Growth regulatory properties of endothelins

Peptides,Vol. 14, pp. 385-399, 1993

0196-9781/93 $6.00+ .00 Copyright@ 1993PergamonPress Ltd.

Printedin the USA.

REVIEW

Growth Regulatory Properties of Endothelins B R U N O B A T T I S T I N I , * P I E R R E CHAILLERflf P E D R O D ' O R L I ~ A N S - J U S T E , * N O R M A N D BRIEREI" A N D P I E R R E SIROIS *l

D@artements de *Pharmacologie, i'd'Anatomie et de Biologie Cellulaire, Facult~ de MOdecine, Universit£ de Sherbrooke, Sherbrooke, P.Q., Canada, JIH 5N4 Received 28 July 1992 BATTISTINI, B., P. CHAILLER, P. D'ORLI~ANS-JUSTE,N. BRIERE AND P. SIROIS. Growthregulatoryproperties ofendothelins. PEPTIDES 14(2) 385-399, 1993.--Endothelinsare produced by endothelialand epithelialcells, macrophages, flbroblasts, and many other types of cells. Their receptors are present in numerous cells, including smooth muscle cells, myocytes, and fibroblasts. Evidence now suggests that the three isoforms of endothelins (ET-1 and the other two related isopeptides, ET-2 and ET-3) regulate growth in several of these cells. Endothelin-I influences DNA synthesis, the expression of protooncogenes, cell proliferation, and hypertrophy. The participation of ET in mitogenesis involves activation of multiple transduction pathways, such as the production of second messengers, the release of intracellular pools of calcium, and influx of extracellular calcium. Moreover, ET-1 acts in synergism with various factors, such as EGF, PDGF, bFGF, TGFs, insulin, etc., to potentiate cellular transformation or replication. Several of these factors may in turn stimulate the synthesis and/or the release of endothelins. The production and release of endothelins are also increased in acute and chronic pathological processes, e.g., atherosclerosis, postangioplastic restenosis, hypertension, and carcinogenesis.It is postulated that endothelins act in a paracrine/autocrine manner in growth regulation and play an important role mediating vascular remodeling in some cardiovasculardiseases. The present review analyses the implication of endothelins (ET-I, -2, and -3) in physiopathologyrelated to their growth regulatory properties. Endothelins ET-1 Growth factors Mitogenesis Gene expression Oncogenes Calcium lnositol phosphate Vascularsmooth muscle cells Fibroblasts

ENDOTHELINS (ET) are members of a family comprising three isoforms called endothelin-l, -2, and -3, which were originally discovered by the group of Masaki in 1988 (150) and described as endothelium-derived contracting factors (EDCF). Vasoactive intestinal contractor (VIC or ET-/3), found in the intestine of the mouse, was also identified as a member of this family (50,106). Endothelins are acidic 21 amino acid polypeptides having two intramolecular disulfide bonds and are formed through an unusual endoproteolytic cleavage from distinct precursors called big-endothelins (bET-1, bET-2, and bET-3). The mature peptides show regional homologies with a group of neurotoxins, including alpha scorpion toxin, omega-conotoxin (145), and the sarafotoxins (S6a, b,c) from the venom of the snake Atractaspis engaddensis (59), that act on voltage-sensitive ion channels. Endothelin-1 has been shown to induce a very potent vasoconstriction, a biphasic effect on blood pressure, as well as inotropic and chronotropic effects (62,120,146). Intravenous infusion ofET- l in forearm vasculature raises blood pressure and produces constriction in isolated human vessels (58). The presence of endothelins in plasma and other biological fluids and their roles in health and diseases have been recently reviewed (6). In brief, conditions such as hypereholesterolemia (2,46), atherosclerosis (67), pulmonary hypertension

DNA synthesis

(123), and scleroderma (52) have been reported to present higher plasma levels of immunoreactive endothelins (IR-ET). Increased plasma IR-ET has been observed during coronary artery bypass (48) related to endothelium injury. Low-density lipoproteins, which accumulate in vessel walls during hypercholesterolemia, have also been shown to activate human macrophages to secrete IR-ET (73), stimulate expression of preproET mRNA and release of IR-ET in cultured human and porcine endothelial ceils (14). Since hypercholesterolemia is an important risk factor for atherosclerosis and since migration and proliferation of vascular smooth muscle cells (VSMC) have been associated with the development of vascular diseases (104,113), endothelins may play an important role in the atherosclerotic process and in the initiation and/or the development of arterial hypertension. This review will emphasize on the possible role for endothelins in mitogenesis and angiogenesis. EFFECT OF ENDOTHELINSON DNA SYNTHESIS Endothelin-1 has been shown to regulate DNA synthesis in various quiescent (Go) types of vascular smooth muscle cells (VSMC): rat (44,61,90), spontaneously hypertensive rats (SHR)

~Requests for reprints should be addressed to Pierre Sirois, Ph.D., Professor and Chairman, Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, P.Q., Canada JIH 5N4.

385

386

BATTISTINI ET AL.

(12,23), a n d rabbit aortic S M C (115); h u m a n S M C (CRL-1692 cell type) (16); porcine a n d bovine p u l m o n a r y artery S M C (PASMC) (40); a n d in two lines of cloned V S M C (83,84) (Table 1). Endothelin-1 stimulated D N A synthesis in cultured neonatal rat cardiomyocytes (51,128). The ability o f ET-1 to induce D N A synthesis has been recorded in various types offibroblasts: normal rat kidney fibroblasts (148), h u m a n breast stromal fibroblasts (111), n o r m a l h u m a n dermal fibroblasts (52), a n d rat-I fibroblasts (72,81). In Swiss 3T3 fibroblasts, Brown a n d Littlewood (15) a n d T a k u w a et al. (130) showed that ET-1 increased D N A synthesis, whereas the results of Fabregat a n d Rozengurt (32) a n d K u s u h a r a et al. (65) did not confirm this observation (Table 2). An increase in D N A synthesis was also reported in other

types o f cells, such as bovine brain capillary endothelial cells (137), astrocytic glial cells o f the rat brain (71,126), rat a n d murine osteoblastic cells ( 112,133), h u m a n melanocytes (143), and rat mesangial cells, a perivascular s m o o t h muscle-like cell type from the kidney glomerulus (3,121,122) (Table 3). In several studies, other endothelin peptides have shown a similar effect on D N A synthesis: rat kidney fibroblasts (ET-3) (148), rat mesangial cells (ET-3) (121), osteoblastic cells from rat calvariae (ET-2, ET-3) (132), rat astrocytes (ET-3) (71,126), a n d h u m a n melanocytes (ET-2, ET-3) (143). Endothelin- 1 and ET-2 were e q u i p o t e n t in p r o m o t i n g D N A synthesis, whereas ET-3 was less active (Tables 1-3). H u m a n big ET-1, the 38 a m i n o acid inactive precursor o f E T - 1 , was substantially less effective in h u m a n me-

TABLE 1 SPECIFIC ANGIOGENIC EFFECTS OF ENDOTHELIN PEPTIDES AND THEIR SYNERGY WITH OTHER GROWTH FACTORS ON VASCULAR SMOOTH MUSCLE CELL CULTURES SMC Rat aortic*

Rat aortic

Rat aortic

Rat aortic* Rat aortict Rabbit aortic t IYB4 clonet A7r5 clonel SHR aortic~t

Porcine PA§ Bovine PA§ Rat myocytes

Agonists ET- 1 EGF PDGF FCS ET-I EGF TGF-a PDGF ET-I ET-2 ET-3 S6b FCS PDGF PDGF PDGF PDGF FCS FCS FCS ET-I ET-I ET-3 ET-1 ET-I PDGF ET-I PDGF ET-I FCS EGF ET-I ET-I ET-I ET-I ET-I¶

[Agonist] + lET- 1] I uM 25 ng/ml 25 ng/ml 10% I nM I0 ng/ml 100 ng/ml 5 ng/ml 100 re'v/ 100 nM 100 nM 100 m'~I 5% 50 ng/ml 50 ng/ml 50 ng/ml 50 ng/ml 2.5% 2.5% 2.5% 100 n M 10 rtM 1000 rL~l 100 nM 1 ng/ml 0.3 ng/ml 1 ng/ml 0.3 ng/ml 100 n~/ 10% 100 rL~l 1000 ng/ml 1 ng/rnl 10 ng/ml 100 nM 1.0 nM

-----1 rL~ 1 I1M I nM -----100 nAl 100 nM ET-2 100 n M ET-3 100 n,~/S6b 100 n~/ 100 nM ET-2 100 n M ET-3 -----1 ng/ml -1 ng/ml ---------

Fold Stimulation Above Basal (Agonist/+ ET- 1) 4.00/-7.33/-5.83/-12.17/-3.25/-3.74/7.79 4.71/11.48 2.02/4.21 1.50/-1.75/-1.50/-1.25/-9.50/-8.50/11.67 8.50/12.67 8.50/I 1.00 8.50/10.00 6.86/10.86 6.86/11.71 6.86/10.86 0.00/-10.5/-5.0/-6.00/-1.23/---/1.39 1.84/---/3.54 3.50/-6.25/-22.50/-1.68/-1.22/1.40/2.15/-2.72/--

Ref.

61

44

140

60

115 83

12

40

51 128

The results are expressed by increases in [3HI Thymidine incorporation (DNA synthesis) or cell number (growth and hypertrophy). * With 1 # M insulin and 5 #g/ml transferrin. t With 5 #g/ml insulin and 5 ug/ml transferrin. ~: With 0.5% fetal bovine serum. § PA: pulmonary artery with 0.5% fetal calf serum. ¶ With I0 #g/ml of insulin and transferrin.

GROWTH REGULATORY PROPERTIES

387

TABLE 2 MITOGENIC PROPERTIES OF ENDOTHELIN PEPTIDES AND THEIR SYNERGY WITH OTHER GROWTH FACTORS ON FIBROBLASTS Fibroblasts Swiss 3T3

Swiss 3T3

Swiss 3T3

Swiss 3T3

Rat-I Rat-I

Human dermal* Rat kidney NRK-49Ft

Human breast stromal

Agonists ET-I PDGF bFGF Insulin ET-I Bombesin IGF-1 ET-I NCS Insulin EGF bFGF PDGF TGF-B Vasopressin Bombesin TPA ET-I VIC EGF Insulin ET-I EGF ET-I NCS Bombesin Serum ET-1 ET-3 hTGF-a hEGF Insulin IGF-I FGF PDGF TGF-~ ET- 1 IGF- 1 EGF FCS Bombesin

[Agonist] + [ET-I] 10 nM" 0.33 nM 0.63 nM 1.7 ~tM 0.5 nM I 0 nM 1 ng/ml 3 rtM 10% 1 ~g/ml 30 ng/ml 0.03 ng/ml 1 ng/ml 0.3 ng/ml 0.1 n3,1 0.3 nM 2 aM 10 nM 10 m~,l 5 nggml 1 ~g/ml 10 nM 10 ng/ml 100 m ~ 10% 617 aM 0.5% 5 ng/ml 5 ng/ml I ng/ml 1 ng/ml 1 ug/ml 2 ng/ml 1 ng/ml 1 ng/ml 1 ng/ml

m

10 n.~l lOnM 10 rL#l

o.oo/-4.59/16.o7 9.12/17.65 3.00/10.83 3.43/--

Ref. 64

130

15.26/--

lOnM

3riM 3nM 3riM 3nM 3nM 3riM 3 m~l 3 m~l

10 n~l VIC/ET-I 10 nM VIC/ET-I 10 m~l

1 ng/ml

ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml

10 m ~

10 ng/ml 10 ng/ml 10% 100 aM

Fold Stimulation Above Basal

10 nM 10 nM lOnM

1.64/ 13.29 1.79/-67.84/-2.41/5.41 5.03/35.97 2.31/27.55 4.08/28.29 6.78t21.52 1.22q7.03 2.84 t7.04 2.89 t23.01 0.0( ~-0.00 t _ 0.20q7.25 1.00 ¢5.00 0.50t-15.00 t29.00 3.33t-6.67t-1.61 t__ 0.18t5.50 1.96t-1.44 t - 7.20 t l 0.80 8.613 t 12.20 1.80 t3.60 2.20/3.80 2.00 t2.70 2.10 t3.40 2.0( t2.80 1.35t-1.71 @05 1.85 t2.43 27.37/-1.31 tl.68

15

32

81 72

52 148

111

The results are expressed by increases in [3H] Thymidine incorporation (DNA synthesis) or cell number (growth and hypertrophy). * With 0.5% serum. ? With 0.1% newborn calf serum.

lanocytes (143). Half-maximal (ECso) and maximal (Amax) concentrations of ET that mediate mitogenic responses are shown in Table 4. Several authors raised questions concerning the efficacy of endothelin peptides as mitogens because the peptides were added to serum-free culture media routinely complemented with insulin and/or transferrin and/or serum (<0.5%) (see Tables 1-4). Weissberg and collaborators (140) suggested that VSMC in culture can synthesize and secrete a low concentration of platelet-derived growth factor (PDGF) that could act as an endogenous mitogen in association with exogenously administered

endothelins. Several studies on rat aortic and h u m a n SMC did not show a significant increase of D N A synthesis while using concentrations of endothelins that reach 100 n M (23,60,101,140). Therefore, ET may not appear to be, per se, a direct mediator of angiogenesis, but rather as a comitogenic factor. However, this cannot explain the discrepancies in D N A synthesis stimulation observed in studies using Swiss 3T3 fibroblasts, since all four reported experiments (15,32,65,130) were performed without the addition of any insulin or other factors that may induce some mitogenic effects. Moreover, Swiss 3T3

388

BATTISTINI ET AL.

TABLE 3 MITOGENIC PROPERTIES OF ENDOTHELIN PEPTIDES AND THEIR SYNERGY WITH OTHER GROWTH FACTORS IN VARIOUS TYPES OF CELLS Type of Cells

Rat mesangial

Rat type- 1 astrocytic glial Calvariae osteoblast-like Rat

Murine Brain capillary endothelial

Mouse C6 glioma Rat cerebellar astroglia

Human melanocytesi-

HeLa HEp-2 Neurite, chick embryonic sensory ganglia:~

Rat FRTL5 thyroid§

Agonists

[Agonist] + [ET-1]

Fold Stimulation Above Basal

ET-I* ET-I* ET-I FBS Insulin ET- 1 ET-3 FBS ET-3

10 m~/ 100 nM 100 nM 2.5% 0.1 ,g/ml 100 rL~l 100 nM 10% 20 ~ t

----100 roll -----

5.80/-9.67/-1.08/-13.00/1.46/3.58 3.00/-1.50/-6.50/-2.24/--

ET- 1 ET-2 E'I--3 ET-I PDGF ET-1 FBS FBS bFGF ET- 1 ET-3 ET- 1

1 m~l 1 m~/ 10 nAl 100 rc~! 3 ng/ml -0. 1% 1.0% 25 ng/ml 0.1 m~t 1000 roll

----10 rim 400 nM 400 aM 400 rtM ----

1 nM

--

ET-3

1 m~I 10% 25 m~/ 10 nM 10 m'd lO nM I0 nM 1 m'~l 10 rL~t 100 pM 16 nM 160 nM 100 m~l 1 ng/ml 10 ng/ml

------lO nM ---100 pM 100 pM -10 ng/ml 100 m~l

1.92/-1.75/-1.64/-2.00/-18.00/30.00 1.77/-1.23/2.77 2.77/3.86 1.32/-1.66/-2.75/-1.15 ~ 1.23 ' - 12.80 ' - 1.92 ' - 2.00 ' - 1.30 ' - 1.00 '-1.79 '2.50 2.00 ' - 1.50 ' - 1.1~ '-1.15 '1.34 0.91 '1.58 0.0£ - 1.2,~ 2.00 1.4C 2.98

FCS ET-I ET-2 ET-3 bET-I bFGF ET- I ET-I ET-1 TPA TPA ET-I IGF-1 IGF-I

Ref

122

3 121

126 132

112 137

71

143

117 47

78

The results are expressed by increases in [SH] Thymidine incorporation (DNA synthesis) or cell number (growth and hypertrophy). * With 0.5% fetal bovine serum. ~"In presence of 10 nM cholera toxin and 0.2% bovine pituitary extract. ~t Increase in neurite length (ram). § With 0.5% calf serum, 0.4 ng/ml cortisol and 5 ug/ml transferrin.

cells do not synthesize growth factors in culture. Phenotypic modulation [a process that involves i m p o r t a n t structural and functional modifications (18)] could explain the difference between these results. It has been reported that the variation o f the phenotypic state (differentiation from a contractile to a synthetic state) o f V S M C strongly reduced the mitogenic activity o f ET-1 (23,115). The binding capacity (Bm,~) o f ET-1 to these subcultured V S M C was also reduced. Interestingly, V S M C derived from S H R exhibit a greater response to ET-1 t h a n cells from normal rats. This may prove to be i m p o r t a n t in the development o f vascular hypertrophy in hypertension (12,23). Previous studies have revealed that V S M C harvested from the

aorta o f S H R exhibit e n h a n c e d growth rates in response to angiotensin II (All) and epidermal growth factor (EGF) (114). Secondly, differences in culture techniques (i.e., explant technique vs. enzymatic digestion) might also account for such discrepancies. The nature o f the substrate appears to play a primary role in altering the shape and growth o f renal ( M D C K ) cells in culture (55). Finally, it has been reported that the stimulation o f growth by exogenous factors is d e p e n d e n t o n cell density (confluency) or the use o f mieromass cultures. Several studies showed the necessity for cell--cell c o m m u n i c a t i o n a n d the stagerelated capacity o f m e s e n c h y m a l cells to proliferate or differentiate. Indeed various cell densities presented different sensi-

GROWTH

REGULATORY

PROPERTIES

389

TABLE 4 HALF-MAXIMAL (ECso) AND MAXIMAL (A,~) CONCENTRATIONS OF ENDOTHELIN-I AND OTHERS RELATED PEPTIDES TO INDUCE MITOGENESIS IN VARIOUS TYPES OF CELLS Stimulation Effect (nM) Types of Cells Rat aortic SMC

Rabbit aortic SMC~

Porcine PASMC § Bovine PASMC§ A7r5 clone t l YB4 clone t Fibroblasts Swiss 3T3

Rat kidney t t Human dermal~¢ Rat mesangial§§ Rat cardiomyocyte Calvariae osteoblast-like Rat

Murine C6 glioma Rat primary cerebellar astroglia Human melanocytes***

HeLa carcinoma HEp-2 Bovine brain capillary endothelial Rat adipocyte precursor t

ECso 30* 0.20 0.10t 25.0 (ET-3) f 0.05 14 (S6b) 189 (ET-3) 312 (bET-l) 42 4.0 <0.40 <0.40

Araax

Ref.

1000 1.0 10 1000 1.0

61 44 90 90 115

400

40

1.2 1.2

83

0.03 0.30 0.10-1.50¶ 0.50# VIC/ET-I 0.40** VIC/ET-I 5.00# S6b 100# ET-3 0.56 0.04 0.90 10.0 ET-3 0.17¶¶ 5.00

100 3.0 10.0 10 2.5 10.00 > 100 2.0 0.4 10.0 100 1.0 100

130 15 64 31 31, 32

<0.05 <0.05 (ET-2) 0.50 (ET-3) 2.5## 0.05 100 (ET-3) 0.10 0.10 (ET-3) 0.10 0.50 (ET-2) 0.50 (ET-3) 0.003 5.00 2-5 1.00

1.0 1.0 10.0 10.0 0.1 1000 1.0 1.0 25.0 10.0 10.0 1.0 10.0 100 100

132

* With 1 ttM insulin and 5 #g/ml transferrin. t With 5 ~g/ml insulin and 5-10 #g/ml transferrin. With 0.5% SVF, 5 #g/ml insulin and 5 tzg/ml transferrin. § With 0.5% fetalcalf serum. ¶ With 0.33 n M P D G F or 0.63 n M b F G F or 1.7 u M insulin. # With 5 ng/ml EGF. ** With l/zg/ml insulin. tt With 0.1% fetalcalf serum. $$ With 0.5% serum. §§ With 0.5% fetal bovine serum. ¶¶ With 10 tzg/ml of insulin and transfcrrin. ## With 3 ng/ml PDGF. *** In the presence of l0 n M cholera toxin and 0.2% bovine pituitary extract.

148 52 121, 122 128 51

I 12 7I 71 143

117 137 I 18

390 tivities to mitogens (53,103). In addition, a recent paper reported that AII-induced (AII is a mitogen for fetal mesangial cells but is only weakly mitogenic for adult mesangial cells) mitogenic response was reduced in confluent culture of fetal cells (98). Therefore, phenotypic modulation, culture techniques or cell density can explain the differences observed in the effect of ET1 on Swiss 3T3 cells. REGULATION OF GENE EXPRESSION, CELL PROLIFERATION. AND HYPERTROPHY BY ENDOTHELINS In normal cells, ET- 1 ( 1 riM) causes a 2-fold increase in the VSM cell numbers (44), promotes proliferation of VSMC [onethird as effective as 10% fetal calf serum (FCS)] (12), stimulates protein synthesis in rat aortic SMC (in the presence or absence of 1 taM insulin in the serum-free medium) (23), and induces hypertrophy of neonatal rat cardiac myocytes that possess endothelin receptors (38,51,119,128). The hypertrophic effect of ET-1 on cardiac myocytes may involve the augmentation of myosin light chain 2, alpha-actin, troponin I, and alpha- plus beta-myosin heavy chain mRNA expression through gene transcription (51,119,139). The ET- 1-induced myocardial protein synthesis was completely blocked by an antisense oligonucleotide against the early growth response gene-1 (Egr-1), which acts as a transcription factor (third messenger) (88). In hVSMC. ET-1 caused both a time- and dose-dependent activation of S6-kinase (101). The activation was lower than that induced by 10% FCS or 100 r ~ TPA, and appeared to be independent of protein kinase C (PKC). In rat adipocyte precursor cells, ET- 1 promoted cell proliferation in a dose-dependent fashion but inhibited adipocyte differentiation (118). Endothelin-1 and ET-2 (10 ru~4) stimulated sustained long-term growth of human melanocytes (4.5-fold increase in cell number for 12 days in culture) and melanin synthesis at 1-50 n M concentrations (143). Endothelin1 also promoted a rapid and transient increase of c-fos and cmyc mRNA levels in rat VSMC (12,61). Protooncogene transcription was stimulated in a concentration-dependent manner and maximal expression was reached with I a M ET- 1 in VSMC (61). C-fos and egr-I expressions were induced by ET-I in cardiomyocytes (51,119). Endothelin-1 and its isopeptide ET-2 increased c-fos mRNA expression in rat FTRL5 thyroid epithelial cells (78). Moreover, ET-1 enhanced the transcription of c-fos, J'ra-1, c-jun, and JunB protooncogenes in rat glomerular mesangial ceils without inducing fosB and junD (121,122). The marked elevation of c-fos steady-state mRNA and protein by ET-1 in these cells was more important when compared to the one induced by ET-3 (121). The expression of c-fos and c-rnyc protooncogenes stimulated by ET-1 was also observed in Swiss 3T3 fibroblasts (130). The expression ofc-fos and c-jun mRNA activated through PKC is one of the earliest genomic responses to cell stimulation by phorbol ester, growth factors, and serum (12l), and is similar to the effect of other calcium-mobilizing agents, such as angiotensin II (57). Endothelin-1 ( 10 ng/ml) also caused a dose-dependent increase in the synthesis rate of oncoproteins and collagen from human dermal fibroblasts (71 + 18% and 60 + 5%, respectively) (52). Moreover, ET-1 presented a direct effect on native collagenase gene expression in mesangial ceils, suggesting the possible implication of ET- 1 as a proinflammatory mediator (121). Other related members of the endothelin family (ET-3, bET- 1, or S6b) were less active on VSMC regarding protooncogene expression (115,148). PRESENCE AND EFFECTS OF ENDOTHELINS IN CARCINOMAS Endothelin- 1 is synthesized and released from two epithelial carcinoma cell lines derived from the human cervix (HeLa) and

BATTISTINI ET AL. larynx (HEp-2) and induces proliferation of these carcinoma cells (117). These results suggest that ET-I may act as an autocrine growth promoter. Endothelin-1 also promoted mitogenesis of C6 glioma cells and primary cerebellar astroglia, which contain ET mRNA (71 ). Endothelin- 1 produced by the glia may act upon both gila and neurons in an autocrine or a paracrine fashion (71). But the cell cycle kinetics of Kirsten MSV-transformed rat kidney fibroblasts (KNRK) remained unaffected by endothelins (148). Endothelin- 1's presence, storage, and/or synthesis in other cancer cell lines or tumors have been demonstrated (lung, pancreas, colon, breast, and human cervix carcinomas) (37,64.111,127,144), whereas no endothelins were released from melanoma (C32, C361). lymphoma ($49). or leukemia cells (HL60) (17,125). A possible role for ET-1 in the growth and/or the differentiation (neoplasia) of tumors and stromal cells surrounding focal tumors has been suggested (37,64). It remains to be established whether ET- 1 acts as an autocrine mitogen or as a paracrine growth promoter for adjacent cells and if it is of some therapeutical relevance. MECHANISMS INVOLVED IN ET-I-INDUCED GROWTH

Endothelin Receptors Endothelial cells were initially shown to synthesize and release ET-I (145). Endothelin production and secretion have since been observed in many types of cells (6). The peptide interacts in an autocrine/paracrine manner with specific high-affinity receptors present on VSMC to induce vasoconstriction/pressor effects (39,68,115) and to regulate growth as reviewed here. Surface receptors that recognize endothelin peptides are also present on Swiss 3T3 cells (32,130), rat cardiomyocytes (43), osteoblastic ceils from rat calvariae (126), and HeLa and HEp-2 cells (117). In Swiss 3T3 fibroblasts, the number of high-affinity receptors for endothelins is even higher than that of any other growth factors and bioactive peptides (such as PDGF, FGF, and EGF) (65). Binding measurements and chemical cross-linking experiments have identified three different endothelin receptors: ETA, ETB, and ETc. However, there has been no report on the isolation of the cDNA encoding a specific receptor for ET-3 (1,108,109). According to the relative binding affinities of the three isopeptides for the receptors, ET-I is equipotent to ET-2 but more potent than ET-3 for ETA, whereas ET-I is equipotent to ET-2 and ET-3 for the ETB subtype. Both ETA and ETa have been found in rat aorta (42) and the ETA receptors are predominant in this tissue (142). Even though ETA and ETa are functionally distinct, they are thought to be a rhodopsin-like receptor type with seven membrane-spanning domains and coupled to guanine-nucleotide regulatory proteins (G-proteins). The receptors have an Nterminal signal sequence, and the cytoplasmic C-terminal tail possesses a number ofserine/threonine residues that are potential substrates for protein kinases (Fig. 1) (108)./3-Adrenergic (/31/ B2), vasopressin (Vi.2), and serotonin (5-HTj.2) receptors are structurally similar (Fig. 1). It is not known whether ET receptor subtypes are differentially coupled to calcium channels or to calcium mobilization from endoplasmic reticulum or other signaling pathways as Vt and 5-HT~ receptors that activate the cAMP pathway, whereas V2 and 5-HT2 receptors induce calcium/phosphoinositide increases (Fig. 1). Cellular generation of cAMP was not induced by ET-1 in 3T3 cells (93). Interestingly, different ET receptor subtypes were proposed to explain the differential regulation and distinct patterns offos andjun expression in rat mesanglal cells (121); it was the first attempt to pinpoint the pathways of nuclear signaling by which ET isopeptides contribute to the regulation of gene expression.

G R O W T H REGULATORY PROPERTIES

391 TGF-B

Ca=+/phosphoinositlde cAMP/cGMP Ca2. channels eeee tyrosine kinases oooo intracellular Ca2.

Bombesin

PDGF

/

I~

.~ NGF-B

•

.

•*

bFGF

Prostacyclin

•

•

Ca 2.

Insulin

•

o o oeo/~

"

O0 ,

ii ~ . .

o

.

•

Oo

EGF (TGF-a)

~ •

•

.

ET-1

•

"//

'O

iotensin

f3-adrenergic Vasopressin serotonin 5-HT.%'~ cAMP

,,

i I Induction of" . . I Immediate early genes~

V1

regulation of DNA synthesis FIG. 1. Cooperative signaling pathways leading to DNA synthesis in VSMC and fibroblasts. Mitogenesis is dependent on both pertussis toxinsensitive and -insensitive pathways. The first one corresponds to the phospholipase C/PKC pathway (blank arrows). The second could be identified to the tyrosine kinase pathway (black dots) or to some calcium-stimulated, PKC-independent effectors (blank dots). Progression factors can generate multiple signals: for example, insulin and EGF are able to exert their signaling through the intrinsic activity of their tyrosine-kinase receptors as PKC is not an obligatory effector of their action (as well as c-myc/c-fosexpression). In contrast, PDGF and ET- I signals are partly blocked by PKC inhibition and pertussis toxin treatment. Both peptides can trigger calcium influx (pointed arrows). For comparison, vasopressin-or neurotransmitterinduced mitogenesis are completely inhibited by pertussis toxin. Competence factors, like PDGF and possibly bombesin, elicit cAMP production through the PGE~-stimulated adenylate cyclase pathway (black arrows). Platelet-derived growth factor is also a factor that does not require the presence ofcomitogens. Bombesin, vasopressin, and ET- 1 stimulate a tyrosine kinase pathway that is specific for neuropeptide-induced mitogenesis.

Transmembrane Signaling Receptor activation of the phospholipase C pathway and alterations in second messenger levels were shown to be key events in the mechanism of action of many mitogens stimulating DNA synthesis and growth of numerous types of cells (8,141) (Fig. 1). Rapid inositol phosphate turnover and calcium mobilization play a fundamental role in the transduction pathway through which growth factors elicit their effects and cell proliferation occurs (97,105). The calcium-mobilizing effects of a given mitogen can be distinguished in terms of kinetics, sensitivity to PKC-mediated feedback inhibition, and signal transduction (32). Like many other vasoactive peptides on VSMC, e.g., angiotensin II (57), various concentrations of ET-I and other related peptides have been reported to increase phosphoinositide (PI) turnover, diacylglycerol (DAG) levels, and/or mobilize calcium from intracellular sources in VSMC (45,61,83,100, ! 01), in rat cardiomyocytes (51), in Swiss 3T3 fibroblasts (15,32,130), and in rat mesangial cells (3,122). The increase in thymidine incorporation ([3H] TdR) is also accompanied by a rise of intracellular levels of calcium in other types of cells (126,132), including carcinoma cell lines (34,117). In these latter cases, the increases in cytosolicfree calcium concentration may be generated in part through

calcium influx (see the next section). Although the transduction signals generated by ET-I and bombesin are similar in Swiss 3T3 fibroblasts, only bombesin stimulated the production of cAMP and caused a 15-fold rise in PI (11,15,129,130). These differences in the kinetics of second messenger metabolism might explain why ET-1 is less potent than bombesin in fibroblasts. The efficacy of ET- 1 in cultured VSMC may well be explained in a similar way (102). This hypothesis has been suggested in rat mesangial cells, where ET-1 stimulated PI turnover more effectively than vasopressin (13). Endothelin-1 can also synergize with TPA, whereas vasopressin cannot (26). Endothelin-1 binding also activates the PKC in promoting DNA synthesis in Swiss 3T3 fibroblasts (15,130) and rat-I fibroblasts (81). Prolonged exposure to TPA markedly reduced cellular PKC activity and affected the ability of ET-1 to stimulate anchorage-independent growth in the presence of EGF in rat-1 fibroblasts (81). By comparison, EGFoinduced anchorage-independent growth of rat-1 fibroblasts is PKC independent, since TPA treatment did not affect its action (81). Endothelin- l's ability to induce second messenger production and increase c-fos and c-jun transcription in rat-I fibroblasts appears to be also independent of cellular PKC activity (81). Conversely, c-fos

392 stimulation by another growth factor, bombesin, is attenuated in PKC-depleted Swiss 3T3 cells (77). In PKC-depleted Swiss 3T3 fibroblasts by pretreatment with 1 #M phorbol 12,13-dibutyrate, the mitogenic influence of ET-1 (10 nM) is attenuated by 60% (130). The ET-induced elevation of c-fos mRNA in VSMC is also attenuated by 67% (12). The PKC inhibitor H7 ( 10-100 ttM) strongly affected the ET-induced cardiac myocyte hypertrophy (128). This PKC inhibitor (7 #M) also partially blocked the stimulatory effect of ET-1 on protein synthesis in rat aortic SMC (23). Pertussis toxin has no effect on EGF and basic fibroblast growth factor (bFGF) effects, which act through tyrosine kinase-dependent mechanisms (20). In contrast, this agent has been shown to induce a concentration-dependent reduction of ET-l-induced polyphosphate metabolism, second messenger production, and mitogenesis in VSMC (at 100 ng/ ml, 37% reduction) (12). However, these pieces of evidence contrast with the inability of the pertussis toxin to affect the ETinduced increase in c-fos mRNA (12). Pertussis toxin also failed to block the ET-induced increase in IP production and calcium mobilization in A10 VSMC (131). Therefore, ET-I action may include pertussis toxin-insensitive processes as commonly observed for growth factors and oncogene products acting through tyrosine kinase-dependent mechanisms, like EGF or FGF, in Chinese hamster lung fibroblasts (20,147). Like PDGF, ET-I may stimulate phospholipase C (PLC) via tyrosine kinase phosphorylation. This mechanism is not involved in vasopressin and bombesin actions (69,138). In cultured rat aortic SMC, ET-I induced a dose- and a time-dependent tyrosine phosphorylation of five different proteins (40, 45, 73, 77, and 79 KDa) in the same manner as PDGF (60). In rat mesangial cells, E'I'-1, vasopressin, and All rapidly enhanced tyrosine phosphorylation of proteins (70, 120, 135, 190, and 225 KDa) (33). These proteins were cytosolic or membrane-associated but not integral to the membrane. Epidermal growth factor, which does not activate PLC in mesangial cells, induced the tyrosine phosphorylation of its own receptor and the five proteins mentioned above (33). In Swiss 3T3 fibroblasts, ET-I and VIC elicited a tyrosine/serine phosphorylation similar to that induced by both bombesin and vasopressin ( ! 50,151). The sharing of signaling pathway between ET- 1 and EGF, such as described in mesangial cells, might explain their synergism as growth regulators. Whether tyrosine kinase activation can fully account for the pertussis toxin-insensitive pathway remains to be determined (12,140). Recently, Schvartz et al. (112) suggested a pivotal role for protein tyrosine phosphorylation in the mitogenic activity induced by ET-1 in osteoblast-like cells.

Influx of Extracellular Calcium Increase in intracellular concentrations of calcium occurs through different mechanisms, including the one defined in the earlier section and calcium mobilization from opening of voltagedependent calcium channels. The mitogenic action of endothelins seems also to depend in part on its capability to mobilize extracellular calcium as previously described for PDGF. Nifedipine, a dihydropyridine-sensitive calcium channel antagonist, was reported to inhibit PDGF-induced stimulation of DNA synthesis in VSMC (83,89). The same calcium blocker ( 1 ttM) completely inhibits both the elevation of intraceUular calcium and the stimulatory effect of ET-I on DNA synthesis into the VSMC (44,83). The pleiotropic actions are also inhibited nonspecifically in the presence of nifedipine or verapamil in human SMC (16). The ability of nifedipine to antagonize ET-1 mitogenicity was again revealed in rat cerebellar glial cells (126). Nicardipine (1 #M), another voltage-dependent calcium channel blocker, has

BATTISTINI ET AL. been shown to affect ET-induced intracellular calcium elevation in VSMC (61) and myocyte contractility (128), but not the protein synthesis rate in rat cardiomyocytes (51). This antagonist (10 riM), as well as the chelating agent EGTA (3 mM) or a polyclonal antibody against ET- 1, completely abolished the ETl dose-dependent increase in cytosolic-free calcium and/or the proliferation of HeLa and/or HEp-2 cells (117). Most of these pieces of evidence support the involvement of extracellular calcium influx in the regulation of DNA synthesis and cell proliferation induced by ET-1. In brief, the mechanism by which ET-I promotes growth involves activation of multiple signal transduction pathways. This assumption is based on the kinetics of calcium mobilization from both intra- and extracellular pools. The pattern of increase in intracellular calcium observed in different types of cells (VSMC, cardiocytes, mesangial cells, fibroblasts, HeLa, A I0) reveals that the initial transient phase (peak) derives from intracellular storage sites; the subsequent sustained phase (plateau) results from extracellular calcium influx (21,93,130,132). A similar dual pattern of calcium mobilization was observed in Swiss 3T3 cells stimulated with bombesin and EGF (96,129). Coupling mechanisms for endothelin receptors might also be different among various cells (133). Moreover, ET- 1 may operate via distinct pathways for the induction of short-term versus longterm events. Both the ET-l-induced DNA synthesis and the synergistic effect on anchorage-independent growth with EGF of rat-1 fibroblasts are highly dependent on PKC activity (longterm). However, in PKC-depleted cells, ET-1 still stimulates production of PI and DAG (second messengers) or c-fos/c-jun transcription (immediate early gene expression) (short-term) (81 ). Whether the pleiotropic effects of ET-1 be related in some aspects to the peculiar structure of its receptors, a protein with seven membrane-spanning domains that have no close resemblance to classical growth factor receptors (Fig. 1) remains to be determined. INTERACTIONSBETWEENGROWTH FACTORSAND ENDOTHELINS

Synergisms of Mitogenic Activities Polypeptide mitogens, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factors (TGF-~, TGF-/5), basic fibroblast growth factor (bFGF), and insulin-like growth factors (IGFs), can stimulate both migration and proliferation (cell division) of VSMC into and/or within the arterial intima (104,113). Circulating vasoactive agents in blood (AII, catecholamines, vasopressin, serotonin) have also been shown to stimulate protein synthesis or cellular proliferation (10,19,86). Synergism of metabolic responses between sets of regulatory peptides and growth factors are well established, although the specific mechanisms are unknown (105,124). Bombesin, vasopressin, PDGF, and TPA (a tumor-promoting phorbol ester: 1 2 - O - t e ~ n o y l p h o r b o l - 13acetate) belong to a group of mitogens whose activities are enhanced by IGF-1. They all activate protein kinase C (PKC) (105). Platelet-derived growth factor, IGF, and EGF function cooperatively to regulate the cell cycle in mouse fibroblasts (124); PDGF renders Go-arrested cells to become competent, while IGF/EGF induces the S-phase, causing the cells to progress toward cell division (44). Interactions between ET-1 and EGF, TGF-a, or PDGF resulted in synergistic stimulatory effects on DNA synthesis in rat aortic SMC (45,140) (Table 1). Endothelin-I (10 nM) was also an effective comitogen with 2.5% FCS or submaximal concentrations of PDGF (5-10 ng/ml) on rat aortic SMC (140). The comitogenic effect of ET-2 was consistently equipotent to ET-

GROWTH REGULATORY PROPERTIES 1, whereas ET-3 was less active (140). In another report, PDGF action, at a concentration that had only a slight effect on DNA synthesis, was enhanced by low concentrations of ET-1 in two types of clonal VSMC (83). In Swiss 3T3 fibroblasts, ET-1 has also been reported to potentiate DNA synthesis and to increase synergistically the [3H] TdR incorporation induced by numerous mitogens in a concentration-dependent manner (Table 2) (15,32,65,130). Agents that either activate PKC (PBt2) or increase the cellular level of cAMP (forskolin and IBMX) also act in synergy with ET-1 or VIC in Swiss 3T3 cells (32). Similar interactions were reported in rat kidney fibroblasts, where the combinations of ET- 1 with several individual growth factors increased DNA synthesis (Table 2) (148). In rat- 1 fibroblasts, ET- 1 stimulated anchorage-independent growth in the presence of EGF (81). Such cooperative cell kinetics (i.e., total [3H] TdR and rate of DNA replication) involving ET-1 were also reported in other types of cells (Table 3). Overall, ET-1 showed a limited synergism with several mitogens on VSMC (from 1.3- to 2.4-fold over) (Table 1), whereas the combined effects were more potent on Swiss 3T3 fibroblasts (from 1.9- to 14-fold) (Table 2). Endothelin-1 strongly potentiates DNA synthesis induced by vasopressin, another vasoconstrictor peptide, but has much less effect in combination with bombesin (Table 2) (15). Interestingly, both vasopressin and bombesin synergize strongly with either insulin or IGF-I to stimulate DNA synthesis (105). The mitogenic effect of TPA on Swiss 3T3 cells and neurite outgrowth from chick ganglia explants were also enhanced by ET-1 (15,47), whereas it is known that vasopressin does not act in synergism with TPA (26). In one report, ET-I has been shown to inhibit TSH and 8-bromo-cAMP-induced DNA synthesis in the rat FRTL5 thyroid epithelial cells (78). However, ET-1 also greatly enhanced IGF-l-induced DNA synthesis and cell replication in the same cells (78). The mechanisms underlying the synergism of ET-I with different competence or progression factors may well be explained by the multiple interactions between the various signaling pathways elicited by these mitogens. There is one reported example where oncogene encodes have been shown to selectively over-express components of cellular growth pathways. The product of v-src oncogene (pp6Ov.src) enhanced the accumulation of lns(l,4,5)P3 and calcium (6- and 3-fold, respectively) induced by ET-I alone in rat-I transformed fibroblasts (74). The enhancement of the signaling response of a mitogenic agonist by oncogene encodes is of interest considering that ET-1 has been proposed to regulate growth in HeLa and HEp-2 transformed cells (117). The regulation of ET-1 gene transcription byfos andjun oncogenes (66), proteins that are themselves regulated by ET- l (l 2,6 l, 12 l, 122), further illustrates the complexity of ET-l actions in various cell types.

Stimulated Expression and Production of ET by Growth Factors Endothelin- I is synthesized and released from various types of cells: endothelial cells (30,150), airway epithelial cells (9,75), human polymorphonuclear leukocytes (116), macrophages (29), fibroblasts (152), rat and human mesangial cells (4,107), etc. (6). In response to tissue injury, circulating plasma levels of IR-ET (6), as well as other factors such as thrombin, adrenaline, TGF/3, PDGF, and interleukin-l/3, are increased. Interestingly, the expression of preproET mRNA has been reported to be stimulated by thrombin, adrenaline, TGF-/3 (63,70,145), and interleukin-1 (149) in porcine aortic endothelial cells (EC). Plateletderived growth factor was unable to modulate ET-1 gene transcription in these cells (63). But coincubation of human

393 platelets (which synthesized PDGF) with bovine pulmonary artery (BPAE) or human umbilical vein (HUVE) endothelial cells was reported to stimulate the expression of ET mRNA and the release of IR-ET (92). Low doses of thrombin (0. I U/ml) or endotoxin (100 ng/ml) incubated with platelets and BPAE cells resulted in significantly higher levels of IR-ET (92). Interleukin1 and EGF have also been shown to modulate ET-l gene transcription in human amnion cells (125). Interleukin-l and TGFfl were found to increase preproET-I mRNA in endometrial stromal cells (27). Transforming growth factor-B, PDGF-A chain, AII, Arg-vasopressin, and ET-l itself generated ET-specific transcripts and stimulated secretion in SHR VSMC up to levels (100 pM) that may have some physiological relevance (39). Endothelin-1 even promoted the expression of transcripts for PDGFA chain, TGF-B, and thrombospondin in quiescent SHR VSMC (39). Thrombin, IL-l, and TGF-/3 significantly increased the amount of ET-1 released from porcine aorta EC, pulmonary artery EC, and human Hep G2 cells in culture (40,63,91,92,110,127,149). Induction of ET-l mRNA expression and secretion by vasopressin and AII was also reported in human VSMC (100) and porcine carotid artery EC (106). There could be a relationship between ET-l and AII considering that ET-I increases the conversion of angiotensin I to AII (56) and that AII, a potent vasoconstrictor, can also stimulate proliferation of human aortic SMC (19). Dose-dependent release of ET from rabbit endometrial cells (94) and human mesangial cells (HMC) (4) was also stimulated by [Phe2,OruS]vasopressin and arginine vasopressin (AVP) or insulin, respectively. Normal HMC cultured with antiendothelin antisera or mAb against ET-1 demonstrated a blunted mitogenic response to AVP (4). Epidermal growth factor (10 nM), bombesin (0.1 #M), cortisol (1 ttM), and dexamethasone (0.1 uM) stimulated IR-ET release from human breast cancer cells (T47D cell line) ( 111 ). Bombesin and cortisol or dexamethasone showed an additive effect on the release of IR-ET. In MDCK ceils, TGF-/3 (0.2 nM), PDGF (10 riM), and NGF (10 nM) enhances IR-ET-1 secretion by 1.4-, 1.9-, and 1.3-fold, respectively (134). The same effect has been reported in HepG-2 cells. Epidermal growth factor (1 nM), TGF-a (4 nM), TGF-/3, bFGF (0.3 nM), PDGF, and nerve growth factor (NGF) increased ET production by 2.2-, 2.9-, 6.7-, 1.5-, 3.3-, and 1.8-fold, respectively (134). In conclusion, many reports indicate that growth peptides and other factors that stimulate cellular proliferation may do so through modulation of transcription and production of endothelin peptides. In return, ET-1 also has the ability to promote the expression of transcripts for several growth factors. There is one reported exception: production of ET-2 in human renal adenocarcinoma cells (ACHN) is strikingly inhibited by EGF, TGF-/3, bFGF, and TGF-a, whereas IGF- 1, -2, insulin, and NGF did not show any inhibitory effects on the secretion of IR-ET-2 in ACHN. It has been suggested that the production of ET-I and ET-2 is regulated differently: EGF could control the ET-2 production at the transcription level of the ET-2 gene (134). INHIBITIONOF THE MITOGENICPROPERTIESOF ENDOTHELINS We have mentioned that voltage-dependent calcium channel antagonists (nicardipine, nifedipine, or veraparnil) partially blocked ET-induced increase in intracellular calcium and mitogenesis in various cells ( 16,44,61,83,126). In Swiss 3T3 cells, [D-Argl,D-PheS,D-Trp7'9,Leu~l]substance P, a broad spectrum neuropeptide antagonist for substance P-related peptides, bombesin, vasopressin, and bradykinin, acted as a peptidic (structurally unrelated) antagonist to ET-1 or VIC. These peptides share the ability to induce PI turnover and increase calcium via

394

BATTISTINI ET AL.

a G-protein signal transduction pathway (Fig. 1). The antagonist blocked ET-I/VIC binding in a competitive and dose-dependent manner. It also inhibited the rapid increase in cytosolic calcium concentration and the DNA synthesis stimulated by ET-1 or VIC in the presence of EGF (31). Thymidine incorporation by VIC was markedly reduced (ECs0 from 0.3 nM to 2 rL~t) in the presence of the same antagonist. The inhibition was competitive and reversible. The antagonist is, however, ineffective against PDGF or vasoactive intestinal peptide (VIP), which may act via tyrosine phosphorylation or cAMP accumulation, respectively (Fig. 1). Insulin- and EGF-induced DNA synthesis were not affected by the antagonist (31). In rat aortic SMC, prostacyclin and its stable analogues (pentanoate derivatives from Teijin Ltd., Tokyo, Japan) inhibited (IC50:1-30 nM) the ET- l-induced DNA synthesis observed in the presence of insulin/transferrin (84). Another endothelium-derived relaxing factor (EDRF), nitric oxide (NO), and nitric oxide-producing vasodilators (glyceD, l trinitrate and sodium nitroprusside), which increase cGMP content, contribute also to the inhibition of ET-1-induced DNA synthesis in rat aortic SMC (clone RACS-1) (82). It is known that prostacyclin inhibits FCS-induced DNA synthesis in VSMC (80,135) and that NO and NO-producing vasodilators also inhibits DNA synthesis and proliferation of cells (36,54,82). Endothelin-I (145), prostacyclin (79), and NO (95) are released from the endothelium. Therefore, EDRF may be effective in preventing SMC proliferation under some pathological situations, such as atherosclerosis (84). The fact that PGI2 and NO increase the levels of cAMP and cGMP, respectively, does not mean that the inhibitory action of these vasodilators is mediated through these messengers. Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells and increases cAMP (153). Another family of vasoactive peptides, atrial natriuretic peptides (1 /zM ANP), acting through cGMP formation (Fig. 1), inhibited the mitogenic activity of ET-I and ET-3 on SHR VSMC (87), whereas they had no effect on protein synthesis stimulated by ET- I on similar preparations (23). Since ET-I can induce the synthesis and release of prostacyclin in isolated vessels (25), nitric oxide (85) and ANP in ventricular cells (119), ET-I can physiologically counterbalance its own growth-promoting activity. The recent utilization of potent and selective antagonists contributes to unraveling the relevance of ET-I in cell growth. Hiley and coworkers (41) have reported that BQ- 123 [cyclo(D-Asp,L-Pro,D-VaI,L-Leu,D-Trp)] was a potent antagonist of ET- 1 (6 nM) responsible for a 50% inhibition of the increase in free intracellular calcium concentration in SKN-MC human neuroblastoma cells. BQ-123 also produced a selective and concentration-dependent inhibition of ET- l-mediated thymidine incorporation (28,90) and PI breakdown (28) in rat aortic SMC. The ICs0 values were 48-100 nM for the [3H] TdR (28,90) and 20 hA! for the PI breakdown (28), which is comparable to the IC5o previously reported (7.4-22 m~/) (35,49). The effect was selective: the increase in DNA synthesis mediated by AII( 1 #M), PDGF (0.35 nM), or thrombin ( 1 U/ml) was not affected by this antagonist (90). ENDOTHELINS AND PATHOLOGICAL PROCESSES

Although the physiological significance of the proliferative effects of numerous neuropeptides remains to be clearly demonstrated, there is increasing interest in the possible involvement ofendothelins in several disease states (24). The hypothesis originally proposed by Ross (104) suggested that the development of vascular lesions is based on the response-to-injury of endothelial cells whereby released growth factors stimulate VSMC proliferation (104, l 13). We have noted in the Introduction that

plasma levels of ET are elevated in various diseases (2,46,52,67,123). Considering the strong and prolonged vasoconstrictor activity induced by ET-l, the release of ET-l at sites of endothelial injury, and its proliferative properties on VSMC (Tables l-4), one may suggest a potential role for ET-l in the development of vascular dysfunction associated with atherosclerosis (67), hypertension-related cardiac hypertrophy, postangioplastic restenosis (76), and/or scleroderma (52) (Fig. 2). The possibility that ET might be involved in conditions such as hypercholesterolemia-related atherogenesis has been proposed (5). Combined with PDGF, ET-1 has been shown to induce proliferation of VSMC (44,140). Interestingly, levels of PDGF and IR-ET are increased and positively correlated with each other in human hypercholesterolemia (5), whereas prostacyclin

[ THROMBIN

egulators of growth factor activity/

/

vasopressin, bombesin, E T - 1

/ ~Competence growth factors/ Go -*G1

~

POGF

\ \ \ G,"Gs

Progression

\

epinephrine serotonin

//

growth factors / /

[neurotransmltters~ \ { /

/

\ \

insulin / IGF-1 / EGF / TGF-I~ //Transport factors TGF-a / / |ransferrjr~ 1

DNA SYNTHESIS HYPERTROPHY PROLIFERATION PROTOONCOGENE EXPRESSION normal carcinoma humanSMC mouse C6 gliorna rat mesang~alcells HeLa rat cardiomyocytes HEp-2 human melanocytes rat adipooyte precursor porcine & bovine PASMC rat calvariae osteoblasts bovine brain capillary EC rat,SHR and rabbit aortic SMC rat primary cerebellar astroglia fibroblasts(rat-l,rat kidney,swiss 3T3, human dermal) /

I

MEDIATE Y ASCUL&R REMODELING IN DISEASES

I

neuronal injury atherosclerosis post-angioplastic restenosis

hvoarten~ion-related cardiac hvtmrtro~hv

]

I

FIG. 2. Schematic representation of the possible interactions between ET- l and other mitogenic substances in promoting DNA synthesis, cell proliferation, and pmtooncogcneexpression. Targets such as VSMC could evolve in various diseases implicating vascular remodeling.

GROWTH REGULATORY PROPERTIES

395

and nitric oxide release are reduced (7,22). Platelets, which release PDGF, can stimulate the expression ofET mRNA and the release of IR-ET from endothelial cells (BPAE and HUVE), suggesting that platelets, interacting with the endothelium, might play a role in vasomotor regulation and growth (92). Activated platelets could release a platelet-derived endothelin regulatory factor (PDERF) that has yet to be identified (92). Cyclosporine, known to increase vascular resistance and to be involved in renal failure (136), has been reported to cause the release of IR-ET and, subsequently, proliferation of human SMC (16). The production and secretion of IR-ET also increased in many conditions related to hypoxia and pulmonary hypertension (6). Since VSMC undergo hyperplasia and hypertrophy in animals suffering from chronic hypoxia and pulmonary hypertension (99), it has been suggested that growth factors and ET could be involved. However, in a recent study using pulmonary arteries under hypoxia, such a role for ET in vascular remodeling of SMC was not supported (40). Still, ET-l could play an important role in the regulation of normal cardiac development or, in extreme cases, in cardiac hypertrophy (139). Endothelial cell injury, with the combined release of ET, growth factors, and other active substances, may well be responsible for the proliferative process observed in several diseases. Disturbances in the control of ET production can also contribute to the pathogenesis of vascular disorders; remaining intact endothelial cells could still produce and secrete ET and mitogenic substances (endothelium-derived growth factors, EDGF). Since ET- l, among other factors, is essentially generated from the vascular endotbelium, the role of the endothelial cells in the regulation of the vascular and perivascular environment becomes predominant. Even though plasma levels of IR-ET are relatively low and the half-life of ET in circulation is extremely short, it is possible that chronic, yet mildly elevated concentrations may become important in long-term vascular remodeling, development of vascular hypertrophy, and hyperplasia (Fig. 2). CONCLUSION AND PERSPECTIVES Endothelins exert various biological effects. When released from endothelial cells, they may act in both paracrine and autocrine manner on adjacent VSMC with effects that include both

vasodilation/vasoconstriction and growth factor activities. Endothelins, and especially ET-!, stimulate DNA synthesis, cell division, and hypertrophy in myocytes, fibroblasts, and many other types of cells. They are effective not only in normal cells but in tumor cells as well. But in several studies, and especially in VSMC cultures, endothelins exert these effects in the presence of insulin or low concentrations of serum. Endothelins themselves elicit relatively weak mitotic response, even though ETI acts in small concentrations in various cells. Therefore, ET-1 may not be classified as a progression factor, like IGF-1 and EGF leading to rapid cell division. The synergistic effects between ET-1 and growth factors suggest a mechanism by which ET-1 acts as a regulator of growth factor activity (Fig. 2). That phenomenon represents a very interesting feature of the endothelin peptides. But the mechanism by which ET- 1 and mitogens produce their potent synergism has yet to be established. Vasopressin and bombesin might also belong to this category of regulators. Endothelin-l, bombesin, and vasopressin induce PI turnover, increase mobilization of calcium from internal stores, and possess specific seven membrane-spanning domain receptors. Endothelin-l's mitogenic action is also dependent in part on extracellular calcium influx. Thus, endothelins act via multiple-signaling pathways. On a functional basis, growth factor PDGF may be classified as a competence factor; it generates multiple intracellular signals, including the production of cAMP, which is, with bombesin, specific for these two factors. Inhibition studies with BQ-123 suggest that ET-I regulates growth in VSMC by selective stimulation of ETA receptors. Further study with selective ETA, ETa, and ETc receptor antagonists will be necessary to provide information on the molecular interactions and signal transduction systems by which endothelin isoforms and receptor subtypes, especially ET-1 and ETA, regulate growth and stimulate mitogenesis. ACKNOWLEDGEMENTS The authors thank Ms. Nathalie Berthiaume for help in the preparation of the manuscript. B.B. is a recipient of a studentship from Le Fonds de la Recherche en Sant6 du Qurbec; P.D.J. is a Scholar of the Canadian Heart & Stroke Foundation (CHSF) and P.S. is a recipient of a ScientistAward from the Medical Research Council (MRC) of Canada. This work was supported by the MRC of Canada and CHSF.

REFERENCES 1. Arai, H.; Hori, S.; Aramori, I.; Ohkubo, H.; Nakanishi, S. Cloning and expressionofa cDNA encodingan endothelin receptor. Nature 348:730-735; 1990. 2. Arendt, R. M." Lampen, U. W.; Heucke, L.; Suhler, K.; Ritter, M.; Richter, W. O. Ir-ET circulates in human plasma: Elevated concentrations in patients with hyperlipoproteinemia. Am. J. Hypertens. 3:93A; 1990. 3. Badr, IC F.; Murray,J. J.; Breyer, M. D.; Takahashi, K.; Inagami,T.; Harris, R. C. Mesangialcell, glomerularand renal vascularresponses to endothelinin rat kidney.J. Clin. Invest.83:336-342; 1989. 4. Bakris, G. L.; Fairbanks, R.; Traish, A. M. Arginine vasopressin stimulates human mesangialcell production ofendothelin. J. Clin. Invest. 87:1158-1164; 1991. 5. Bath, P. M.; Martin, J. F. Serum PDGF and ET concentrations in human hypercholesterolemia. J. Intern. Med. 230:313-317; 1991. 6. Battistini,B.; D'OflEans-Juste,P.; Sirois, P. Endothelins:Circulating plasma levels and presence in other biological fluids. Lab. Invest. (in press). 7. Beetens,J. R.; Coene, M. C.; Verheyen,A.; Zonnekeyn, L.; Herman, A. G. Biphasicresponse ofintimal prostacyclin production during the development of experimental atherosclerosis. Prostaglandins 32:319-334; 1986.

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