BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

48,

8-18

(1992)

Increased EGF Binding and EGFR mRNA Expression in Rat Aorta with Chronic Administration of Pressor Angiotensin II MOHINDER P. SAMBHI, NARAYAN SWAMINATHAN, HONG WANG, AND HONGMEI Hypertension Research Administration

RONG

Laboratory, Hypertension Section, Department of Medicine, Medical Center, Sepulveda; and UCLA School of Medicine, Los Angeles, California 91343

Veterans

Received January 14, 1992, and in revised form March 27, 1992 This study examines the changes in the mRNA expression of epidermal growth factor (EGF), EGF receptor (EGFR), platelet derived growth factor (PDGF-B), and transforming growth factor /3 (TGF-P,) before and after sustained pressor infusion of angiotensin II (Ang II) for 4 weeks. A threefold increase occurred in the levels of EGFR mRNA (17,240 I 827 vs 6403 ? 1372 units, P < 0.01) and TGF-Pl mRNA (1644 k 584 vs 475 + 30 units, P < 0.01) only in the aorta and not in the heart and kidney tissues. This increase in both of the above mRNA transcripts highly correlated (r = 0.96 and 0.92, P < 0.01) with the elevation of blood pressure. The specific binding of ‘%labeled EGF to aortic membranes also increased (11,429 ? 728 vs 8630 2 420 cpm/mg protein, P < 0.05) with a parallel increase in the protein tyrosine kinase activity of the membranes indicating that the enhanced EGFR mRNA expression resulted in increased activity of a functional receptor. No significant changes were observed in either EGF mRNA or PDGF-B mRNA levels. These findings suggest that EGFR and TGF-/3, participate in the long-term progressive pressor response to Ang II and thus potentially in the progression and the maintenance of chronic hypertension. 0 1992 Academic Press. Inc.

It is increasingly being appreciated that humoral factors (endocrine, paracrine, and autocrine) independent of blood pressure play a prominent role in the regulation of vascular growth. Angiotensin II (Ang II), a vasopressor agonist, has been shown to have a direct stimulating effect on the growth of smooth muscle cells (SMC) in culture derived from rate (1) and human (2) aorta. The concept that Ang II may induce vascular mitogenesis in vivo has been supported by observations on renal vasculature surrounding a renin-secreting tumor (3) and on the vascular hypertrophy observed in Bartters’s syndrome (4) as well as in familial chloride diarrhea syndrome (5). In recent years, a role for polypeptide growth factors such as the epidermal growth factor (EGF), the platelet derived growth factor (PDGF), and the insulinlike growth factor has been proposed in stimulating the proliferative cellular response (6). The vasopressor agonist Ang II shares cellular effects with PDGF 8 0885-4505/92 Copyright All rights

$5.00

0 1992 by Academic Press. Inc. of reproduction in any form reserved

ANGIOTENSIN

II AND GROWTH

FACTOR

EXPRESSION

9

and EGF including vasoconstriction (7,8), rise of intracellular Ca*+, and stimulation of Na’/H+ antiporter (9,lO). Naftilan et al. (11) demonstrated that the addition of Ang II to rat aortic SMC stimulated mRNA expression of PDGF-A chain and c-myc proto-oncogene. We and others (12,13) have shown that the addition of EGF in a dose-dependent manner, leads to a selectively enhanced stimulation of DNA synthesis in cultured SMC from spontaneously hypertensive rat (SHR) aorta, compared with that from the normotensive control Wistar Kyoto (WKY) rat. In addition, we found that the EGF-induced increased DNA synthesis in the SHR-derived aortic SMC cultures parallels an enhanced receptor tyrosine kinase (RTK) activity of the EGF receptor (EGFR) (14). Sarzani et al. (15) compared the expression of several growth factors in the aorta of normotensive and DOG/salt hypertensive rats and found only the transforming growth factor /3 (TGF-0,) to be overexpressed in the hypertensive rats. Barrett and Benditt (16) have reported that the gene transcript levels of PDGF-B chain are elevated in human atherosclerotic lesions compared to normal blood vessels. In the present study we have examined the effect of long-term sustained infusions of Ang II on the steady-state mRNA expression of the widely expressed cellular proto-oncogene, EGFR and of its principal ligand EGF in the rat aorta. In addition, in the same tissue, we have studied the expression levels of TGF-/3, and PDGF-B gene transcripts. The expression levels of the same transcripts were also examined in the renal and cardiac tissues of the animals. MATERIALS

AND METHODS

Angiotensin II Infusions

Angiotensin II (human, octapeptide) was purchased from Bachem Inc., (Torrance, CA). Alzet minipumps (Alza Corp., Palo Alto, CA) delivering 2.99 ,ul/h for 4 weeks were implanted intraperitoneally into 20-week-old adult male SpragueDawley rats. Each pump contained 2.2 ml of Ang II solution (4 mg/ml) in normal saline as vehicle (n = 8). Under these conditions, the pump delivered an approximate dosage of 500 ng/min/kg body weight. The Ang II dose and the 4week period of infusion were selected as optimal from earlier studies reported in the literature (17,18). Control group was installed with minipumps filled with vehicle (n = 3). Systolic blood pressures (tail cuff method) were recorded at the beginning (Ang II, 115 ? 7; vehicle, 116 ? 6 mm Hg) and after a period of 4 weeks (Ang II, 181 ? 10.8; vehicle, 122 ? 8.7 mm Hg, P < 0.001). The body weights were also recorded before and after the infusion period. The animals were sacrificed and the tissues were excised and analyzed as described below. Removal of Tissues

All fat and appendages were removed from aorta, heart, and kidneys. The tissues were weighed and homogenized. The aorta was taken from the beginning of the aortic arch to the abdominal bifurcation. Since Sarzani et al. (15) had demonstrated that constitutive expression of several growth factors occurred in the rat aortic wall from which endothelium had been scraped, we have used the entire aortic wall in the present studies. The heart was stripped of atria and

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retained both ventricles. The aortae, due to the small sample size, were pooled as required from more than one animal (generally two animals) before further processing. Extraction of the Total RNA from the Tissues Total RNA was prepared from the tissues using guanidine thiocyanate-acidified phenol extraction as described by Chromczynski and Sacchi (19). Preparation of Probes EGFR probe. Plasmid pE7 was purchased from ATCC (American Type Culture Collection, Bethesda, MD), and digested with EcoRI-HindIII. The resulting 0.70kb fragment, representing the 3’ tyrosine kinase portion of the message, was cloned in pGEM-4Z vector. EGF probe. Plasmid phEGF121 was purchased from ATCC and digested with EcoRI and the resulting 0.56kb fragment was cloned in pGEM-3Z vector. PDGF-B probe. Plasmid pSM-1 was purchased from ATCC and cleaved with BamHI. The resulting 2-kb fragment was cloned in PGEM-3Z vector. TGF-/3 probe. Plasmid phTGF+, was purchased from ATCC and cleaved with EcoRI and the resulting 2-kb fragment was cloned in PGEM-3Z vector. Labeling the Probes Labeling was performed according to the protocol described in the Promega manual. In a total volume of 20 ~1 the reaction mixture contained 4 ~1 of 5 x transcription buffer (0.2 M Tris-HCl, pH 7.5, with mM MgCl*, 10 mM spermidine, and 50 mM NaCl), 2 ~1 of 100 mM DTT, 0.8 ~1 of RNasin ribonuclease inhibitor (25 units/p1 stock), 4 ~1 of 2.5 mM each of CTP, UTP, and GTP), 2.4 ,ul 100 PM ATP, 1 pg of linearized plasmid DNA in water, 5 ~1 of 10 mCi/ml [32P]ATP, and 1 ~1 (10 units) of T7 RNA polymerase. The reaction mixture was incubated for 60 min at 37°C. The reaction was terminated by adding 2 units of RQl DNase and incubating for 15 min at 37°C. The solution was extracted with 200 ~1 of phenol:chloroform followed by 200 ~1 of chloroform. The labeled RNA was isolated by precipitation by 3 M sodium acetate ethanol. Quantitation of mRNA The specific mRNAs were quantitated by slot blot hybridization essentially as described by Davis et al. (20). RNA isolated from the tissues (0.03 to 0.50 pg/slot) was vacuum filter blotted to nitrocellulose after denaturation in 6~ SSC, 2.2 M formaldehyde at 65°C for 15 min. The slots were washed with 20 x SSC and dried in vacua at 80°C for 2 h and were hybridized to [32P]ATP-labeled probes. Hybridization conditions were 5 x SSPE, 50% formamide, 5 x Denhardt’s, 0.5% SDS, 50 pg/ml salmon sperm DNA, and 5% polyethylene glycol at 42°C for 1820 hr. Filters were given two high stringency washes in 0.1 X SSC, 0.1% SDS at 58°C for 30 min, and once in 0.1 x SSC for 10 min. Filters were wrapped in cellophane and autoradiographed for 1 to 5 days with Kodak XAR-5 film for varying time periods and scanned with a densitometer (Hoffer Scientific) and the filters were cut and counted in a Beckman LS-7000 liquid scintillation counter.

ANGIOTENSIN

II AND GROWTH

FACTOR

0

n

11

EXPRESSION

Vehicle AnglI

”

EGFR EGF FIG. 1. Quantification of mRNA expression in aorta. Total RNA extracted from the aortic tissue of the rats after 4 weeks of implantation of minipumps with vehicle or angiotensin II was analyzed by slot blot analysis, using hybridization with specific labeled RNA probes for EGFR, EGF, PDGF, and TGF-j3, mRNAs. The counts from the slot blots were plotted against the applied RNA concentrations. The means of the slopes of the lines are shown as bars with their standard deviations. Closed bars, Ang II treated; open bars, vehicle.

Plots of the concentration of RNA blotted versus counts per minute (cpm) for each concentration were analyzed by linear regression using Datamanager, an IBM computer-based statistics package. Only plots with regression coefficients, r > 0.90, P < 0.05 were compared and the significance of the difference in the slopes determined. Comparison of the ratios of the significantly different slopes (P < 0.05) were used to estimate the relative abundance of the specific mRNA in total RNA from the tissues. EGF-Binding Studies

The binding of 1’51-labeled EGF to aortic membranes scribed by Swaminathan and Sambhi (21).

was performed

as de-

Protein Tyrosine Kinase Activity

The receptor tyrosine kinase activity of the aortic membranes according to the method of Akiyama et al. (22).

was determined

RESULTS The animals receiving Ang II infusions at an approximate dose of 500 ng/kg/min for 4 weeks developed a significant increase in systolic BP (181 + 10.8 mm Hg) compared with those receiving vehicle (122 ? 8.7 mm Hg, P < 0.001). The body weight of animals in the two groups at 4 weeks did not differ from each other (431.4 2 44.4 vs 382.7 +- 57.5 g, NS). Figure 1 shows the quantified levels of the mRNA transcripts, hybridized with the four labeled probes (specific for EGF, EGFR, TGF-Pi, and PDGF-B mRNAs) examined in the aortic tissue of animals receiving Ang II or vehicle infusions. At 4 weeks Ang II caused a highly significant (up to threefold) increase in the steadystate mRNA expression of EGFR (17,240 -+ 827 vs 6403 -+ 1372 arbitrary mRNA units, P < 0.01). The levels of EGF mRNA, in animals receiving Ang II were

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TABLE 1 EGF Binding and Receptor Tyrosine Kinase Activity in Aorta Receptor tyrosine kinase activity Infusion

n

EGF binding

Control

+ EGF

+ Genistein

Vehicle Ang II

3 8

8,630 k 420 11,429 2 728*

852 -c 9.5 1,232 k 213*

3,225 f 102 4,335 2 172*

903 + 93 1,421 f 121;

Note. The EGF binding and the receptor tyrosine kinase activity were determined as described under Materials and Methods. The results shown are the means f SD of triplicate determinations on membrane homogenates of aortic tissue samples from each animal. EGF binding data is shown as cpm of ‘*%labeled EGF bound/mg of membrane protein. Receptor tyrosine kinase activity was measured with and without the addition of EGF (1 pg/ml) and/or genistein (1 pg/ml). The results are expressed as cpm in “P-labeled precipitated proteins/100 pg of menbrane protein. * Significance, P < 0.01.

also higher than those receiving vehicle (1495 + 599 vs 543 + 117 arbitrary mRNA units) but the difference did not reach statistical significance in the current experiments. PDGF-B mRNA did not show significant changes. The observed increase in the prevalence of EGFR mRNA transcripts in the aortic tissue of Ang II vs vehicle infused rats was further supported by the 12?labeled EGF-binding studies. The B,,, for the EGF binding was greater in the aortic tissue of Ang II-infused animals compared to vehicle-infused animals (11,429 + 728 vs 8630 + 420 cpm/mg protein, P < O.OS), with no significant change in kd values (0.10 +: 0.01 vs 0.11 2 0.01 nM), indicating an increase in the number of EGF-binding sites (Table 1). We further examined the protein tyrosine kinase activity in the aortic membrane preparations from Ang II and vehicle-infused rats (Table 1). The phosphorylation in TCA precipitated proteins, (incubated with [Y-~*P]ATP increased three- to fourfold by the addition of EGF and was inhibited up to 70% by added genistein (1.0 pug/ml), a specific inhibitor of tyrosine kinase (21). The receptor tyrosine kinase activity of aortic homogenates from Ang II-infused rats was higher than that of controls (Table 1) thus suggesting that the increased prevalence of EGFR mRNA transcripts resulted in the formation of a whole membrane-bound functional EGFR protein molecule and this functional activity was significantly enhanced in Ang II-infused animals. The expression levels of TGF-PI mRNA (Table 2) were also significantly elevated in the aorta of animals receiving Ang II infusions (1644 & 584 vs 474 + 30, P < 0.05). The levels of TGF-/3i mRNA in the heart and kidney tissues, however, showed no difference between the Ang II-treated and the vehicle-infused groups. Figure 2 examines the relationship of the EGFR mRNA transcript levels with the corresponding BP levels of the animals. Whereas a highly significant correlation emerges between BP and the EGFR mRNA in the aortic tissue (r = 0.96, P < O.Ol), the relationship is entirely nonexistent in the heart and the kidney tissue. A similar pattern of high correlation between BP and the quantified TGF-PI mRNA units in the aortic tissues was also observed (r = 0.92, P < 0.01).

ANGIOTENSIN

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EXPRESSION

TABLE 2 Effect of Chronic Ang II Infusion on TGFj31 Gene Expression (Arbitrary

mRNA Units)

Tissue

Ang II infusion

Vehicle infusion

P value

Aorta Kidney Heart

1644 2 584 2759 2 1003 6906 t 1087

474 2 30 3032 _’ 9.5 6841 2 1043

-co.05 N.S. N.S.

Note. In the aorta the mRNA expression levels of TGF-j31 (quantified by counting) in the individual animals correlated (r = 0.92, P < 0.01) with systolic BP (mm Hg). No correlation between the two parameters existed in the heart or kidney tissues.

DISCUSSION Our studies demonstrate that in the intact animal, a long-term systemic infusion of Ang II leading to sustained hypertension at 4 weeks was accompanied by a significant upregulation of the gene expression in the aortic tissue of two cellular proto-oncogenes. A marked upregulation was observed in the gene expression of the potent vascular mitogen EGFR (8) and a modest but significant upregulation in the same tissue in the gene expression of TGF-Pi, a potent desmoplastic agent. The expression levels of other growth factors examined including EGF and PDGFB were not influenced. Significant changes in the other two tissues studied were not observed. Studies on the constitutive expression of EGFR in the rat kidney have shown the presence of multiple transcripts of varying sizes (23). Similar studies in our own laboratory on aorta showed two bands at 6.4 and 2.8 kb (unpublished observations). Since in the present study we had to analyze individual organs, as far as possible, from each animal to examine the expression of several growth factors and to determine their correlation with blood pressure, only slot blots (which require a far smaller sample of RNA compared to Northern blots) could be performed. However, studies on EGF binding and protein tyrosine kinase activity supported the thesis that the quantified parameter represented a functional receptor. It should be recognized that further studies are needed to characterize the encoded receptor protein and its morphological site(s) of expression in the aortic wall. In the aortic tissue, the elevation in the quantified levels of mRNA transcripts of EGFR and TGF-Pi in all of the experimental animals significantly correlated with the levels of BP recorded before sacrifice. The significance of the observed correlation, however, should be interpreted cautiously because of the relatively small sample size. Nevertheless the existence of strong correlations in the aortic tissue coupled with their total absence in other tissues, the heart, and kidney is noteworthy. From the present studies it may be concluded that the observed stimulation in the growth factor gene expression may in part be secondary to the concomitant elevation of BP with Ang II infusions. Further work using subpressor infusions of Ang II, and antihypertensive agents is needed in order to dissect the potential

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P

d

c ,o

15

120

140

160

140

160

lb0 BPmm

o Vehicle OAngII

0’

120

(80 BPmm

3o

c oVeh& 25 l AngII t

T 9

i

20.

s 15. 0” d

P

p

f

i

IO-

OT

120

140

160

180

BPmm

2. Blood pressure and EGFR expression. Correlation of systolic BP (mm Hg) in individual animals or group of animals at 4 weeks of infusion (Ang II, closed circles; vehicle, open circles) with the quantified levels of EGFR mRNA expressed as relative mRNA arbitrary units (see Materials and Methods) in heart (A), aorta (B) and kidney (C) tissues. The correlation coefficients for the three tissues were; aorta: r = 0.96, P < 0.01, y = 132.3 + 165.6.x; heart: r = -0.53, N.S., y = 347.2 - 94.1 X; kidney: r = - 55, N.S., y = 313.2 - 84.9 x. The aortae from Ang II-infused animals were pooled in pairs. In the case of aortae from the control animals, two were pooled and one was analyzed as a single sample. The kidneys and hearts from each animal were analyzed separately. FIG.

ANGIOTENSIN

II AND

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EXPRESSION

1.5

mitogenic or trophic effects of Ang II per se vs elevated BP in the vasculature of the intact animal (17). It should be pointed out, however, that an adequate body of available evidence exists to support the notion that the vascular proliferative effects attributable both to Ang II and to EGFR can be potentially exerted independently of the prevailing BP levels in the intact animal. Dickinson and Lawrence (24) showed that Ang II administered chronically in dogs at a dose which was not initially pressor led gradually to well-marked hypertension. This slow pressor effect of low dose of Ang II and the ability of Ang II to increase the slope of its own pressor dose-response curve has been amply confirmed in subsequent studies on experimental animals and in man (25,26). It has been argued, however, that vasopressor agonists such as Ang II, designed for relatively acute presor homeostasis, by themselves are not sufficient to mimic the natural history of the slow gradual progression of essential hypertension, and the involvement of unknown trophic or growth factors is essential (27,28). Our studies propose that the potent protooncogenes EGFR as well as the TGF-j3, together constitute plausible candidates for such a role. Likewise evidence exists that EGF-mediated vascular proliferative response can occur independently of systemic BP elevations. EGF effects have been implicated in the genesis of the proliferative changes observed in equine distal digital peripheral vascular disease (29). We have recently demonstrated that the EGFR gene is overexpressed in the aorta from an adult spontaneously hypertensive rat compared to the aorta from its normotensive counterpart, the Wistar Kyoto rat (21). Lowering of blood pressure in the SHR by a 4-week treatment with the vasodilator hydralazine, however, did not alter the quantified mRNA transcript levels of EGFR in the aorta. Also, Dahl rats made hypertensive with salt feeding did not show an increase in EGFR mRNA transcripts in the aortic tissue (30). These findings lead us to conclude that the regulation of EGFR gene expression in the aortic tissue is not entirely a secondary result of changes in blood pressure. Other than vascular proliferative response, Ang II and EGFR appear to mediate a number of similar cellular effects that include a stimulation of Na+/H+ exchange (14,15), actions on the distal nephron to influence sodium (31) and water (32) transport, and a stimulation of the arachidonate prostaglandin system metabolism in the aortic tissue (l&33,34). Taken together, these findings suggest that the two important vasoconstrictor mitogens Ang II and EGFR may share, at least in part, some of the mechanisms of their cellular effects. The significance of the observed increased expression in these experiments of the multifunctional TGF-PI also remain to be elucidated. TGF-PI peptides appear to regulate growth and differentiation of a wide variety of cell types, generally stimulating mesenchymal cells, promoting the production of extracellular matrix, and inhibiting the proliferation of most epithelial and endothelial cells (35-37). The cellular mode of action of TGF-Pi and the signal transduction pathways are unknown. Complex and bifunctional interactions of TGF-Pi have been described at the transcriptional level with other growth factors. TGF-P1 (a potential inhibitor of EGF) has been shown to increase the synthesis of EGFR in response to its ligand (38). TGF-PI is secreted from the cells in a high molecular weight, latent form which

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is biologically inert and the physiological mechanisms for its activation in vivo are unknown (39,40). Increased TGF-& mRNA transcripts have been demonstrated (15) in the aortic tissue of deoxycortisone acetate/salt hypertensive rats, a model in which circulating levels of ang II are suppressed. Accordingly it may be speculated that TGF-P1 provides a protective mechanism for the arterial wall against increased transmural stress of rising intra-arterial pressure. Additionally, TGF-/3i may function as a potential mediator of an upregulation of EGFR and thus lead to an integrated vascular proliferative response. The precise function of growth factors examined in this study and their in vivo relationship and interactions remain to be defined. Nevertheless, the observed response to the potent vasopressor agonist Ang II, known to be involved in the mechanisms of several forms of chronic hypertension, represents findings of considerable relevance and interest. Our studies suggest that EGFR- and TGF-&mediated effects may potentially participate in the genesis and the maintenance of hypertension. REFERENCES 1. Geisterfer AAT, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aorta smooth muscle cells. Circ Res 62749-756, 1988. 2. Campbell-Boswell M, Robertson Jr AL. Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro. Exp Mol PathoZ35:265-276, 1981. 3. Lindop GM, Lever AF. Anatomy of the renin-angiotensin system in the normal and pathological kidney. Histopathology 10~335-362, 1986. 4. Fujita T, Sakaguchi H, Sibagaki M, Fukui T, Nomura M, Sekiguichi S. The pathogenesis of Bartter’s syndrome. Functional and histological studies. Am J Med 63~467-474, 1977. 5. Pasternack A, Perheentupa J. Hypertensive angiopathy in familial chloride diarrhoea. Lancer 2:3047-1049, 1966. 6. Deul TF. Polypeptide growth factors: roles in normal and abnormal cell growth. Annu Rev Cell Biol3:443-492, 1987. 7. Berk BC, Alexander RW, Brock TA, Gimbrone Jr MA, Webb RC. Vasoconstriction: A new activity for platelet derived growth factor. Science 232:87-90, 1986. 8. Berk BC, Brock TA, Webb RC, Taubman MB, Atkinson WJ, Gimbrone MA, Alexander RW. Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aorta contraction. J Clin Invest 75:1083-1086, 1985. 9. Rozengurt E, Heppel LA. Influence of EGF on the sodium hydrogen transport across membranes. Proc Nat1 Acad Sci USA 72~4492-4495, 1975. 10. Vallega GA, Canessa ML, Berk BC, Brock TA, Alexander RW. Vascular smooth muscle Na+H’ exchanger kinetics and its activation by angiotensin II. Am J Physiol 254:C751-C758, 1988. 11. Naftilan AJ, Pratt RE, Dzau VJ. Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells. J Clin Invest 83~1419-1424, 1989. 12. Scott-Burden T, Resink TJ, Baur U, Burgin M, Buhler FR. Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats. Hypertension 13:295304, 1989. 13. Suithichaiyakul T, Clegg KB, Sambhi MP. Selectively enhanced stimulation of DNA synthesis by EGF in vascular smooth muscle cells from pre- and post-hypertensive SHR. Clin J Exp Hypertens AI2(3):307-316, 1990. 14. Clegg KB, Sambhi MP. Inhibition of epidermal growth factor-mediated DNA synthesis by a specific tyrosine kinase inhibitor in vascular smooth muscle cells of the spontaneously hypertensive rat. J Hypertens Suppl7(6):S144-S145.

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