Vascular eicosanoids and platelet-aortic wall interactions in spontaneously hypertensive rats

202 ( 1991) 3 1 I -3 I6 0 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50 ADONIS 001429999100616S European Jowm7l of Phartnacology,

311

EJP 52031

Marcello

D. Lograno

’ Phartnnco-Biological School of Pharmacy,

‘, Cristina Department,

Mosconi

2: Franca Marangoni and Claudio Galli 2

Phurtnacological

Utrkersify of Milan, Milan,

2, Lucia Medini

2, Gianluca

Grassi 3

Unit, Utksersity of Bari, Bari, Italy, 3 it~stitutc of Pharm cological Sciences. School qf Medicine, Uttfi,. nity of Milan, Milm.

Italy and .’ Depurtnzetzt of Pharmacology,

Received

14 May 1991. accepted

Italy

2 Juty 1991

We studied the aggregation of collagen and ADP-stimulated platelet-rich plasma (PRP) and the formation of thromboxane B, (TxB,) by collagen-stimuklted PRP in spontaneously hypertensive rats (SHR) and in W&tar-Kyoto control rats (WKY). :n addition, WC evaluated the inhibition of the aggregation of PRP following homologous or heterologous perfusions through isolated aortas, the release of 6-keto-prostaglandin (PG)F,, from these arteries perfused with PRP, and the sensitivity of PRP to the antiaggregatory activity of the stable PGI, analogue, iloprost, in both SHR and WKY. The lower activities (aggregation induced by ADP and collagen, collagen-stimulated TxB, production) of SHR platelets, -were not accompanied by morphological differences from WKY platelets. These changes were associated with a greater release of arterial 6-keto-PGF,,, with greater platelet antiaggregatory activity of the arterial wall and with higher sensitivity of platelets to iloprost. The lower reactivity of platelets to aggregating agents, and the grcatcr sensitivity to prostacyclin, associated with a greater production of arterial prostacyclin were the major changes observed in SHR animal: These alterations in the SHR vs. normotensivc WKY may lead to an enhanced risk of hemorrb Igc in the hypcrtensivc state. Hypertension;

Platclct aggregation; Platclct thromboxanc;

1. Introduction Altered eicosanoid production or activity in kidneys and in the circulation may play a role in the pathogenesis of hypertension (Greene and Dunn, 1984; Uehara et al., 1983; Grose et al., 1983; Shibouta et al., 1981). Enhanced urinary excretion of thromboxane B, (TxB2 1, the stable metabolite of TxA,, has been reported in patients with essential hypertension (Grose et al., 1983) and in spontaneously hypertensive rats (SHR) (Purkerson et al., 1986). In addition, treatment with selective inhibitors of thromboxane synthetase, such as OKY046, reduced blood pressure in SHR (Purkerson et al., 1986; Gomi et al., 19891, with a predominant effect during the developmental stage of hypertension (Stier and Itskovitz, 1988). On the other hand, indomethacin treatment of SHR enhances the development of the hypertensive state (Stier and Itskovitz, 19881, suggesting that production of cyclooxygenase metabolites dif-

Correspondence to: C. Galli, Institute of Phermacolog~c.rl Sciences, School of r’harmacy. University of Mihm. Via Balzarctti 9. 20133 Milan. Iluly. Tel. 3~~.2.20.4X.X3.flO,fax 30.2.20.40.4~~.1,1.

Prostacyclin (aortic); Platclct inhibition (by prostacyclin)

ferent from thromboxane may play a protective role in the development of spontaneous hypertension in the rat. This may be due to prostacyclin production since increased formation of prostaglandin (PG)I 2 (PGI> 1 by aortic tissue of developing hypertensive rats (PaceAsciak and Carrara, 1979) and enhanced activity of PGI, synthetasc in the aortic wall of SHR (Uehara et al., 1987) have been described. Vessel walls from SHR appear also to produce more TxA, (Tasbe et al., 19821, although the significance of this finding is questionable. In contrast with the evidence of enhanced synthesis of the proaggregatory thromboxanc, at least in the kidney of SHR, the aggregatory response of platelets from SHR to agents such as thrombin and collagen, which trigger thrombovane formation, is progressively reduced during the development of hypertension (Tomita et al., 1984, l985), with an inverse correlation between changes in blood pressure and platelet aggregability. Defective Cal+ function (Tomita et al., 1984) and/or appearance of exhausted platelets (Tomita et al., 1985) have been suggested to be responsible for platelet hypoaggregability in SHR. The number of platclcts also is reduced in SHR (Tomita et al., 1984).

Together. these observations indicate that alterations of eicosanoid metabolism of a different nature are present in hypertension. They appear to play a role in the onset of the hypertensive state, but may also be part of adaptation processes. In addition to changes in eicosanoid production, modifications of the responses of cells such as platelets to the activity of prostacyciin may further r;dduIate functional ceil and vascular parameters during hypertension. In order to explore some of these aspects, we have measured both the aggregation and thromboxane formation by stimulated plateiet-rich plasma IPRP), and the effects of the perfusion of isolated aortas with PRP on the release of prostacyciin and on the inhibition of PRP aggregation in SHR and in controi rats. More specifically, we assessed the ability of aortas from SHR and control rats to inhibit the aggregation of PRP perfused through the arteries under both homologous tie. contro1 or SHR platelets through the corresponding aortas) and heterologous (i.e. SHR platelets through control aortas. and vice versa) conditions. Under the same conditions we measured the release of 6-ketoPGF,, from the perfused vessels and the response of PRP from SHR to the antiaggregatory activity of prostacyciin. Our study shows that, in SHR, both the reduced aggregatory response of stimulated platelets and the decreased thromboxane fo~atio~ by stimulated platelets without morphological evidence of platelet defects are associated with enhanced sensitivity to the antiaggregatory activity of the vessel wail and with an enhanced r&ease of prostacycIin by isolated aortas. In addition, platelets from SHR rats are more sensitive to the antiaggregatory activity of a stable prostacyciin anaiogue. The lower sensitivity of SHR piateiets to aggregating agents, coupled with higher sensitivity to prostacyclin, appeared to be the prevailing factors responsible for the greater inhibitory activity of arterial walls on SHR platelets.

2. Materials and methods 2.1. Experima~tai animals

MaIe spontaneously hypertensive rats (SHR) aged 12 weeks and with an average weight of 250-300 g and, as corresponding controls, normotensive Wistar-Kyoto rats (WKY), were purchased from Charles River. They were housed in the animals care facility and fed iaboratory rat chow and water ad Iibitum. 2.2. Materials ADP was from Sigma Chemical Company, St. Louis (MO, USA); Collagen from Semmeiwciss, Mascia

BruneIIi (Milano, Italy); itoprost from Schering (Berlin, Germany). 2.2. I. Preparation 3f ~~~~p~e~

Blood was drawn by cardiac puncture from anesthetized animals into plastic syringes with sodium citrate (3.13% 9 : I) as anticoagulant. Aortic segments were removed from the arch to the ihac bifurcation, carefully cleaned and kept at 4°C for a few minutes until used for perfusion studies. PRP was prepared by centrifugation of blood and PRP was diluted with autologous platelet-poor plasma (PPP) to a concentration of 5 x 10” platelets/PI. Ahquots of PRP samples were used for: (a) platelet aggregation studies, (b) measurements of TxB, formation after stimulation, (cl assay of the sensiti‘:ity to the inhibitory effects of iioprost, (d) perfusion through isolated aortas. Samples of washed platelets were also prepared from transmission electron microscopy examination. 2.3. Measurements 23.1. Platelet pnr~meter~ 2.3.1.1. Electron microscopy methods. Platelets were prepared by centrifugation of blood in the presence of PGE, (lo-” M, final concentration) in order to minimize actisation, and pellets were fixed in 2.5% glutaraidehyde in 0.1 M phosphate buffer pH 7.2 for 4 h at 4” C, rinsed in the same buffer and post-fixed in buffered 1% osmium tetroxide at 4°C for 1 h. The sampies were dehydrated in a series of graded ethano1 solutions and propylene oxide and were embedded in Epon 812 resin mixture. The samples were then incubated at 60”~ for 48 h for pofymerization, Thin sections were prepared and sections were double-stained with uranyi acetate and lead citrate, taken up on copper grids and viewed with Philips CMlfl transmission electron microscopy. 2.3.1.2. Piatdet aggregatiott. Aggregatory responses of PRP obtained from SHR and controls after coIIagen and ADP stimulation were qdantitated by measuring the amplitude of the aggregation curves, expressed as percent of maxima! light transmittance (PPP), obtained with increasing concentrations of the agents. Assays were carried out with 250 ~1 aliquots of PRP containing 5 X IO” pIateiets/~i in an ELVI Logos (MiIano, Italy) aggregometer. 2.3.1.3. TxB, formation in PRP. TxB, production in PRP samples after stimulation with collagen was evaluated by measuring TxB, levels in PRP which was preincubated (37 ’ C, 1100 rpm stirring) for 1 min, after 10 min of incubation with 10 and 20 @g/ml of the agent. The reaction was stopped with 25 voIumes of methanol and TxB, was measured by EIA (PradeIIes et al., 1985) after evaporation of the solvent and addition of the appropriate buffer.

313

2.3.1.4. Assay of the sensitislity of PRP to the inhibitory effects of iloprost. The antiaggregatory activity

% aggregation

of iloprost was assessed by determining the percentage inhibition of collagen (15 ~g/mi)-induced PRP aggregation in the presence of increasing concentrations of iioprost, and by evaluating the IC,, (concentration inhibiting the aggregatory response at the 50% level). 2.3.2. Aortic parameters 2.3.2.1. Prostacyclin release from perfused aortic segmen ts. Prostacyciin production by aortic tissue was

evaluated by measuring the concentration of 6-ketoPGF,, in PRP (3 x lo5 piatelets/~i) perfused through the isolated aortic segments (Gaiii et al., 19811 under conditions of homologous (control or SHR PRP through the homologous aortas) and heteroiog3us (control PRP through SHR aortas, and vice versa) perfusions. The compound was measured by EIA (Pradeiies et al., 1985) after solvent extraction from the PRP samples, in the first l-ml fraction, which contains more prostacyciia than the subsequent fractions (Gaiii et al., 1981). 2.3.2.2. Antiaggregatory acticity of aortic walls. The platelet antiaggregatory activity of the aortas was evaiuated as inhibition of the collagen (8 pg/mil-induced aggregation of PRP, expressed as percentage inhibition of the aggregation measured after PRP perfusion in comparison with that of non-perfused PRP. In addition we evaluated the inhibition of aggregation of PRP from WKY rats after perfusion through WKY aortas and, vice versa, the aggregation of WKY PRP perfused through SHR aortas. 2.4. Statistical analyses SHR and WKY values in figs. 4 and 5, and in tables 1 and 2 were compared using an unpaired, Student’stest.

WKY SHR

-

’1 0

5

10

g/ml

15 Collagen

Fig. 1. Dose-response cutves for aggregation of collagen-stimulated PRP from SHR and WKY. The values are expressed as percentages of maximal aggregation, corresponding to maximal light transmittance (PPP). The values are averages+S.E.M. of determinations carried out in six animals/group. All values in the SI’R curves are significantly fat least P < 0.01) different from controls at each collagen concentration.

difference was found between SHR and WKY ceils, which might explain the functional changes occurring in SHR samples (fig. 3). Formation of TxB,, the stable TxA, metaboiite, by collagen-stimulated PRP (fig. 4) was lower in SHR rats than in WKY rats, at a IO-pg/mi concentration of the agent, whereas there was no difference between SHR and WKY samples at 20 pg/mi collagen. The ability of the aortas to inhibit the aggregation of platelets interacting with the surface of the arterial wall was assessed by measuring the percentage reduction of collagen-induced PRP aggregation after homoiogous and heteroiogous perfusion through the isolated vessel, in comparison with the aggregation of non-per% aggregation

T

50 1

3. Results The aggregatory responses of PRP from SHR to increasing concentrations of collagen and ADP were markedly lower than those of PRP from controls (WKY) (figs. 1 and 2). At the lowest concentrations of the agents, PRP aggregations of SHR were more than 90% lower for collagen (fig. 1) and more than 70% lower for ADP (fig. 2) when compared to WKY, whereas at the highest collagen (20 pg/mi) and ADP (10 PM) concentrations the reductions vs. WKY were about 40% for collagen and about 55% for ADP. In order to detect possible morphological modifications of SHR platelets in comparison with the controls, washed cells were prepared and were examined by transmission electron microscopy. No morphological

----c 1

3

5

7

SHR

11

9 ADP

PM

Fig. 2. Dose-response curves for aggregation of AD?stimulated PRP from SHR and WKY. The values are expressed as percentages of maximal aggregation, corresponding to maximal light transmittance (PPP). The values are averagesfS.E.M. of determinations carried out in six animals/group, All values in the SHR curves are significantly fat Icast P < 0.01) different from controls, at each ADP concentration.

TABLE

1

Percentage inhibition of collagen-induced PRP aggregation after perfusion through isolated aortas. SHR, spontaneouslj hypertensive rats: WKY, Wistar-Kyoto rals. The values are the averages+_S.E.M. of four determinations. Values sharing the same superscript are significantly different from each other at the following levels: (I P < 0.05; P.“.h P < 0.001. SHR/WKY

PRP * Fig. 3. Ultrastructural morphology of washed platelets 15500X magnification) from control WKY (A) qd SHR (B). A marginal drprre of cell activation and degranulation ‘was observeti in both preparations. without appreciable differences hetween the two samples.

Aortas WKY SHR

WKY

SHR

35 + 3 .‘.(’ 48 I 4 hJr

67 _+ 3 ;‘+

1.37

SHR/WKY

9X+2 h.p

I.91 2.09

1.47

* 8 inhibition vs. non-perfused PRP.

fused PRP (table 1). The concentration of collagen selected for the aggregation of all PRP sampies before and after perfusion was 8 pg/ml, since, afte: homologotis perfusions with concentrations of collagen as high as i5 pg/ml. SHR platelets were Ml inhibited, and the aggregation of WKY PRP was not further enhanced. Instead, when WKY PRP, perfused through the corresponding aorta, was stimulated with 4 pg/ml collagen, about 70% inhibition of aggregation was observed. After homologous perfusions, WKY and SHR PRP were inhibited by 35 and of 98% respectively, whereas the inhibition of WKY PRP perfused through SHR aortas and of SHR PRP perfused through WKY aortas was 48 a;rd 67%, respectively. Analysis of PRP response with the various types of perfusions indicates that inhibition of SHR PRP was greater than the inhibition seen with WKY samples (1.91- and 2.09-fold in SHR vs. WKY, respectively) when different platelets (i.e. WKY vs. SHR) were perfused through the same

TX&? ng 1 ml PRP

type of aorta (WKY or SHR) than when the same type of platelet (WKY or SHR) was perfused through different types of aortas (WKY vs. SHR) (1.37 and 1.47, respectively). The release of 6-keto-PGF,, from the PRP-perfused aortas was measured unde: the same experimental conditions and the results are reported in table 2. Levels of the PGI, metsbolite were about 3-fold higher in SHR PRP than in WKY PRP, after homologous perfusions. In heterologous perfusions, concentrations of 6-keto-PGF,, were greater in SHR than in WKY when the same PRP was perfused through the two different aortas (2.06 and 1.9, respectively), than when the two different PRP were perfused through the same aorta (1.62 and 1.50, respectively). Perfusions of SHR PRP, although less responsive than WKY to aggregating agents, always stimulated a release of 6-keto-PGF,,, from any type of aorta that was greater than the release induced by WKY PRP. Finally, we explored the sensitivity of PRP from the two groups of animals to the antiaggregatory activity of the stable prostacyclin analogue, iloprost. The IC,, for inhibition of PRP aggregation induced with 15 pg/ml collagen (fig. 5) were 2.7 X lo-’ M and 2.2 x lo-” M, in the SHR and WKY platelets, respectively, i.e. sensi-

TABLE

2

Levels of h-kelo-PGF,,,

10

20

The values are the averages + S.E.M. of four determinations. Values with the same superscript are significantly different from each other at P < 0.05.

Collagen( p g I ml) Fig. 3. Concentrations (ng/ml) of TxBz in PRP (5 x 10’ platelets/~l) from SHR and WKY rats. IO min after stimulation (37°C. 1100 rpm) with collagen at the concentrations indicated. Samples were preincubated for I min before stimulation. TxB, was measured hy the EIA. The values are the averages*S.E.M. of determinations carried out in six animals/group. * Significantly different from control values at P < 0.05.

in PRP perfused through isolated aortas.

PRP (ng/ml)

SHR/WKY

WKY

SHR

Aortas WKY SHR

3.2 f 0.6 “,” 6.6 * I .4 “.fi

5.2 f 0.7 ” ‘J.‘)k I.6 fl

SHR/WKY

2.06

1.90

I .62 I.50

315 IlOp3Sl

(molar concentration;

Animal groups Fig. 5. Inhibiting concentrations at 50% level UC,,) for iloprost, of collagen (15 ~g/ml)-stimulated aggregation of PRP from SHR and control rats. The values are the averages+ S.E.M. of determinations carried out in six animals/group.

tivity to the agent was about 100 times greater for the SHR than for WKY samples.

4. Discussion Alterations of platelet and vessel wall function and eicosanoid formation in hypertension have been reported, the most consistent findings being enhanced urinary excretion of thromboxane metabolites (Grose et al., 1983; Purkerson et al., 1986), reduced platelet aggregation (Tomita et al., 1984, 1985) and enhanced prostacyclin formation (Pace-Asciak and Carrara, 1979; Uehara et al., 1987). In addition, the activity of protein kinase C (PKC) in vessels (Turla and Webb, 1987; Mackay and Cheung, 1987; Shibata et al., 1990) and in platelets (Takaori et a1.,1986) is altered in SHR. These observations indicate that various regulatory processes in cell activation are impaired by the hypertensive state. Our results confirm previous observations concerning the production of vascular eicosanoids in SHR. In addition, new information was obtained on the sensitivity of SHR platelets to exogenous prostacyclin, and on the responsiveness of normal and SHR aortas to SI-JR PRP, in terms of prostacyclin release and antiaggregatory activity. Platelets from the SHR were less responsive than those of WKY to aggregating agents and this difference could not be attributed to platelet exhaustion, on the basis of morphological examinations. Aggregatory responses to both collagen and ADP were reduced and the dose-response relationships suggest that reduced affinity for the agents and, to some extent, reduction of the maximal response were present in SHR vs. WKY platelets. A reduced responsiveness of washed SHR platelets to thrombin and Ca” ionophore, in addition to ADP and collagen, was described previously (Tomita

1984, 1985), suggesting that the defect can be specifically attributed to a faulty platelet function. Under our conditions, using PRP, the reduction of platelet responsiveness might also be due to the prolonged exposure of SHR platelets, which are hypersensitive to PGI,, to higher concentrations of this eicosanoid. The reduced aggregability of SHR platelets was associated with a reduced formation of thromboxane after collagen stimulation when the lowest concentration of the agent was used. Since stimulation with 20 pg/ml collagen resulted in lower aggregation of SHR platelets VS. WKY (fig. 11, with no difference in thromboxane formation (fig. 31, it would appear that the reduced aggregation of SHR platelets primarily involves processes other than the thromboxane pathway. The modifications of aortic prostacyclin production and of the inhibitcry activity of endogenous and exogenous prostacyclin on platelets, from SHR were quite complex. The reported (Pace-Asciak and Carrara, 1979; Uehara et al., 1987) enhanced production of aortic PGI, was in fact associated with a greater inhibition of the aggregation of PRP after perfusion through the aortas. The inhibition of SHR PRP perfused through any type of aorta was similarly and significantly greater than the inhibition of WKY platelets perfused through any type of aorta. Furthermore, the inhibition of SHR platelets perfused through WKY aortas (which produced less prostacyclin than the SHR artery) was greater than that of WKY platelets perfused through SHR aortas. Release of prostacyclin from perfused SHR aortas was also always greater than that from WKY arteries, irrespective of the type of PRP perfused. Finally, in spite of the fact that SHR platelets were less active than normal ones, their perfusion stimulated PGI, release from any kind of aorta more effectively when SHR PRP were perfused. Since elevated prostacyclin release from arterial walls is generally associated with enhanced platelet activity, it would appear that, when SHR PRP was perfused, factors other than platelet function contributed to stimulate prostacyclin release from the aorta. In conclusion, the greater sensitivity of SHR platelets to the antiaggregatory activity of the arterial walls and the greater production of PGIz by SHR aortas appeared to be additive in determining the (complete) inhibition of SHR platelets perfi;sed through SHR aortas. Platelets from SI-IR were indeed much more sensitive to exogenous PGI,. In contrast with the findings in SHR, we have previously observed, with the same experimental model, that enhanced production of aortic PGI, was associated with increased platelet aggregability in hypercholesterolemic rabbits VS. controls (Tremolo et al., 1982) and that a greater release of PGI, from the aorta and a greater sensitivity of PRP to PGI, in aged rats (Giani et al., 1985) than in young rats were both associated with increased platelet aggreet al.,

gabihty. The combined increases of arterial prostacychn ~r~~ction and of PRP sensitivity to PGI 2 in SHR. rather than a compensatory effect, could represent a greater risk for some of the consequences of the hppertensive state. In fact, the above alterations associated with the reduced number of platelets in SHR (Tomita 9841, together with mechanical damage of arterial, especially cerebral, walls due to the high blood pressure, might contribute to cerebral hemorrhage, which is a frequent event in hypertension.

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