PAF antagonists inhibit pulmonary vascular remodeling induced by hypobaric hypoxia in rats.

Chronic hypoxia causes pulmonary hypertension and pulmonary vascular remodeling in rats. Because platelet-activating factor (PAF) levels increase in lung lavage fluid and in plasma from chronically hypoxic rats, we examined the effect of two specific, structurally unrelated PAF antagonists, WEB 2170 and BN 50739, on hypoxia-induced pulmonary vascular remodeling. Treatment with either agent reduced hypoxia-induced pulmonary hypertension and right ventricular hypertrophy at 3 wk of hypoxic exposure (simulated altitude 5,100 m) but did not affect cobalt (CoCl2)-induced pulmonary hypertension. The PAF antagonists had no effect on the hematocrit of normoxic or chronically hypoxic rats or CoCl2-treated rats. Hypoxia-induced pulmonary hypertension was associated with an increase in the vessel wall thickness of the muscular arteries and reduction in the number of peripheral arterioles. In WEB 2170-treated rats, these changes were significantly less severe than those observed in untreated chronically hypoxic rats. PAF receptor blockade had no acute hemodynamic effects; i.e., it did not affect pulmonary arterial pressure or cardiac output nor did it affect the magnitude of acute hypoxic pulmonary vasoconstriction in awake normoxic or chronically hypoxic rats. Isolated lungs from chronically hypoxic rats showed a pressor response to the chemotactic tripeptide N-formyl-Met-Leu-Phe (fMLP) and an increase in the number of leukocytes lavaged from the pulmonary circulation. In vivo treatment with WEB 2170 significantly reduced the fMLP-induced pressor response compared with that observed in isolated lungs from untreated chronically hypoxic rats. These results suggest that PAF contributes to the development of chronic pulmonary hypertension induced by chronic hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary vascular reactivity in Fischer rats

We previously reported that Fischer (F) rat lungs developed more extensive injury when challenged with oxidants than age-matched Sprague-Dawley (SD) rat lungs. We now describe a reduced pulmonary vascular response to alveolar hypoxia and angiotensin II (ANG II) in F compared with SD rats. The comparative studies were performed with isolated lungs perfused with salt solution or blood, catheter-implanted awake rats, and isolated main pulmonary arterial rings. Isolated lungs from F rats perfused with either blood or salt solution had reduced vasoconstriction in comparison with lungs from SD rats when exposed to alveolar hypoxia or challenged with ANG II. Instrumented awake F rats had a smaller mean increase in total pulmonary vascular resistance (PVR) than SD rats (35 vs. 94 mmHg.min.l-1, P less than 0.05) when challenged with 8% oxygen. The contractile response of isolated pulmonary artery but not thoracic aortic rings to KCl and ANG II was reduced in F compared with SD rats. In addition, F rats exposed to 4 wk of hypobaric hypoxia developed less pulmonary hypertension and right ventricular hypertrophy (when corrected for the hematocrit) than SD rats. We conclude that the oxidant stress-sensitive inbred F rat strain is characterized by a lung vascular bed that is relatively unresponsive to vasoconstricting stimuli. The mechanism underlying this genetic difference in lung vascular control remains to be defined.     

Enantiodifferentiation of dihydropyridine PAF antagonists

The PAF antagonist activity of a series of enantiomeric dihydropyridines is described. In the first example, 1, the PAF antagonist activity and calcium channel blocking activity reside in opposite enantiomers. Subsequent examples also display enantioselectivity and the SAR of the series is described.     

Pulmonary vascular reactivity and hemodynamic changes in elastase-induced emphysema in hamsters.

Changes in pulmonary hemodynamics and vascular reactivity in emphysematous hamsters were studied in an isolated lung preparation perfused at constant flow with blood and 3% dextran. Hamsters were treated with intratracheal porcine pancreatic elastase at 70 days of age, and experimental studies were conducted at 1, 3, and 8 mo after treatment. Baseline pulmonary arterial pressure in elastase-treated lungs was increased compared with saline-treated control lungs 1 mo after treatment, but this increase did not progress at 3 and 8 mo. Increases in pulmonary arterial pressure in elastase-treated lungs were temporally correlated with the morphological development of emphysema and right ventricular hypertrophy; both of these were evident at 1 mo after treatment and showed little change thereafter. Pressor responses to hypoxia and angiotensin II were not different between elastase-treated and control lungs at 1 and 3 mo. At 8 mo, however, pressor responses in emphysematous lungs to 0% O2 (but not to angiotensin II) were significantly increased. This was the result of a lack of the normal age-related fall in the hypoxic pressor response. Our results suggest that the right ventricular hypertrophy found in these emphysematous animals results from a chronically increased pulmonary vascular resistance. Furthermore, increases in pulmonary vascular resistance in the early development of emphysema are likely a result of the loss of vascular beds and supporting connective tissue.     

PAF antagonists inhibit monocrotaline-induced lung injury and pulmonary hypertension.

Lung platelet-activating factor (PAF) levels increased in some rats at 1-3 wk after subcutaneous injection of monocrotaline (MCT). We tested the effect of specific PAF antagonists, WEB 2086 and WEB 2170, on MCT-induced lung injury and subsequent pulmonary hypertension and right ventricular hypertrophy. Treatment with either agent decreased MCT-induced pulmonary hypertension and right ventricular hypertrophy at 3 wk after injection. Treatment with WEB 2170 reduced MCT-induced pulmonary vascular leak at 1 wk after injection, and WEB 2086-treatment exclusively during the early leak phase also decreased MCT-induced right ventricular hypertrophy at 3 wk. Treatment with WEB 2170 between the 3rd and 4th wk after MCT injection inhibited the progression of right ventricular hypertrophy at 4 wk. These results suggest that PAF contributes to the early pulmonary vascular leak, and this leak phase is important for the development of pulmonary hypertension and right ventricular hypertrophy in MCT-treated rats. Furthermore, it appears that PAF action contributes to the maintenance of a chronic inflammatory process that involves the synthesis of other lipid mediators (prostaglandins and leukotrienes) and leads to pulmonary hypertension. We conclude that PAF has a role in the MCT-induced inflammatory lung injury and pulmonary hypertension.     

Synthesis of bicyclic thiazolidine PAF antagonists

PAF antagonist 1 is susceptible to thiazolidine ring fragmentation in vitro and in vivo. The search for a more stable compound prompted the synthesis of a series of bicyclic analogs. Three classes of bicyclic thiazolidines (2: X = 0, CH₂, NCH₃) were prepared using a common synthetic pathway which generated all the possible diastereomers. The most potent PAF antagonists were the oxygen-substituted analogs which possessed receptor binding affinities largely dependent on stereochemistry.     

Platelet-activating factor antagonists increase vascular reactivity in perfused rat lungs

Platelet-activating factor (PAF) administered to the pulmonary circulation in low dose (nanogram) has vasodilatory properties. Therefore, we investigated whether endogenous PAF plays a role in the control of tone in the pulmonary circulation. The PAF receptor antagonists, SRI 63-441 (2.6 X 10(-4) M) and L659,989 (1 X 10(-5) M), were the major investigative tools. In isolated perfused rat lungs, both agents caused a persistent increase in base-line perfusion pressure (Ppa), potentiated angiotensin II (ANG II) vasoconstriction, and potentiated hypoxic vasoconstriction (HPV). This potentiation of ANG II and HPV was found to be independent of circulating blood elements. Vasodilation in the presence of PAF blockade was also impaired. The combination of cyclooxygenase inhibition and PAF receptor blockade had an additive effect on ANG II vasoconstriction but did not cause more potentiation of HPV than achieved with PAF antagonism alone. In vivo, SRI 63-441 (10 mg/kg) caused only a transient increase in base-line Ppa without altering ANG II and hypoxic vasoconstriction. These findings support a vasodilatory role for endogenous PAF in the pulmonary circulation.     

PAF increases vascular permeability in selected tissues: effect of BN-52021 and L-655,240

The effect of the potent inflammatory mediator, platelet activating factor (PAF) was studied on the vascular permeability of selected rat tissues using the extravasation of Evans blue dye (EB) as a marker. EB (20 mg/kg) was injected in the caudal vein together with increasing doses of PAF (0.1, 1.0 and 5.0 micrograms/kg). The animals were killed and the dye was extracted in selected organs using formamide (4 ml/g wet weight tissues) and the content was expressed as EB micrograms/g dry weight. Extravasation of EB varied markedly from one tissue to another and increased as a function of time (from 0 to 60 min). PAF (5.0 micrograms/kg) increased the pancreas and duodenum vascular permeability by 15 and 5 fold respectively. At the doses of 0.1 and 1.0 microgram/kg, PAF induced a slight increase (P less than 0.01) of the vascular permeability of the heart 5 min after the injection. The PAF antagonist BN-52021 (2 and 10 mg/kg) produced a dose-dependent inhibition of the PAF effects on the pancreas, heart and duodenum. Maximum inhibition (approximately 100%) was achieved at the dose of 10 mg/kg. This antagonist given in the absence or the presence of PAF reduced the lung plasma extravasation below control levels. A thromboxane antagonist, L-655,240 (1.0 and 5.0 mg/kg) also inhibited PAF-induced increases in vascular permeability in heart, duodenum and pancreas. It also reduced below control levels the EB extravasation in kidneys, spleen and lungs. Maximum inhibition (50% for the duodenum, and 40% for the pancreas) was achieved at the dose of 5.0 mg/kg.     

Pulmonary hypertension and pulmonary vascular reactivity in beagles at high altitude

It is unclear whether dogs develop pulmonary hypertension (PH) at high altitude. Beagles from sea level were exposed to an altitude of 3,100 m (PB 525 Torr) for 12-19 mo and compared with age-matched controls remaining at low altitude of 130 m (PB 750 Torr). In beagles taken to high altitude as adults, pulmonary arterial pressures (PAP) at 3,100 m were 21.6 +/- 2.6 vs. 13.2 +/- 1.2 Torr in controls. Likewise, in beagles taken to 3,100 m as puppies 2.5 mo old, PAP was 23.2 +/- 2.1 vs. 13.8 +/- 0.4 Torr in controls. This PH reflected a doubling of pulmonary vascular resistance and showed no progression with time at altitude. Pulmonary vascular reactivity to acute hypoxia was also enhanced at 3,100 m. Inhibition of prostaglandin synthesis did not attenuate the PH or the enhanced reactivity. Once established, the PH was only partially reversed by acute relief of chronic hypoxia, but reversal was virtually complete after return to low altitude. Hence, beagles do develop PH at 3,100 m of a severity comparable to that observed in humans at the same or even higher altitudes.     

Pulmonary vascular reactivity and permeability to alveolar hypoxia in the dog

Hemodynamics and vascular permeability were studied during acute alveolar hypoxia in isolated canine lung lobes perfused at constant flow with autogenous blood. Hypoxia was induced in the presence (COI + Hypox, n = 6) or absence (Hypox, n = 6) of cyclooxygenase inhibition (COI) with indomethacin or meclofenamate. Hypoxic ventilation reduced blood PO2 from 143 to 25-29 Torr without a change in PCO2. During hypoxia a capillary filtration coefficient (Kf) was obtained gravimetrically as an index of vascular permeability to water. In COI + Hypox, pulmonary arterial pressure (Pa) increased from 11.5 +/- 0.7, post-COI normoxia, to a peak of 22.1 +/- 2.3 during hypoxia (P less than 0.01) without a change in capillary pressure (Pc). In contrast, hypoxia changed neither Pa nor Pc in Hypox relative to an untreated normoxic control group (Normox, n = 6, P greater than 0.05). Kfs (means +/- SE in ml.min-1.Torr-1.100 g-1) for Normox (0.070 +/- 0.014), Hypox (0.082 +/- 0.024), and COI + Hypox (0.057 +/- 0.017) did not differ from one another (P greater than 0.05). Although COI markedly enhanced the pressor response to acute alveolar hypoxia, hypoxia increased neither Pc nor vascular permeability regardless of COI.     

Endothelium independent protective effect of NG-monomethyl-L-arginine on endotoxin-induced alterations of vascular reactivity.

The effects of NG-monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide (NO) synthesis was tested on the endotoxin-induced alterations of alpha-adrenoceptor function. In isolated aorta, there was no significant difference in the tension induced by phenylephrine (PE, 10 microM) on rings removed from control and endotoxin injected rats (10 mg/kg, ip). However, a lack of tonicity of the contraction was observed in rings of shocked rats (8 +/- 2.9 and 86 +/- 4.6% relaxation at 105 min for sham and shocked rings respectively). The gradual tension decrease to PE was more potent in rings possessing endothelial cells. However, in both preparations, the loss of tonicity was significantly inhibited by NMMA (30 microM). In endothelium-free rings, L-arginine (100 microM) potentiated the loss of tonicity to PE and reversed the inhibitory effect of NMMA. NMMA, like methylene blue, was also able to restore the PE-contraction. The results indicate that the endotoxin-induced alterations of vascular reactivity may be due, in part, to NO formation from L-arginine independent of the endothelium.     

Regulation of platelet-activating factor (PAF) activity in human diseases by phospholipase A2 inhibitors, PAF acetylhydrolases, PAF receptor antagonists and free radical scavengers.

The aim of this review is to present recent findings indicating the likely involvement of platelet-activating factor (PAF) in human diseases, and possible ways of alleviating its harmful effects. PAF is a potent proinflammatory mediator and promotes adhesive interactions between leukocytes and endothelial cells, leading to transendothelial migration of leukocytes, by a process of juxtacrine intercellular signalling. This process leads to activation of leukocytes and the release of reactive oxygen radicals, lipid mediators, cytokines and enzymes. These reaction products subsequently contribute to the pathological features of various inflammatory diseases. The reactive oxygen radicals cause low density lipoprotein (LDL) oxidation which mediates the development of atherosclerosis. Oxidized LDL may damage cellular and subcellular membranes, leading to tissue injury and cell death. Among the therapeutic approaches considered are agents that inhibit/degrade proinflammatory mediators and thereby have anti-inflammatory and/or anti-atherogenic potential. These include inhibitors of phospholipase A2 activity, PAF-acetylhydrolases, PAF antagonists and free radical scavengers/antioxidants, the latter protecting against oxidized LDL-induced cytotoxicity.     

Platelet-activating factor (PAF) enhances tumor necrosis factor production by alveolar macrophages. Prevention by PAF receptor antagonists and lipoxygenase inhibitors

The effect of platelet-activating factor (PAF) on TNF production by rat alveolar macrophages (AM) and the role of endogenous leukotriene B4 (LTB4) in this regulation were examined. When AM were cultured with PAF alone, no change in TNF production was observed. However, the concomitant addition of PAF and muramyl dipeptide to AM cultures markedly enhanced (2- to 3-fold) TNF production in a concentration-dependent fashion with peak effect at 10(-10)M PAF. This enhancement occurred when muramyl dipeptide and PAF were present together at the initiation of the 24-h culture. Stimulation of TNF production by PAF was blocked by specific, but structurally different PAF receptor antagonists, BN 52021, CV3988 and WEB 2086. Additionally, the stereoisomer of PAF, [S]PAF, and the biologically inactive precursor/metabolite of PAF, lyso-PAF failed to induce significant enhancement in TNF production. In parallel, addition of PAF to AM triggered LTB4 release in a concentration-dependent manner. Inhibition of 5-lipoxygenase by nordihydro-guaiaretic acid or AA-861 blocked the PAF-induced augmentation of both TNF and LTB4 production. This was partially reversed by addition of exogenous LTB4. Collectively, these data suggest that PAF enhances TNF production by interaction with a specific putative receptor and by subsequent induction of endogenous 5-lipoxygenase activity in AM.     

Interactions of PAF and its antagonists in the guinea pig atrial myocardium

The effects of platelet activating factor (PAF) and three its antagonists on the transmembrane intracellular potentials and stimulated (0.5 Hz) contraction amplitude (CA) of the left auricle has been studied. PAF (1-5 X 10(-7) M) was added to the standard Tyrode solution or the same perfusing solution with 15 mM K+, and 6 mM Ca++ (t = 30 degrees C, pH = 7,2). PAF induced the straight cardio-depressing action: the CA always was suppressed during 20 min. The electrical activity was depressed in parallel; in the Tyrode the action potential (AP) duration was lowered, but in the atrial depolarized preparations PAF resulted in a decrease of the slow calcium potential (Ca-AP) amplitude from 100% to 20.2 +/- 2.0%. After 20 min PAF-acting the perfusing solutions contained also one of PAF-antagonists. Antagonist U-66985 led to the weakening of the PAF-depressing effects in the myocardium U-66985 is also able to increase electrical and mechanical activity in myocardium depressed by the blood serum from patients with virulent infections.