Endotheliumderived Mediators and HPV

Although there is strong evidence that the pulmonary vascular endothelium is important, possibly crucial, for development of sustained HPV, and that its action appears to be via an increase in vascular smooth muscle Ca2+ sensitivity mediated via RhoA/Rho kinase, the mediator responsible has not been identified (Fig. 2D). Moreover, the pulmonary endothelium also has a powerful modulatory influence on pulmonary vasomotor tone and vascular resistance via the action of numerous other mediators, and this may be modified during hypoxia. In the following section we briefly review the influence of key endothelium-derived mediators on HPV (Fig. 3), and discuss the possible identity of the putative mediator underlying the endothelium-dependent component of sustained HPV.

Figure 2: Schematic of potential pathways underlying sustained, endothelium-dependent HPV. A: Hypoxia causes an increase in VSM [Ca2+]j via mechanisms independent of the endothelium; these are discussed elsewhere in this volume. B: Ca2+ sensitivity is regulated by the balance between phosphorylation of MLC by MLCK and its dephosphorylation by SMPP-1M; ROK inhibits SMPP via its regulatory subunit, thus enhancing MLC phosphorylation. PKC also inhibits SMPP-1M, but this effect is dominated by the Rho/Rho kinase pathway. C: ET-1 causes activation of PKC which, in turn, suppresses opening of voltage gated and other K+ channels and causes depolarization; PKC also activates Ca2+ channels. Note that other priming agents also activate PKC and/or cause depolarization (see text). D: During sustained HPV an unidentified endothelium-derived mediator ("X") causes RhoA and ROK translocation to the membrane where ROK activates, leading to Ca2+ sensitization. PLC: phospholipase C; MLCK: myosin light chain kinase; ROK: Rho kinase; SMPP-1M: myosin phosphatase.

Figure 2: Schematic of potential pathways underlying sustained, endothelium-dependent HPV. A: Hypoxia causes an increase in VSM [Ca2+]j via mechanisms independent of the endothelium; these are discussed elsewhere in this volume. B: Ca2+ sensitivity is regulated by the balance between phosphorylation of MLC by MLCK and its dephosphorylation by SMPP-1M; ROK inhibits SMPP via its regulatory subunit, thus enhancing MLC phosphorylation. PKC also inhibits SMPP-1M, but this effect is dominated by the Rho/Rho kinase pathway. C: ET-1 causes activation of PKC which, in turn, suppresses opening of voltage gated and other K+ channels and causes depolarization; PKC also activates Ca2+ channels. Note that other priming agents also activate PKC and/or cause depolarization (see text). D: During sustained HPV an unidentified endothelium-derived mediator ("X") causes RhoA and ROK translocation to the membrane where ROK activates, leading to Ca2+ sensitization. PLC: phospholipase C; MLCK: myosin light chain kinase; ROK: Rho kinase; SMPP-1M: myosin phosphatase.

5.1. Cyclooxygenase Products

The cyclooxygenase pathway synthesizes a wide range of vasoactive products, with both vasoconstrictor and vasodilator properties. Numerous studies have failed to show that any vasoconstrictor prostanoid is involved in HPV, although prostacyclin may act as a physiological brake (1). It has been proposed that inhibition of prostacyclin production during hypoxia may form an important component of HPV (6), but inhibition of the cyclooxygenase pathway has little if any effect on HPV in isolated small arteries of rat or pig (21, 22).

Pgf2a Pathway

Figure 3: Pulmonary arterial endothelium-derived mediators affecting vascular tone. P450: Cytochrome P-450 monooxygenase; NOS: NO synthase; COX; cyclooxygenase; ECE; endothelin-converting enzyme; LO: lipoxygenase; LTC4/D4: cysteinyl leukotrienes C4 and D4; ROK: Rho kinase. Note a: P450 mono-oxygenase is expressed in both endothelium and VSM. Note b: Vasoconstrictor prostanoids are also produced by other cell types, and are increased in inflammation; thromboxane and PGF2a have been implicated in the pulmonary vasculature. Note c: ET-1 also acts via ETB receptors on the endothelium to activate NOS and COX; atypical ETB receptors may promote constriction in VSM. The evidence that ET-1 production is increased in acute hypoxia is open to question, and there is strong evidence that it is not. Note d: Leukotriene production by the endothelium may only be significant in inflammation.

Figure 3: Pulmonary arterial endothelium-derived mediators affecting vascular tone. P450: Cytochrome P-450 monooxygenase; NOS: NO synthase; COX; cyclooxygenase; ECE; endothelin-converting enzyme; LO: lipoxygenase; LTC4/D4: cysteinyl leukotrienes C4 and D4; ROK: Rho kinase. Note a: P450 mono-oxygenase is expressed in both endothelium and VSM. Note b: Vasoconstrictor prostanoids are also produced by other cell types, and are increased in inflammation; thromboxane and PGF2a have been implicated in the pulmonary vasculature. Note c: ET-1 also acts via ETB receptors on the endothelium to activate NOS and COX; atypical ETB receptors may promote constriction in VSM. The evidence that ET-1 production is increased in acute hypoxia is open to question, and there is strong evidence that it is not. Note d: Leukotriene production by the endothelium may only be significant in inflammation.

5.2. Lipoxygenase Products

The lipoxygenase pathway synthesizes a variety of inflammatory mediators from arachidonic acid, the most important in this context being the cysteinyl leukotrienes. These are powerful vasoconstrictors, and there is also evidence that they activate RhoA/Rho kinase (31). However, several studies have dismissed them as playing any role in HPV whatsoever (36, 41).

5.3. Cytochrome P-450 Products

The cytochrome P-450 mono-oxygenase system consists of a family of enzymes that are expressed in both pulmonary vascular smooth muslce and endothelium, and synthesize hydroxyeicosatetraenoic acids (HETEs) and cis-epoxyeicosatrienoic acids (EETs) (14). Both HETEs and EETs are normally regarded as pulmonary vasodilators (14), and there is little evidence for any major role for cytochrome P-450 products in HPV (42). Nevertheless, as hypoxia inhibits the production of 20-HETE it has been proposed that this effect could provide an important contribution to HPV by relieving a vasodilator influence (14).

5.4. Nitric Oxide

Endothelium-derived nitric oxide is an important modulator of pulmonary vascular tone and resistance. There is little if any evidence that it is involved in the mechanisms of HPV per se, but changes in its synthesis during hypoxia undoubtedly have a major modulatory influence in the intact animal and perfused lung. Inhibition of nitric oxide synthase (NOS) is almost universally found to potentiate HPV in such preparations, even though it may have little effect on pulmonary vascular resistance during normoxia (1). Results derived from isolated arteries are less consistent, but often there is little or no effect of NOS inhibition on HPV except when hypoxia is severe (1). The overall consensus is that production of NO by the lung decreases in moderate hypoxia (19), as might be expected because 02is a substrate for NOS. Nevertheless, it has been proposed that nitric oxide may act as a physiological brake on HPV (19), possibly at the level of the small pulmonary arteries and as a result of increased shear stress due to hypoxia-induced narrowing of these arteries (1).

5.5. Endothelin 1

ET-1 has been mooted as either the primary mediator, or an important priming factor, in HPV. The actions of this 21-amino acid polypeptide on the pulmonary vasculature are extraordinarily complex and context-dependent. It is generally held that ET-1 acts mainly via ETA receptors on the pulmonary VSM to cause constriction, and via receptors on the endothelium to release nitric oxide and prostacyclin to cause relaxation (Fig. 3), with the latter effect predominating if ET-1 is applied in the presence of pre-existing tone (5). However this picture is complicated by the presence, at least in some species, of atypical ETB receptors which mediate contraction (23), and by the reported ability of ET-1 to release vasoconstricting prostanoids (4).

The role of ET-1 in acute HPV remains controversial, since the substantial literature which supports its involvement is balanced by an equally persuasive body of work which denies it. These reports can be summarized by noting that selective ETA or mixed ETA/ETB blockers inhibit HPV in some studies, but not in others of apparently similar quality (1). Recently, however, several papers have appeared which go some way towards resolving this debate.

Sato et al. (30) noted that studies supporting a role for ET-1 in HPV have mainly been carried out in intact animals, while those showing a lack of effect have typically been conducted in perfused lungs. They therefore examined the effects of ET-1 antagonists on HPV in conscious catheterized rats, as well as in isolated blood-perfused and salt-perfused rat lungs. In each case, blockade ofthe ETa receptor greatly attenuated HPV. HPV was, however, restored in salt perfused lungs exposed to combined ETA/ETB blockade when hypoxia was applied in the presence of angiotensin II, and also when the lungs had been pretreated with the KATP channel antagonist glibenclamide. Based on the results of this elegant study, they proposed that ET-1 acts on pulmonary VSM to tonically inhibit KATP channels, thereby causing a small degree of ongoing depolarization which allows other processes stimulated by hypoxia to cause constriction (Fig. 2C). Angiotensin II could also play a similar role in 'enabling' HPV, thus explaining the observation of many investigators that HPV is greatly enhanced in isolated lung preparations if they are first primed with this drug (and see section 3 above). The work of Sato et al. (30) suggests, however, that ET-1 may be a more physiologically relevant priming agent.

A similar proposal was advanced by Sham et al. (32), who found that the very small contraction and rise in [Ca2+]j induced by hypoxia in VSM cells isolated from distal porcine pulmonary arteries was greatly increased if cells were primed with a low concentration (0.1 pM) of ET-1, which itself had no effect. In a subsequent study of HPV in porcine small pulmonary arteries using a perfusion myograph (22), they observed that HPV was entirely abolished by removal of the endothelium. This effect was mimicked by the ETA antagonist BQ-123, whereas blockade of cyclooxygenase with indomethacin had no effect, and inhibition of eNOS with L-NAME greatly enhanced HPV. Application of ET-1 (0.1 pM) to endothelium-denuded arteries was then able to restore the response to hypoxia. Again, ET-1 was suggested to be acting as a priming agent rather than specific mediator of HPV.

Johnson et al. (15) have very recently examined the effect of BQ-123 on pulmonary hemodynamics in non-sedated young human volunteers under normoxic and moderately hypoxic conditions (arterial Po2 = 100 and 49 mmHg, respectively). BQ-123 caused a significant fall in pulmonary vascular resistance (PVR) under normoxic conditions, but did not cause any inhibition of the percentage increase in PVR elicited by hypoxia. Although these results support the concept that ET-1 supports a degree of basal tone in the human pulmonary vasculature, they argue against the proposal that this ET-1 mediated tone is an obligatory priming factor for HPV, at least in healthy young individuals. They do not, however, rule out the possibility that some degree of pretone contributed by other factors (including ET-1 acting via ETB receptors) is required for the full expression of the hypoxic response in humans, or that ET-1 may play more of a role in conditions such as heart failure or primary pulmonary hypertension, in which its plasma levels and/or receptor expression may be elevated.

Possible subtleties which may complicate the role of ET-1 in HPV are evident in the report of Ambalavanan et al. (2), who studied HPV in chronically instrumented and sedated 3-4 week old piglets. They found that the ETA receptor blockers EMD 122946 and BQ 610 partially suppressed the moderate hypoxia-elicited rise in PVR. The eNOS inhibitor L-NAME, however, caused a large increase in PVR, after which hypoxia had no further effect. The rise in PVR caused by L-NAME was not reversed by EMD 122946. Based on their results, the authors proposed that HPV in their model was being caused by a fall in NO release. They suggested that the partial reversal of HPV by EMD 122946 and BQ 610 was occurring because blockade of ETA receptors was unmasking or enhancing an ETB-mediated release of NO, rather than by preventing a direct constriction. One implication of this intriguing hypothesis, which is that combined ETA/ETB receptor antagonists should not be useful in preventing HPV, is however not supported by several other studies, in which non-selective antagonists ET-1 antagonists were effective in suppressing HPV (13).

On the face of it, these reports suggest that ET-1 is not the unique stimulus of HPV, at least in humans, rats, and piglets. Instead, ET-1 may be one of several priming factors which potentiate HPV non-specifically (see section 3) (39), depending on the extent to which it is contributing to basal pulmonary tone or excitation in a particular species and under the specific conditions obtaining at the time of study. It is clear, however, that the effects of ET-1 on the pulmonary vasculature are complex, and it is not unlikely that future studies will fuel rather than resolve the ongoing debate concerning its role in HPV.

5.6. Other Potential Mediators of Endothelium-dependent HPV

We originally suggested that the endothelium released a substance during sustained HPV that effectively altered the Ca2+-tension relationship of the underlying VSM in 1995 (26). Since that time there has been a significant amount of work to support this hypothesis. So far however, no known endothelium-derived mediator has been shown to play an indispensable role in sustained HPV, and the identity of the mediator responsible for endothelium-dependent Ca2+ sensitization of the VSM during hypoxia remains unknown. Although ET-1 is the candidate of choice for many, and may indeed play an important role as an endogenous facilitator or priming agent for HPV (see above), blockade of all endothelin receptors does not even slightly diminish HPV

(7, 8, 10, 18). Therefore, there is clear evidence that the endothelium must release another vasoconstrictor mediator that is distinct from ET-1, and which is not a product of the cyclooxygenase or lipoxygenase pathways.

An important study in support of this hypothesis was published by Gaine et al. (10), who showed that removal of the endothelium from pig pulmonary artery rings abolished the slowly developing and sustained component of HPV, but that this component could be fully restored in the presence of pulmonary valve leaflets as a source of pulmonary endothelial cells. Neither indomethacin nor the ETA antagonist BQ123 had any effect on either the initial sustained component of HPV before endothelium removal, nor that after restoration in the presence of the valve leaflets (10). This strongly suggests the action of an endothelium-derived factor that is not ET-1 or a cyclooxygenase product.

Further evidence for a specific mediator of sustained HPV and the associated Ca2+-sensitization comes from our recent study on the properties of a factor derived from the effluent of hypoxic-perfused rat lungs (29). Lungs were perfused under normoxic and hypoxic conditions, and the factor extracted and partially purified. There are several distinct similarities between the effects of this factor and the sustained, endothelium-dependent component of HPV. The factor induced a slowly developing vasoconstriction in pulmonary arteries, which was insensitive to L-type channel blockade and not associated with a rise in [Ca2+]i; it also significantly potentiated the action of another vasoconstrictor. Both of these results imply that it induces an increase in Ca2+ sensitivity in the VSM. Importantly, the vasoconstriction induced by the factor was completely unaffected by either indomethacin or combined ETA/ETB blockade, strongly suggesting that it is neither a cyclooxygenase product nor ET-1. An interesting observation was that the factor has very little effect on mesenteric arteries, and that when similar experiments were performed with effluent from hypoxic perfused mesenteric beds no equivalent vasoconstrictor activity could be discovered (29). This raises the possibility that this factor not only has a specific action in the pulmonary circulation, but also is specifically produced there.

Evans and Dipp (8) have recently reported the results of a similar study using the effluent from perfused lungs, where they additionally showed that the vasoconstriction induced in endothelium-denuded pulmonary arteries by effluent from hypoxic lungs was suppressed by the Rho kinase inhibitor Y27632. Moreover, the Y27632 concentration-inhibition curve for this effect was almost identical to those observed for both sustained HPV in endothelium-intact pulmonary arteries and the hypoxic pressor response in perfused lungs; it is particularly noteworthy that Y27632 was considerably less effective in inhibiting the vasoconstrictor response to ET-1 (8). Taken together, these two reports strongly suggest that during hypoxia the lungs produce a mediator distinct from ET-1 which has very similar properties to those that would be predicted for the proposed endothelium-derived mediator of sustained HPV, in that both apparently act via a Rho kinase-mediated pathway to enhance the Ca2+ sensitivity of the VSM (Fig. 2D). It remains to be seen however whether the factor derived from the effluent of hypoxic perfused lungs is indeed produced by the endothelium. Moreover, attempts to identify the factor have proved to be technically very difficult.

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  • marroc
    Do vasoconstrictors inhibit HPV or potentiate HPV?
    6 years ago

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