification was performed with the primers listed in Table 1. For all primer sets 1× PCR buffer, 1.0 ␮L RT product, 2.5 ␮l of the appropriate dilution of competitor, 500 nM of sense and antisense primers, 0.25 mM of dNTP and 2.5 U of Platinum TaqTM (Invitrogen Inc.) along with 2.0 mM MgCl2 for ET-1, ET-3, VEGF or 1.5 mM MgCl2 for endothelial nitric oxide synthase (eNOS), and iNOS to a final volume of 25 ␮l. 40 cycles of amplification were used for ET-3 while all other genes were amplified for 30 cycles; 92 ◦ C for 45 s, 60 ◦ C for 45 s and 72 ◦ C for 1 min, followed by a final extension of 72 ◦ C for 10 min. Preliminary experiments confirmed that amplification was in the linear phase of the PCR reaction. 2.5. Quantification The PCR products were analysed on a 2% agarose gel in 1× TBE buffer. The gels were stained with ethidium bromide and visualised with ultra violet light. For quantification, the ratio of optical density and area of the target gene band and its competitor band were assessed using MochaTM densitometry software (Jandel Scientific, CA, USA). Due to competition of target and competitor DNA to be amplified with the same primers, the ratio between the two amplified products reflects the original amounts of target cDNA and its competitor. 2.6. Cell culture To further explore a possible mechanism of iNOS expression, endothelial cell culture experiments were performed with previously established techniques within our laboratory. [23] A HUVEC cell line was purchased from the American Type Tissue Collection (Catalog # CRL-2480, Manassas, VA, USA). Cells were grown in 25 cm2 tissue culture flasks in a humidified 37 ◦ C, 5% CO2 incubation chamber, with all experiments repeated in triplicate. VEGF signal inhibitor SU5416 (Sugen Inc., 5 mM final concentration), MEK1/2 inhibitor U0126 (Promega, Madison WI, USA., 10 ␮M final concentration), recombinant human VEGF (Sigma, 50 ng ml−1 final concentration), d-glucose (25 mM final concentration) l-glucose (25 mM final concentration as a control) were added at 80% confluence. Cells were analysed after 48 h of glucose incubation, as our previous stud-

ies have indicated that gene expression in endothelial cells occurs after such an incubation [14]. RNA was extracted and analysed by real time PCR (see below). 2.7. Real time PCR After RNA isolation and first strand cDNA preparation, real time quantitative PCR using the Roche LightCycler system (Roche Diagnostics Canada, Lavalle, PQ, Canada) was performed. PCR reactions were carried out in mirco-capillary tubes (Roche Diagnostics Canada) to a final volume of 20 ␮l. The reaction mixture consisted of 1.0 ␮l cDNA, 1.0 ␮l both forward and reverse 10 ␮M primers, 1.6 ␮l 25 mM MgCl2 , 5.4 ␮l molecular grade H2 O and 10.0 ␮l SYBR Green Taq ReadyMix, Capillary Formulation (Sigma Aldrich Canada, Oakville ON, Canada). The primer sequences for human ␤-actin were: sense 5 -CCTCTATGCCAACACAGTGC-3 , antisense 5 -CATCGTACTCCTGCTTGCTG-3 . The cycling parameters for ␤-actin were [temperature (◦ C); hold time (s); ramp rate (◦ C/s); denaturation step [95/30/20], followed by 35 cycles of amplification: denaturation [95/0/20], annealing [58/5/20], elongation [72/8/20] and signal acquisition [83/1/20]. Melting curve analysis consisted of a three step process with continuous signal acquisition: step 1 [95/0/20], step 2 [67/15/20], step 3 [95/0/0.1]. The primer sequences for human iNOS were: sense 5 -TGCAGACACGTGCGTTACTCC-3 , anti-sense 5 -GGTAGCCAGCATAGCGGATG-3 [24]. The cycling parameters for iNOS were: denaturation step [95/30/20], followed by 55 cycles of amplification: denaturation [95/0/20], annealing [57/5/20], elongation [72/9/20] with continuous signal acquisition. The melting curve analysis consisted of three steps with continuous signal acquisition: step 1 [95/0/20], step 2 [65/15/20] and step 3 [95/0/0.1]. To optimise the amplification of the genes, melting curve analysis was used to determine the melting temperature between specific products and primer– dimers. mRNA was quantified with the standard curve method. Standard curves for iNOS and ␤-actin were constructed using different amounts of standard template. The data was normalised to ␤-actin in order to account for differences in reverse transcription efficiencies and template amounts within each reaction mixture [25].

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2.8. Immunohistochemistry Retinal sections were immunocytochemically stained for albumin. Five micrometer thick retinal tissue sections from formalin fixed, paraffin embedded blocks were transferred to positively charged slides to be used for staining. A polyclonal rabbit anti-human albumin antibody (DAKO Diagnostics. Mississauga, ON, Canada) (1:400) was used along with the streptavidin biotin reaction Vectastain Elite Kit (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine was used as a chromogen. Slides were counterstained with hematoxylin. For negative controls, the primary antibody was replaced with non-immune rabbit serum as well as PBS without immunoglobulin. The experiments were repeated three times with slides analysed, in a masked fashion by two investigators unaware of the treatment. Slides were arbitrarily scored as to the extravascular compartment staining using a graded scale ranging from zero (no stain) to four (intense stain). 2.9. Statistical analysis All values are displayed as mean ± S.E.M. Values were analysed with ANOVA followed by analysis between groups with the unpaired Student’s t-test with Bonferoni corrections. Qualitative data were analysed by the Chi-squared test. A P value of 0.05 or less was considered significant.

3. Results 3.1. Clinical monitoring All diabetic animals had higher blood glucose levels, higher glycated haemoglobin levels and reduced body weight gain compared to age matched,

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non-diabetic control animals. Treatment with SU5416 had no effect in modifying blood glucose and body weight changes [Table 2]. Twenty four hours post injection plasma levels of SU5416 high dose group was 29.86 ± 2.75 ng ml−1 , which is within the therapeutic range. SU5416 levels in the low dose group was determined to be 9.48 ± 2.75 ng ml−1 . 3.2. Permeability alterations in the retina In non-diabetic control animals albumin positivity was limited to the lumen of the microvasculature, with very little or no positivity (range score 0–1) in the extravascular component. Poorly controlled diabetic animals demonstrated increased immunoreactivity throughout the retina and the intensity of the staining was greater in the extravascular region (range score 3–4) compared to control (P < 0.001). High dose SU5416 treatment prevented diabetes induced increased permeability and the immunostaining pattern was similar to the control animals (score range 0–1). No extravascular albumin staining was seen in the lens or cornea, as an internal control (Fig. 1). 3.3. Blood flow alteration Resistivity index as calculated from Doppler measurement, was significantly elevated in poorly controlled diabetic rats compared to non-diabetic animals. High dose SU5416 therapy significantly reduced the retinal RI value compared to poorly diabetic animals and control animals. Low dose SU5416 treatment however failed to achieve a therapeutic effect. (Fig. 2) No significant changes in either mitral valve or pulmonary artery RI values due to diabetes or SU5416 were observed. [RI of pulmonary artery was 0.88 ± 0.01 (control), 0.89 ± 0.02 (diabetes), 0.94 ± 0.02 (diabetes with high dose SU5416),

Table 2 Clinical parameters (average ± S.E.M. for each group)

Body weight (g) Blood glucose (mmol/l) Glycated haemoglobin (%) a

Non-diabetic control (n = 6)

Poorly controlled diabetes (n = 6)

Poorly controlled diabetes with SU5416 (n = 12)

399.4 ± 10.66 5.5 ± 0.22 5.2 ± 0.77

347.9 ± 11.04a 22.6 ± 1.15a 12.2 ± 1.09a

288.3 ± 24.61a 17.6 ± 3.92a 9.8 ± 1.17a

Denotes significant difference from non-diabetic control (P < 0.05).

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Fig. 1. Immuno-histochemical staining of the retina for albumin to detect capillary permeability. (a) From a non-diabetic control rat, (b) poorly controlled diabetic rat, (c) poorly controlled diabetic rat with SU5416 treatment and (d) negative control. No permeability alteration was seen in the adjoining lens (L). (Arrows indicate microvessels, original magnification of (a); (b); (d) = 100×; (c) = 200× (n = 6 for samples a, b, c.)

0.91±0.01 (diabetes with low dose SU5416). RI of the mitral valve was 0.91±0.01 (control), 0.94±0.01 (diabetes), 0.94±0.00 (diabetes with high dose SU5416), 0.91 ± 0.03 (diabetes with low dose SU5416).] 3.4. Retinal mRNA expression In vivo, poorly controlled diabetes increased the mRNA expression of ET-1, ET-3, and VEGF 164 in the retina. eNOS, iNOS, VEGF 120 and VEGF 188 expression remained unchanged in poorly controlled diabetes. SU5416 treatment had no effect on ET-3 and eNOS mRNA expression in diabetic animals. However, iNOS expression was significantly augmented by

both doses of SU5416 treatment. SU5416 high dose further augmented VEGF 120 and VEGF 164 mRNA expression in the retina (Fig. 3). The ET-1 mRNA expression following treatment of diabetic animals with both low and high doses of SU5416 remained significantly elevated compared to non-diabetic animals. Although the levels were slightly higher than untreated diabetic animals, statistical significance was not achieved. 3.5. Mechanism of SU5416 iNOS upregulation As our studies on the retina demonstrated an inducible effect of SU5416 on iNOS expression (Fig. 3),

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Fig. 2. Resistivity index (RI) as calculated from colour Doppler ultrasound of the central retinal artery (see Section 2). Poorly controlled diabetes significantly increased the RI value, while treatment of diabetic rats with high dose SU5416 significantly reduced the RI. Low dose SU5416 had no effect on RI. (n = 6 for all groups.) (∗) Indicates significant difference from non-diabetic controls (P < 0.05); (†) indicates significant difference from poorly controlled diabetes (P < 0.05).

we further explored the mechanism of such phenomenon using cultured endothelial cells. Previous studies in kidney and neuronal tissues [26–28] showed iNOS mRNA expression may be partially mediated through a MAP kinase pathway. To determine whether iNOS mRNA upregulation due to SU5416 occurred through a MAPK signalling pathway, selective inhibition of MAPK was used in a cell culture (HUVEC) system. HUVEC cells treated with 25 mM glucose suppressed iNOS expression. However, SU5416 in addition to 25 mM glucose did increase iNOS expression. The MAPK inhibitor U0126 suppressed the expression of iNOS in spite of SU5416 treatment (Fig. 4).

4. Discussion In the present study we demonstrated the important role of VEGF by itself and its interaction with other vasoactive substances in the pathogenesis of diabetes induced early functional changes in the retina. Increased vascular permeability and microvascular blood flow abnormalities are characteristic features of diabetic retinopathy [1,2]. VEGF is a potent factor causing increased vascular permeability. Increases in vascular permeability have been demonstrated in STZ diabetic rats after 1 week of diabetes [29]. Quantitatively, increased vascular permeability as demonstrated by increased albumin permeation has been shown to increase 2.9 fold after 1 week and 10.7

fold after 4 weeks of diabetes [30]. In this study we have demonstrated that increased permeability occurs in association with increased VEGF mRNA expression in the retina and such increases in permeability may be prevented using a specific blocker of VEGF signalling. SU5416 was developed as a chemotherapeutic agent used to inhibit angiogenesis [21,22,31]. This compound has demonstrated to be very effective in blocking VEGF signalling in other systems [21,22,30,31]. Furthermore, SU5416 has demonstrated an ability to cross the blood brain barrier [32]. However, this is the first report where this compound has been used to prevent diabetes induced microvascular abnormalities. The method used for demonstration of increased permeability is simple, reliable and has been used previously by other investigators [33]. VEGF may interact with ICAM-1 in mediating increased permeability in diabetes [30]. ICAM-1 expression can be induced through VEGF stimulation [34,35]. Increased ICAM-1 expression is associated with a breakdown of the blood retinal barrier by way of increased leukocyte adhesion [30]. Furthermore, the inhibition of ICAM-1 has demonstrated a reduction of the blood retina barrier breakdown [30]. PKC activation is an important mechanism leading to VEGF up-regulation in diabetes [7,11,36]. However, it has previously been shown that augmented polyol pathway activity, non enzymatic glycation and oxidative stress may also regulate VEGF expression [37].

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Fig. 3. Diagrammatic representation of retinal mRNA expression of (a) ET-1; (b) ET-3; (c) iNOS; (d) eNOS and (e) VEGF. Diabetes lead to increased expression of ET-1, ET-3 and VEGF mRNA but not eNOS or iNOS mRNA. SU5416 treatment on the other hand lead to increased iNOS mRNA expression. (n = 6 for all groups.) (∗) Indicates significant difference from non-diabetic controls (P < 0.05).

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Fig. 4. Diagrammatic representation of iNOS mRNA analysis from HUVEC cells as analysed by real time PCR. (a) Treatment of cells with 25 mM d-glucose lead to decreased iNOS mRNA expression. SU5416 treatment along with 25 mM d-glucose lead to increased iNOS mRNA expression which was normalised by treatment with a MAPK inhibitor. (b) Represents an amplification plot of human iNOS. (c) Represents a melting curve analysis plot from human iNOS demonstrating the specificity of the PCR product. (∗) Indicates significant difference from 5 mM glucose (P < 0.05); (†) indicates significant difference from 25 mM glucose treatment (P < 0.05).

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It is of further interest that this study has demonstrated an interaction of VEGF with other vasoactive factors which are important in mediating blood flow within the retina. We and others have previously demonstrated increased ET-1 expression which is a key factor in the production of retinal vasoconstriction [13,38,39]. We used colour Doppler ultrasound based RI measurement as an indicator of retinal capillary vasoconstriction. As RI values across the mitral valve or pulmonary artery were not altered, the present data suggest that changes seen in this study reflect retinal microvasculature changes. Diabetic animals as previously reported, demonstrated increased RI in the retina, in association with increased ET expression [13,38]. VEGF signal inhibition with high dose SU5416 was found to abolish diabetes induced retinal vasoconstriction in spite of increased ET-1 expression. Interestingly, the same treatment also lead to increased iNOS expression. Hence, it is conceptually possible that increased NO production secondary to SU5416 treatment may have counterbalanced the ET induced vasoconstriction. On the other hand, animals treated with low dose SU5416 did not normalise the diabetes-induced, increased RI. Although iNOS mRNA expression of high and low dose SU5416 were similar, the ET-1 mRNA level was highest in the low dose SU5416 animals. The exact cause of this phenomenon is not clear. Due to the intricate regulatory mechanisms between these vasoactive factors, it is theoretically possible that partial VEGF signal blockade with SU5416 may have further augmented ET-1 expression which prevented the vasodilatory effects of NO. However, it is also possible that other yet unidentified mechanisms which are not mediated by NO may also be responsible. These ideas will require further confirmation. It has been demonstrated that VEGF may increase NO production [16,17]. We have previously ascertained that although treatment of non-diabetic animals with NO blocker produced no change in RI, the treatment of diabetic animals with NO donor prevented a diabetes induced RI increase [40]. In addition, ET and VEGF have a co-stimulatory relationship [15]. In the present study however, VEGF signal inhibition was not effective in preventing diabetes induced ET-1 up-regulation in the retina, suggesting several mechanisms may be involved in the up-regulation of ETs in diabetes [41]. As eluded to earlier ET-1

and NO also have a counter-inhibitory relationship [19,42]. To further characterise the mechanism of increased iNOS mRNA expression observed in the diabetic animals treated with SU5416 we investigated endothelial cells with respect to MAPK signalling. Mitogen activated protein kinase (MAPK) signalling consists of a three protein system signal cascade: the MAPK protein and upstream MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK) [43]. There are three MAPK pathways: the extracellular signal regulated kinases (erk1 and 2), the c-Jun-N terminal kinase 1 (JNK 1) and the p38 MAPK [44]. In diabetes, MAPK proteins are activated due to the direct effects of hyperglycemia, glucose induced oxidative stress, and AGE interactions [45–48]. In our endothelial cell system, the SU5416 induced up-regulation of iNOS mRNA was normalised with the use of a MAPK inhibitor, suggesting that the effects of VEGF receptor signal inhibition may be partially mediated through a MAPK signal cascade. In keeping with this notion it has previously been demonstrated that MAPK signalling is important in the regulation of NOS expression [26]. In summary, we have demonstrated that VEGF is an important mediator of increased vascular permeability in diabetes. In addition, VEGF by its interaction with ET and NO may be of importance in mediating retinal blood flow abnormalities in diabetes. Furthermore, MAPK signalling may be significant in the regulation of iNOS. VEGF signal inhibition may offer an important therapeutic modality for diabetic retinopathy. Acknowledgements This work was supported in part by grants from the Canadian Diabetes Association in memory of Glenn W. Liebrock as well as the Lawson Health Research Institute Internal Research Fund. The authors wish to thank K. Mukherjee for the histological preparations and immunostaining.

References [1] G.L. King, M. Brownlee, The cellular and molecular mechanisms of diabetic complications, Endocrinol. Metab. Clin. North Am. 25 (1996) 255–270.

M. Cukiernik et al. / Diabetes Research and Clinical Practice 65 (2004) 197–208 [2] S. Chakrabarti, M. Cukiernik, D. Hileeto, T. Evans, S. Chen, Role of vasoactive factors in the pathogenesis of early changes in diabetic retinopathy, Diabetes Metab. Res. Rev. 16 (2000) 393–407. [3] I. Spyridopoulos, C. Luedemann, D. Chen, M. Kearney, D. Chen, T. Murohara, N. Principe, J.M. Isner, D.W. Losordo, Divergence of angiogenic and vascular permeability signalling by VEGF: inhibition of protein kinase C suppresses VEGF-induced angiogenesis, but promotes VEGF-induced, NO-dependent vascular permeability, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 901–906. [4] T.V. Petrova, T. Makinen, K. Alitalo, Signalling via vascular endothelial growth factor receptors, Exp. Cell Res. 253 (1999) 117–130. [5] L.P. Aiello, J.S. Wong, Role of vascular endothelial growth factor in diabetic vascular complications, Kidney Int. Suppl. 77 (2000) S113–S119. [6] N. Ferrara, Role of vascular endothelial growth factor in regulation of physiological angiogenesis, Am. J. Physiol. Cell Physiol. 280 (2001) C1358–C1366. [7] L.P. Aiello, S.E. Bursell, A. Clermont, E. Duh, H. Ishii, C. Takagi, F. Mori, T.A. Ciulla, K. Ways, M. Jirousek, L.E. Smith, G.L. King, Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor, Diabetes 46 (1997) 1473–1480. [8] D.R. Senger, S.J. Galli, A.M. Dvorak, C.A. Perruzzi, V.S. Harvey, H.F. Dvorak, Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid, Science 219 (1983) 983–985. [9] C.A. Do, P. Ramos, A. Reis, R. Proenca, J.G. Cunha-vaz, Breakdown of the inner and outer blood retinal barrier in streptozotocin-induced diabetes, Exp. Eye Res. 67 (1998) 569–575. [10] T. Murata, K. Nakagawa, A. Khalil, T. Ishibashi, H. Inomata, K. Sueishi, The relation between expression of vascular endothelial growth factor and breakdown of the blood-retinal barrier in diabetic rat retinas, Lab. Invest. 74 (1996) 819– 825. [11] P. Xia, L.P. Aiello, H. Ishii, Z.Y. Jiang, D.J. Park, G.S. Robinson, H. Takagi, W.P. Newsome, M.R. Jirousek, G.L. King, Characterization of vascular endothelial growth factor’s effect on the activation of protein kinase C, its isoforms, and endothelial cell growth, J. Clin. Invest. 98 (1996) 2018– 2026. [12] R.G. Tilton, T. Kawamura, K.C. Chang, Y. Ido, R.J. Bjercke, C.C. Stephan, T.A. Brock, J.R. Williamson, Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor, J. Clin. Invest. 99 (1997) 2192–2202. [13] D. Deng, T. Evans, K. Mukherjee, D. Downey, S. Chakrabarti, Diabetes-induced vascular dysfunction in the retina: role of endothelins, Diabetologia 42 (1999) 1228–1234. [14] S. Chen, M.D. Apostolova, M.G. Cherian, S. Chakrabarti, Interaction of endothelin-1 with vasoactive factors in mediating glucose-induced increased permeability in endothelial cells, Lab. Invest. 80 (2000) 1311–1321.

207

[15] A. Cruz, C. Parnot, D. Ribatti, P. Corvol, J.M. Gasc, Endothelin-1, a regulator of angiogenesis in the chick chorioallantoic membrane, J. Vasc. Res. 38 (2001) 536–545. [16] J. Kroll, J. Waltenberger, VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR), Biochem. Biophys. Res. Commun. 252 (1998) 743– 746. [17] A. Bouloumie, V.B. Schini-Kerth, R. Busse, Vascular endothelial growth factor up-regulates nitric oxide synthase expression in endothelial cells, Cardiovasc. Res. 41 (1999) 773–780. [18] N. Ghiso, R.M. Rohan, S. Amano, R. Garland, A.P. Adamis, Suppression of hypoxia-associated vascular endothelial growth factor gene expression by nitric oxide via cGMP, Invest. Ophthalmol. Vis. Sci. 40 (1999) 1033–1039. [19] E.R. Levin, Endothelins, N. Engl. J. Med. 333 (1995) 356– 363. [20] P.M. Vanhoutte, Endothelin-1. A matter of life and breath, Nature 368 (1994) 693–694. [21] T.A. Fong, L.K. Shawver, L. Sun, C. Tang, H. App, T.J. Powell, Y.H. Kim, R. Schreck, X. Wang, W. Risau, A. Ullrich, K.P. Hirth, G. McMahon, SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types, Cancer Res. 59 (1999) 99–106. [22] L. Angelov, B. Salhia, L. Roncari, G. McMahon, A. Guha, Inhibition of angiogenesis by blocking activation of the vascular endothelial growth factor receptor 2 leads to decreased growth of neurogenic sarcomas, Cancer Res. 59 (1999) 5536–5541. [23] S. Chen, S. Mukherjee, C. Chakraborty, S. Chakrabarti, High glucose-induced, endothelin-dependent fibronectin synthesis is mediated via NF-kappa B and AP-1, Am. J. Physiol. Cell Physiol. 284 (2003) C263–C272. [24] J. Dotsch, N. Hogen, Z. Nyul, J. Hanze, I. Knerr, M. Kirschbaum, W. Rascher, Increase of endothelial nitric oxide synthase and endothelin-1 mRNA expression in human placenta during gestation, Eur. J. Obstet. Gynecol. Reprod. Biol. 97 (2001) 163–167. [25] S. Chen, Z.A. Khan, M. Cukiernik, S. Chakrabarti, Differential activation of NF-kappa B and AP-1 in increased fibronectin synthesis in target organs of diabetic complications, Am. J. Physiol. Endocrinol. Metab. 284 (2003) E1089–E1097. [26] M. Poljakovic, J.M. Nygren, K. Persson, Signalling pathways regulating inducible nitric oxide synthase expression in human kidney epithelial cells, Eur. J. Pharmacol. 469 (2003) 21–28. [27] A. Lahti, H. Kankaanranta, E. Moilanen, P38 mitogen-activated protein kinase inhibitor SB203580 has a bi-directional effect on iNOS expression and NO production, Eur. J. Pharmacol. 454 (2002) 115–123. [28] I.O. Han, H.S. Kim, H.C. Kim, E.H. Joe, W.K. Kim, Synergistic expression of inducible nitric oxide synthase by phorbol ester and interferon-gamma is mediated through NFkappaB and ERK in microglial cells, J. Neurosci. Res. 73 (2003) 659–669.

208

M. Cukiernik et al. / Diabetes Research and Clinical Practice 65 (2004) 197–208

[29] T. Qaum, Q. Xu, A.M. Joussen, M.W. Clemens, W. Qin, K. Miyamoto, H. Hassessian, S.J. Wiegand, J. Rudge, G.D. Yancopoulos, A.P. Adamis, VEGF-initiated blood-retinal barrier breakdown in early diabetes, Invest. Ophthalmol. Vis. Sci. 42 (2001) 2408–2413. [30] K. Miyamoto, S. Khosrof, S.E. Bursell, R. Rohan, T. Murata, A.C. Clermont, L.P. Aiello, Y. Ogura, A.P. Adamis, Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 10836– 10841. [31] D.B. Mendel, R.E. Schreck, D.C. West, G. Li, L.M. Strawn, S.S. Tanciongco, S. Vasile, L.K. Shawver, J.M. Cherrington, The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function, Clin. Cancer Res. 6 (2000) 4848–4858. [32] T. Takamoto, M. Sasaki, T. Kuno, N. Tamaki, Flk-1 specific kinase inhibitor (SU5416) inhibited the growth of GS-9L glioma in rat brain and prolonged the survival, Kobe J. Med. Sci. 47 (2001) 181–191. [33] S.A. Vinores, C. Gadegbeku, P.A. Campochiaro, W.R. Green, Immunohistochemical localization of blood-retinal barrier breakdown in human diabetics, Am. J. Pathol. 134 (1989) 231–235. [34] M. Lu, V.L. Perez, N. Ma, K. Miyamoto, H.B. Peng, J.K. Liao, A.P. Adamis, VEGF increases retinal vascular ICAM-1 expression in vivo, Invest. Ophthalmol. Vis. Sci. 40 (1999) 1808–1812. [35] K. Miyamoto, S. Khosrof, S.E. Bursell, Y. Moromizato, L.P. Aiello, Y. Ogura, A.P. Adamis, Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1), Am. J. Pathol. 156 (2000) 1733–1739. [36] P. Xia, T. Inoguchi, T.S. Kern, R.L. Engerman, P.J. Oates, G.L. King, Characterization of the mechanism for the chronic activation of diacylglycerol–protein kinase C pathway in diabetes and hypergalactosemia, Diabetes 43 (1994) 1122– 1129. [37] I.G. Obrosova, A.G. Minchenko, V. Marinescu, L. Fathallah, A. Kennedy, C.M. Stockert, R.N. Frank, M.J. Stevens, Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats, Diabetologia 44 (2001) 1102–1110.

[38] T. Evans, D.D. Xi, K. Mukherjee, D. Downey, S. Chakrabarti, Endothelins, their receptors, and retinal vascular dysfunction in galactose-fed rats, Diabetes Res. Clin. Pract. 48 (2000) 75–85. [39] S. Dallinger, G.T. Dorner, R. Wenzel, U. Graselli, O. Findl, H.G. Eichler, M. Wolzt, L. Schmetterer, Endothelin-1 contributes to hyperoxia-induced vasoconstriction in the human retina, Invest. Ophthalmol. Vis. Sci. 41 (2000) 864– 869. [40] M. Cukiernik, S. Mukherjee, D. Downey, S. Chrakabarti, Heme oxygenase in the retina in diabetes, Curr. Eye Res. 27 (2003) 301–308. [41] S. Chakrabarti, M. Cukiernik, S. Mukherjee, S. Chen, Therapeutic potential of endothelin receptor antagonists in diabetes, Expert. Opin. Investig. Drugs 9 (2000) 2873–2888. [42] D. Fukumura, T. Gohongi, A. Kadambi, Y. Izumi, J. Ang, C.O. Yun, D.G. Buerk, P.L. Huang, R.K. Jain, Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 2604–2609. [43] S. Kumar, J. Boehm, J.C. Lee, p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases, Nat. Rev. Drug Discov. 2 (2003) 717–726. [44] G. Pearson, F. Robinson, G.T. Beers, B.E. Xu, M. Karandikar, K. Berman, M.H. Cobb, Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions, Endocr. Rev. 22 (2001) 153–183. [45] D.M. Cohen, Mitogen-activated protein kinase cascades and the signalling of hyperosmotic stress to immediate early genes, Comp. Biochem. Physiol. A Physiol. 117 (1997) 291– 299. [46] D.R. Tomlinson, Mitogen-activated protein kinases as glucose transducers for diabetic complications, Diabetologia 42 (1999) 1271–1281. [47] M. Haneda, S. Araki, M. Togawa, T. Sugimoto, M. Isono, R. Kikkawa, Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions, Diabetes 46 (1997) 847–853. [48] X. Wang, J.L. Martindale, Y. Liu, N.J. Holbrook, The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival, Biochem. J. 333 (Pt 2) (1998) 291–300.