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Cardiovascular Research 2004 61(2):333-338; doi:10.1016/j.cardiores.2003.11.009
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Copyright © 2004, European Society of Cardiology

Effects of chronic PGHS-2 inhibition on PGHS-dependent vasoconstriction in the aged female rat

Stephen J Armstrong, Yi Xu and Sandra T Davidge*

Department of Ob/Gyn and Physiology, 232 Heritage Medical Research Centre, Perinatal Research Centre, University of Alberta, Edmonton, AB, Canada T6G 2S2

* Corresponding author. Tel.: +1-780-492-1864; fax: +1-780-492-1308. sandra.davidge{at}ualberta.ca

Received 21 July 2003; revised 21 October 2003; accepted 14 November 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: In menopause, aging and decreased estrogen levels are risk factors contributing to impaired vascular function. Previously, in a young ovariectomized rat model, we demonstrated an increase in prostaglandin H synthase (PGHS)-dependent vasoconstriction that could be prevented by estrogen replacement. Subsequently, we found that, with aging, estrogen acts through suppression of the PGHS-2 isoform. Hypothesis: Chronic PGHS-2 inhibition reduces PGHS-dependent vasoconstriction in aging. Methods: Ovariectomized, aged (12 months) Sprague–Dawley rats were treated with a placebo (n = 7), or the PGHS-2 inhibitor (NS-398, s.c. 3 mg/kg) for 1 week (n = 6) or 4 weeks (n = 6). Methacholine (endothelium-dependent dilator) and phenylephrine (adrenergic constrictor) were used to assess vascular function in the absence or presence of the nonselective PGHS inhibitor, meclofenamate (1 µmol/l) or the specific PGHS-2 inhibitor NS-398 (10 µmol/l). Results: One week of chronic PGHS-2 inhibition abolished a PGHS-dependent shift in methacholine-induced relaxation, while modulation was still observed in phenylephrine constriction. Surprisingly, 4 weeks of PGHS-2 inhibition enhanced PGHS-dependent modulation of vasoconstriction (P<0.05). PGH2/thromboxane inhibition (U-51605, 50 µmol/l) mimicked the results observed with PGHS inhibition among the groups. PGHS-2 expression increased with chronic PGHS-2 inhibition compared to control (P<0.05). Conclusions: Our data indicate a paradoxical increase in PGHS-dependent vasoconstriction and PGHS-2 expression with prolonged inhibition of PGHS-2 activity. Hence, inhibitors of PGHS-2 activity may not be beneficial in counteracting the vascular dysfunction seen with aging and menopause.

KEYWORDS Aging; Mesenteric arteries; PGHS-2 inhibition; Prostaglandin; Female


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Aging and estrogen deficiency are both risk factors for cardiovascular disease, the leading cause of death among postmenopausal women [1]. Aging is associated with an increase in vasoconstriction due, in part, to alterations in the vascular endothelium [2]. The enzyme prostaglandin H synthase (PGHS) has been shown to be important in this process. Two isoforms, PGHS-1 and PGHS-2, metabolize arachidonic acid to produce the intermediate PGH2. Subsequently, eicosanoids such as prostacyclin and thromboxane are formed. Normally, prostacyclin, which elicits vasodilation, would balance or supercede the constriction promoted by thromboxane [3]. In aging, however, the production of prostacyclin is reduced [4] and thromboxane is preferentially generated [5].

Prior to menopause, women are protected from age-related vascular dysfunction, in part, by the actions of estrogen [6]. One effect of estrogen is to inhibit PGHS-dependent vasoconstriction in young, ovariectomized rats [7]. Recently, we demonstrated that PGHS-2 protein expression is upregulated with aging in female rat mesenteric arteries, and that vessel tone is enhanced through this pathway [8]. Moreover, estrogen replacement in an ovariectomized, aged rat suppressed vasoconstriction dependent upon the PGHS-2 pathway [9]. This was associated with a reduction in PGHS-2 protein expression in the vasculature from estrogen-replaced animals [9]. Estrogen has also been shown to reduce PGHS-2 mRNA expression in bovine chondrocytes [10]. Since estrogen replacement is contraindicated for some women, specific PGHS-2 inhibition may be an alternative therapy to provide vascular benefits.

Selective inhibitors of PGHS-2 activity have been developed to treat chronic inflammatory conditions while attempting to reduce the gastrointestinal toxicity observed with traditional NSAIDs [11,12]. The rationale behind selective PGHS-2 inhibition is to target those eicosanoids involved in the inflammatory response and bypass the prostaglandins that protect against gastric ulceration [13]. These drugs have been used successfully in conditions such as acute lung injury [14], arthritis [15] and colon cancer [16]. However, the chronic effects of PGHS-2 inhibitors on vascular responses in an aging model are not known.

These data led to our hypothesis that chronic PGHS-2 inhibition should facilitate enhanced vasorelaxation by reducing PGHS-dependent vasoconstriction in the aged, ovariectomized rat.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal model
Female Sprague–Dawley rats were obtained from Charles River and were housed in our facilities until 11–12 months of age. Rats were ovariectomized (OVX) and given a placebo pellet (Innovative Research of America; n = 7), or a daily (each morning) subcutaneous injection of the PGHS-2 inhibitor, NS-398 (3 mg/kg), for 1 week (n = 7) or 4 weeks (n = 6) prior to experimentation. Ovariectomy was performed in order to control for the potential variability in estrogen levels that is characteristic of animals approaching reproductive senescence (constant estrous). The NS-398 dose was chosen based on effective PGHS-2 inhibition for up to 24 h from previous studies in the rat [17,18]. Rats were killed by exsanguination while under anesthesia (sodium pentobarbitol, ~60 mg/kg-body wt.). A blood sample was taken from the chest cavity (after extraction of the heart) and serum obtained by centrifugation. To confirm a successful ovariectomy, samples were snap-frozen (–80 °C) for subsequent measurement of 17 β-estradiol levels via a radioimmunoassay technique (Diagnostic Products). Animal protocols were approved by the University of Alberta Animal Welfare Committee which conforms to NIH guidelines.

2.2. Vessel preparation
A portion of the mesentery was excised and immersed in ice-cold N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]-buffered physiological saline solution (HEPES-PSS) which contained the following (in mmol/l): NaCl 142, KCl 4.7, MgSO4 1.17, Ca2Cl 1.56, KH2PO4 1.18, HEPES 10, and glucose 5.5. Resistance-sized arteries (diameter ~250 µm) were dissected and connected to the myograph system (Kent Scientific) as previously described [9]. Mesenteric arteries were chosen due to their substantial contribution to vascular resistance in rats [19]. Force production by these arteries was recorded on a data acquisition system (Workbench, Strawberry Tree).

2.3. Experimental design
Phenylephrine was administered in the initial curves to determine the concentration needed for 50% maximal constriction (EC50) of each segment. This provided a preconstriction base line from which vasorelaxation curves could be measured. Each phenylephrine curve was quantified as a percent of its own control. Methacholine was added (1 nmol/l to 1 µmol/l) to assess endothelium-dependent relaxation. An intact endothelium within each blood vessel was verified by the presence of relaxation to methacholine. The nonselective PGHS inhibitor, meclofenamate (1 µmol/l; Cayman Chemical, Ann Arbor, MI), the specific PGHS-2 inhibitor, NS-398 (10 µmol/l), or the stable PGH2 analogue, U-51605 (50 µmol/l; Cayman Chemical), which is capable of inhibiting PGH2- and thromboxane-dependent constriction, were incubated in different baths, 15 min before various curves, to study the functional significance of eicosanoids in these arteries. This design enabled us to determine whether or not chronic PGHS-2 inhibition was successful in the animals and/or if it altered PGHS-(2)-dependent vascular effects. We hypothesized that the rats that were administered chronic NS-398 treatment would have little or no change in vascular function with addition of the PGHS pathway inhibitors to the bath.

2.4. Western immunoblot
Mesenteric arteries from individual animals were dissected, pooled and homogenized in eppendorf tubes (containing a protease inhibitor cocktail to inhibit serine, cysteine and aspartic proteases to prevent degradation; Sigma, Saint Louis, MO) using a small tissue homogenizer. The Bradford assay was employed to measure protein concentration [20]. Western immunoblots were performed as previously described [21]. Polyclonal antibodies for PGHS-1 and PGHS-2 (Cayman Chemical) and monoclonal antibodies for {alpha}-actin (Boehringer Mannheim) were used. Bands were quantified by densitometric analysis and normalized to {alpha}-actin. PGHS-1 expression was detectable, however the intensity was not sufficient for analysis.

2.5. Data analysis
Data from each dose–response curve was fitted to the Hill equation, and a straight line generated by linear least-squares regression analysis. EC50 was determined from this line and the mean±S.E. calculated from the curves. ANOVA was used for statistical analysis among groups as well as for Western immunoblot bands. Post hoc analysis was performed using Tukey's or Student–Newman–Keuls tests. Tests were considered significant at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Body weights were not significantly different among the groups. The plasma estradiol levels were below <5 pg/ml (detectable limit of assay) in all animal groups.

Contrary to our hypothesis, methacholine-induced relaxation was not significantly altered among all the groups. However, the influence of PGHS in modifying vascular responses varied among the groups. Meclofenamate (PGHS inhibitor, 1 µmol/l) enhanced methacholine-induced relaxation in the placebo, aged group (Fig. 1A, P<0.05) but did not alter relaxation in the 1-week treatment group (Fig. 1B, P = 0.733), suggesting the presence of PGHS-dependent vasoconstriction in the aged rat which could be prevented by chronic PGHS-2 inhibition for 1 week. Interestingly, in the 4-week treatment group, meclofenamate enhanced relaxation (Fig. 1C, P<0.05), suggesting the reoccurrence of PGHS-dependent vasoconstriction. Similar findings were observed with the specific PGHS-2 inhibitor, NS-398 (10 µmol/l) (Fig. 1). Furthermore, inhibition of downstream eicosanoids with the stable PGH2 analogue, U-51605, had similar effects to meclofenamate and NS-398 (Table 1), suggesting that PGH2/thromboxane is involved in the PGHS-dependent constriction.


Figure 1
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  		Fig. 1 Methacholine concentration response curves from mesenteric arteries of aged, ovariectomized rats. Panel A: Placebo (n = 7), Panel B: in vivo PGHS-2 inhibition (NS-398, 3 mg/kg) for 1 week (n = 7), Panel C: in vivo PGHS-2 inhibition for 4 weeks (n = 6) in the absence (bullet) and presence ({circ}) of 1 µmol/l meclofenamate or 10 µmol/l NS-398 ({bigtriangledown}) in the bath. *EC50 P<0.05 vs. methacholine alone.

 

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  		Table 1 EC50 values of methacholine-induced vasorelaxation and phenylephrine-induced vasoconstriction in the absence and presence of PGHS pathway inhibitors in rats given 1 or four weeks of chronic PGHS-2 inhibition or placebo treatment

 
Phenylephrine elicited a similar dose response among the animal groups. In the placebo group, the presence of the nonselective PGHS inhibitor or the specific PGHS-2 inhibitor resulted in a trend towards an increase in the dose required to elicit 50% constriction with each drug (Fig. 2A). The 1-week treated group exhibited a significant increase in EC50 with meclofenamate or NS-398 in the bath (Fig. 2B), indicating a role for PGHS-dependent constriction with 1 week of chronic PGHS-2 inhibition. Four weeks of NS-398 treatment also revealed a significant increase in EC50 for phenylephrine after PGHS inhibition in the bath (Fig. 2C). The increase in PGHS-dependent constriction in the 4-week group was the most marked indicating an increase of the PGHS vasoconstrictor pathway over time with chronic PGHS-2 inhibition.


Figure 2
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  		Fig. 2 Phenylephrine concentration response curves and EC50 bar graphs from mesenteric arteries of aged, ovariectomized rats. Panel A: Placebo (n = 5–7), Panel B: in vivo PGHS-2 inhibition for 1 week (n = 5–7), Panel C: in vivo PGHS-2 inhibition for 4 weeks (n = 6), in the absence (bullet) and presence ({circ}) of meclofenamate (Meclo, 1 µmol/l) or NS-398 ({bigtriangledown}, 10 µmol/l). *EC50 P<0.05 vs. phenylephrine alone.

 
In support of this, Western immunoblots revealed a time-dependent increase in PGHS-2 expression between the 1- and 4-week treatment with the PGHS-2 inhibitor (Fig. 3, P<0.05). PGHS-1 expression was not altered among the groups (data not shown).


Figure 3
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  		Fig. 3 Panel A: Representative Western blot for PGHS-2 expression in mesenteric arteries from aged, ovariectomized placebo-treated rats (n = 7), and aged, ovariectomized rats given the PGHS-2 inhibitor for 1 (n = 7) or 4 weeks (n = 6). Panel B: Ratio of the densitometric analysis of PGHS-2 bands normalized to {alpha}-actin. Bar and error represents the mean+S.E.M. of samples. Different letters denote significant differences at P<0.05.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our data indicate that although the PGHS-2 pathway impairs vascular function in the aging rat model, long-term use of specific inhibitors of PGHS-2 activity may not have vascular benefits. Chronic inhibition of PGHS-2 activity with NS-398 paradoxically caused an increase in PGHS-2 expression and PGHS-dependent vasoconstriction in arteries from the aged, female rat.

A similar feedback effect has been observed in studies using aspirin. Aspirin induced a significant increase in PGHS-2 mRNA and protein expression in the superficial mucosa of the rat stomach [22]. Furthermore, aspirin enhanced PGHS-2 expression when administered to placental cytotrophoblasts [23]. In contrast, aspirin given immediately prior to lipopolysaccharide challenge suppressed PGHS-2 mRNA expression in murine peritoneal macrophages [24]. These studies indicate differential responses for the actions of aspirin on PGHS-2 levels. Our study also demonstrated a time-dependent feedback response to induce the PGHS-2 enzyme in animals given a specific inhibitor of PGHS-2 activity.

Contrary to our hypothesis, chronic PGHS-2 inhibition did not enhance endothelium-dependent relaxation nor did it blunt adrenergic vasoconstriction in the aged rat. However, the role of PGHS-dependence in the vascular responses was shifted among the treatment groups. As previously reported, the PGHS-dependent portion of the age-related constriction was substantial in the ovariectomized, placebo-treated animals [9]. Moreover, 1 week of PGHS-2 inhibition abolished this PGHS-sensitive component of methacholine relaxation, albeit with a role still detectable in phenylephrine constriction. Interestingly, however, an extended 4-week period of chronic treatment markedly enhanced the PGHS-dependent portion of the constriction, shifting the balance back towards the placebo controls.

PGHS-2 is capable of producing both vasodilator and vasoconstrictor prostanoids. The ability of the enzyme to elicit a downstream effect is largely dependent upon the activity of terminal synthases, such as prostacyclin or thromboxane synthase. Some reports have concluded that PGHS-2 inhibition may be mediating its vascular effects by blocking prostacyclin production [4,25,26]. However, in the female aging rat model, PGHS-dependent constriction predominates. Indeed, our data revealed an enhanced relaxation to methacholine and a blunted constriction to phenylephrine after non-selective PGHS blockade with meclofenamate and specific PGHS-2 inhibition with NS-398. Furthermore, inhibition of PGH2/thromboxane with U-51605 demonstrated a similar increase in methacholine-induced relaxation, which was suggestive of a role for these constrictor eicosanoids in the placebo and 4-week treated animals. Prostacyclin exhibited a minimal role in this modulation. This could be due to the nitration and inactivation of prostacyclin synthase by peroxynitrite (the reactive oxygen species produced by the reaction between nitric oxide and superoxide) [27]. Indeed, vascular aging has been shown to be associated with enhanced peroxynitrite formation [28] and our lab demonstrated that peroxynitrite decreased the protein mass of prostacyclin synthase in bovine microvascular endothelial cells [29]. Interestingly, peroxynitrite has been shown to induce PGHS-2 in human endothelial cells [30]. Ultimately, a feed forward loop could occur whereby there is an increased production of PGHS-2 (which itself can be a source of superoxide anion) leading to further peroxynitrite production, inactivation of prostacyclin synthase and hence PGHS-2-dependent constriction.

Interestingly, although there was a difference in modulation by PGHS-dependent constrictors among the groups, relaxation to methacholine alone was not different. We speculate that the enhanced PGHS-dependent constriction may be compensated for by a concomitant increase in an undetermined vasodilator pathway. Indeed, there could be an enhancement of vasodilation due to other downstream effects on arachidonate metabolism. Since our in vitro protocol involved inhibiting PGHS activity, the available arachidonic acid could be shunted down an alternative pathway, such as lipoxygenase or cytochrome P450. Indeed, recent work in rat mesenteric and bovine coronary arteries revealed arachidonic acid-induced vasodilation dependent upon downstream lipoxygenase metabolites (e.g. 12-HETE) [31], or epoxygenase products (e.g. 14,15-EET) [32], respectively. The latter (EETs) are potential candidates for the endothelium-derived hyperpolarizing factor (EDHF). However, diminished EDHF-mediated arterial relaxation has been observed in aging [33] and ovariectomized rats [34]. Hence, arachidonate metabolism may favour production of the HETEs, although these can also be dependent upon PGHS activity for their formation. Indeed, 20-HETE, one vasoconstrictor eicosanoid implicated in hypertension, could be produced dependent upon the PGHS pathway [35] or be formed independently of PGHS-2 across different arterial beds [36]. A role for this type of eicosanoid metabolite would need to be verified in our aging model. It remains to be determined whether or not these mechanisms occur in aging humans.

We speculate that the increased levels of the PGHS-2 enzyme after 4 weeks of chronic PGHS-2 inhibition is due to a reduction in the degradation of existing protein rather than through de novo synthesis. This is due to the fact that PGHS can undergo suicide inactivation after a set number of cycles [37]. Since NS-398 specifically inhibits PGHS-2 activity this could prevent the suicide inactivation of this isoform.

Recently, two randomized trials named the Celecoxib Long-term Arthritis Safety Study (CLASS) [11] and the Vioxx Gastrointestinal Outcomes Research (VIGOR) Study [12] were performed on large populations (>8000) to assess the efficacy of the PGHS-2 inhibitors, celecoxib and rofecoxib (Vioxx), on gastrointestinal and other outcomes. For cardiovascular outcomes, the CLASS study showed no significant change in cardiovascular events between celecoxib and the traditional NSAIDs [38]. Interestingly, a study of rats revealed that celecoxib treatment led to increases in blood pressure [39]. Moreover, the VIGOR study demonstrated a significant increase in the development of hypertension in patients taking rofecoxib compared with the non-selective PGHS inhibitor, naproxen [38]. By contrast, a study of forearm blood flow response to acetylcholine in young, healthy humans given either rofecoxib or naproxen for 1 week revealed no significant changes in endothelial-dependent vasodilation [40]. Indeed, our data indicated no change in vasodilation to methacholine with chronic PGHS-2 inhibition. However, we demonstrated a paradoxical increase in PGHS-dependent vasoconstriction with prolonged PGHS-2 inhibition.

In summary, our study revealed direct vascular effects after chronic inhibition of PGHS-2 with a selective inhibitor. Importantly, these findings provide a possible mechanism for the adverse vascular function. Moreover, the increased expression of PGHS-2 in the resistance vasculature may be a mechanism for the hypertension observed in the VIGOR study [12]. That is, chronic inhibition of PGHS-2 activity appears to have a detrimental feedback effect on vascular function leading to greater PGHS-2 expression and enhanced PGHS-dependent constriction. Further studies to understand the feedback effects of aspirin and PGHS-2 inhibitors will be important to understand the long-term effects of these widely used drugs. We speculate that the aging population will be contraindicated for selective PGHS-2 inhibition. Moreover, since chronic PGHS-2 inhibition is currently used in cancer therapy and for treatment of inflammatory conditions, caution needs to be observed regarding the vascular effects. Hence, inhibitors of PGHS-2 activity may not be the panacea for reducing vascular complications in conditions where PGHS-dependent constriction predominates. Alternative approaches to reduce expression of PGHS-2, whether directly or indirectly, are necessary, and could be achieved by targeting other initiating factors.


   Acknowledgements
 
The Canadian Institute for Health Research (CIHR) supported this study. S.T. Davidge is a Canada Research Chair in Women's Cardiovascular Health and an Alberta Heritage Foundation for Medical Research (AHFMR) Senior Scholar. S.J. Armstrong is a recipient of a Studentship from CIHR/Canadian Hypertension Society/Pfizer. We would like to thank Neelam Kainth, Kenman Gan and Jennifer Au for their technical assistance.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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