Role of endothelin-1 in lung disease.

Endothelin-1 (ET-1) is a 21 amino acid peptide with diverse biological activity that has been implicated in numerous diseases. ET-1 is a potent mitogen regulator of smooth muscle tone, and inflammatory mediator that may play a key role in diseases of the airways, pulmonary circulation, and inflammatory lung diseases, both acute and chronic. This review will focus on the biology of ET-1 and its role in lung disease.

Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension.

BACKGROUND: The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice. METHOD: Mice were exposed to sea level, ambient conditions (5280 feet) or severe altitude (17,000 feet) for 6 weeks from birth, and hemodynamics and lung NOS expression were assessed. RESULTS: Hypoxic mice developed severe pulmonary hypertension (right ventricular systolic pressure [RVsP] 60 mmHg) as compared with normoxic mice (27 mmHg). Using quantitative reverse-transcription PCR, it was found that expressions of eNOS and inducible NOS (iNOS) increased 1.5-fold and 3.5-fold, respectively, in the lung. In addition, the level of lung eNOS protein was increased, neuronal NOS (nNOS) protein was unchanged, and iNOS was below the limit of detection. Immunohistochemistry demonstrated no change in lung iNOS or nNOS staining in either central or peripheral areas, but suggested increased eNOS in the periphery following hypoxia. CONCLUSION: In mice, hypoxia is associated with increases in lung eNOS, possibly in iNOS, but not in nNOS; this suggests that the pattern of lung NOS expression following hypoxia must be considered in studies using genetically engineered mice.

p27(Kip1) is important in modulating pulmonary artery smooth muscle cell proliferation.

Vascular remodeling due to pulmonary arterial smooth muscle cell (PASMC) proliferation is central to the development of pulmonary hypertension. Cell proliferation requires the coordinated interaction of cyclins and cyclin-dependent kinases (cdk) to drive cells through the cell cycle. Cdk inhibitors can bind cyclin-cdk complexes and cause G(1) arrest. To determine the importance of the cdk inhibitor p27(Kip1) in PASMC proliferation we studied [(3)H]thymidine incorporation, changes in cell cycle, cell proliferation, and protein expression of p27(Kip1) following serum stimulation in early passage rat PASMC. p27(Kip1) expression decreased to 40% of baseline after serum stimulation, which was associated with an increase in both [(3)H]thymidine incorporation and the percent of cells in S phase. p27(Kip1) binding to cyclin E decreased at 24 h, and this correlated with an increase in phosphorylation of retinoblastoma both in vivo and in vitro. Overexpression of p27(Kip1) decreased [(3)H]thymidine incorporation and reduced cell counts at 5 d compared with controls. PASMC obtained from p27(Kip1-/-) mice showed a 2-fold increase in [(3)H]thymidine incorporation (at 24 h) and cell proliferation compared with p27(Kip1+/+) PASMC when cultured in 10% fetal bovine serum (FBS). These results suggest an important role for p27(Kip1) in regulating PASMC mitogenesis and proliferation.

Tuberous sclerosis gene products in proliferation control.

Two genes, TSC1 and TSC2, have been shown to be responsible for tuberous sclerosis (TSC). The detection of loss of heterozygosity of TSC1 or TSC2 in hamartomas, the growths characteristically occurring in TSC patients, suggested a tumor suppressor function for their gene products hamartin and tuberin. Studies analyzing ectopically modulated expression of TSC2 in human and rodent cells together with the finding that a homolog of TSC2 regulates the Drosophila cell cycle suggest that TSC is a disease of proliferation/cell cycle control. We discuss this question including very recent data obtained from analyzing mice expressing a modulated TSC2 transgene, and from studying the effects of deregulated TSC1 expression. Elucidation of the cellular functions of these proteins will form the basis of a better understanding of how mutations in these genes cause the disease and for the development of new therapeutic strategies.

Gene therapy for pulmonary diseases.

Gene therapy for pulmonary disease has attracted a great deal of attention since the first report of successful gene delivery 10 years ago. Potential indications for gene therapy include chronic illnesses such as cystic fibrosis and alpha(1)-antitrypsin deficiency, and acute illnesses such as acute transplant rejection and chemotherapy-induced lung injury. The key technological impediment to successful gene therapy is vector optimization. Viral vectors, including adenovirus and adeno-associated virus, have relatively low efficiency in vivo. In addition, adenovirus has been associated with a brisk inflammatory response and limited duration of expression in the lung. Nonviral vectors, particularly liposomes, have also been tried, with limited expression efficiency and some toxicity. Although work is ongoing to improve adenoviral and adeno-associated viral vectors and test other viral and nonviral vectors, an ideal vector has not yet been identified. Several important barriers to successful gene therapy, including the host inflammatory response, promotor down-regulation, tissue-specific targeting, and physical barriers to gene delivery in the airway, will need to be overcome. Despite these daunting problems, several human gene therapy trials have been completed, using adenovirus, adeno-associated virus, and liposomes. In general, these trials have been focused on safety, and have shown that there is dose-dependent inflammation in response to adenovirus. Adeno-associated virus appears to cause little inflammation. Demonstration of successful gene delivery and transcription has been quite variable in human trials. In general, the level of expression of transgene appears to be quite low. In summary, although there is great promise for gene therapy in the lung, significant challenges remain in translating this technology to successful human therapy.

Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry.

Endothelial nitric-oxide synthase (eNOS), a Ca(2+)/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through its N-myristoylation, the significance of membrane association in locating eNOS near sources of Ca(2+) entry is uncertain. To assess the Ca(2+) source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca(2+) sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca(2+) concentration, demonstrating preferential sensitivity to Ca(2+) originating from intracellular pools (MHA) or from capacitative Ca(2+) entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca(2+) influx via capacitative Ca(2+) entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca(2+) release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca(2+)](i) due to the Ca(2+) ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca(2+) influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.

Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice.

The pulmonary endothelin (ET) system has been implicated in the pathogenesis of chronic lung diseases such as pulmonary hypertension, asthma, chronic obstructive lung disease, idiopathic pulmonary fibrosis, and bronchiolitis obliterans. However, the etiologic role of ET-1 in these diseases has not yet been established. We recently demonstrated that ET-1 transgenic mice, generated using the human prepro-ET-1 expression cassette including the cis-acting transcriptional regulatory elements, had predominant transgene expression in lung, brain, and kidney. We used these mice in the present study to analyze the pathophysiologic consequences of long-term pulmonary overexpression of ET-1. We found that ET-1 overexpression in the lungs did not result in significant pulmonary hypertension, but did result in development of a progressive pulmonary fibrosis and recruitment of inflammatory cells (predominantly CD4-positive cells). Our study provides evidence that a long-term activated pulmonary ET system, without any other stimuli, produces chronic lymphocytic inflammation and lung fibrosis. This suggests that overexpression of ET-1 may be a central event in the pathogenesis of lung diseases associated with fibrosis and chronic inflammation, such as pulmonary fibrosis and bronchiolitis.

Mechanism of hypoxic pulmonary vasoconstriction involves ET(A) receptor-mediated inhibition of K(ATP) channel.

There is controversy on the role of endothelin (ET)-1 in the mechanism of hypoxic pulmonary vasoconstriction (HPV). Although HPV is inhibited by ET-1 subtype A (ET(A))-receptor antagonists in animals, it has been reported that ET(A)-receptor blockade does not affect HPV in isolated lungs. Thus we reassessed the role of ET-1 in HPV in both rats and isolated blood- and physiological salt solution (PSS)-perfused rat lungs. In rats, the ET(A)-receptor antagonist BQ-123 and the nonselective ET(A)- and ET(B)-receptor antagonist PD-145065, but not the ET(B)-receptor antagonist BQ-788, inhibited HPV. Similarly, BQ-123, but not BQ-788, attenuated HPV in blood-perfused lungs. In PSS-perfused lungs, either BQ-123, BQ-788, or the combination of both attenuated HPV equally. Inhibition of HPV by combined BQ-123 and BQ-788 in PSS-perfused lungs was prevented by costimulation with angiotensin II. The ATP-sensitive K(+) (K(ATP))-channel blocker glibenclamide also prevented inhibition of HPV by BQ-123 in both lungs and rats. These results suggest that ET-1 contributes to HPV in both isolated lungs and intact animals through ET(A) receptor-mediated suppression of K(ATP)-channel activity.

Nitric oxide synthase in pulmonary hypertension: lessons from knockout mice.

Nitric oxide (NO) is implicated in a wide variety of biological roles. NO is generated from three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) all of which are found in the lung. While there are no isoform-specific inhibitors of NOS, the recent development and characterization of mice deficient in each of the NOS isoforms has allowed for more comprehensive study of the importance of NO in the lung circulation. Studies in the mouse have identified the role of NO from eNOS in modulating pulmonary vascular tone and in attenuating the development of chronic hypoxic pulmonary hypertension.

Vascular inflammation inhibits gene transfer to the pulmonary circulation in vivo.

We report gene transfer to the normal and injured murine pulmonary circulation via systemic (intravascular) and airway (intratracheal) delivery of novel polycationic liposomes (imidazolium chloride, imidazolinium chloride-cholesterol, and ethyl phosphocholine). With use of the reporter genes chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP), intravascular injection of lipid-DNA complexes resulted in gene expression primarily in the lung, with lesser expression in the heart (11% of lung, P < 0.05) and spleen (8% of lung, P < 0.05). Histochemical staining for the hpAP reporter gene showed localized transgene expression in the microvascular endothelium. Monocrotaline (80 mg/kg body wt sc) treatment produced endovascular inflammation and reduced lung CAT activity (2 days postintravascular transfection) by 75 +/- 8 and 86 +/- 6% at 7 and 21 days, respectively, after monocrotaline (P < 0. 05). Despite the apparent decrease in functional CAT protein, Southern blot analysis suggested maintained plasmid delivery, whereas quantitative PCR (TaqMan) showed decreased CAT mRNA levels in monocrotaline mice. In contrast, intratracheal delivery of lipid-DNA complexes showed enhanced CAT expression in monocrotaline mice. Transfection in alternate pulmonary vascular disorders was studied by inducing hypoxic pulmonary hypertension (4 wk at barometric pressure of 410 mmHg). Efficiency and duration of gene transfer, assessed by CAT activity, were similar in pulmonary hypertensive and normal lungs. We conclude that imidazolinium-derived polycationic liposomes provide a means of relatively selective and efficient gene transfer to the normal and injured murine microvascular circulation, although translation of transgene mRNA may be reduced by preexisting endothelial injury.

Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation.

Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation. eNOS null mice had augmented HPV (225 +/- 65% control, P < 0.02, mean +/- SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 +/- 1.2 mmHg, P < 0.001) and, to a lesser extent, in iNOS-deficient (37.2 +/- 0.8 mmHg, P < 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 +/- 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 +/- 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically.

Enhanced ET(A)-receptor-mediated inhibition of K(v) channels in hypoxic hypertensive rat pulmonary artery myocytes.

Endothelin (ET)-1 has been implicated as a critical mediator in the pathogenesis of hypoxic pulmonary hypertension. We questioned whether, during exposure to chronic hypobaric hypoxia, rat pulmonary artery smooth muscle cells (PASMC) became sensitized to ET-1. Two effects of ET-1, inhibition of voltage-gated K(+) (K(v)) channels and release of intracellular Ca(2+), were studied using whole cell patch clamp and single cell indo 1 fluorescence, respectively. In both normotensive and chronically hypoxic-hypertensive PASMC, ET-1 caused concentration-dependent inhibition of voltage-gated K(+) current [I(K(v))], with maximum inhibition of 12-18% seen at a concentration of 0.1-1 nM. Although the chronically hypoxic-hypertensive PASMC was no more susceptible to ET-1-mediated I(K(v)) inhibition, a switch in coupling between ET-1 and I(K(v)) from ET(B) to ET(A) receptors occurred. This switch in receptor coupling, combined with reduced I(K(v)) density and increased ET-1 production in the hypoxic rat lung, may help explain the ability of ET(A)-receptor blockers to attenuate the development of hypoxic pulmonary hypertension in vivo.

Hypoxia inhibits increased ETB receptor-mediated NO synthesis in hypertensive rat lungs.

Although hypertensive lungs of chronically hypoxic rats express increased levels of nitric oxide (NO) synthases (NOSs) and produce increased amounts of NO-containing compounds (NOx) during normoxic ventilation, the level of NO production during hypoxic exposure is unclear. Because hypoxia inhibits NO synthesis in normotensive lungs, we investigated whether hypoxic ventilation inhibited NO synthesis in isolated hypertensive lungs and chronically hypoxic rats. Measurement of perfusate NOx concentration in hypertensive lungs from male rats exposed to 4 wk of hypobaric hypoxia showed that basal NOx production was reduced during hypoxic (0% O2) vs. normoxic (21% O2) ventilation. Similarly, plasma NOx concentration was lower in chronically hypoxic rats breathing 10% O2 than in those breathing 21% O2. Hypoxic inhibition of lung NOx production was not prevented by supplementary L-arginine or tetrahydrobiopterin and was not mimicked by inhibition of Ca2+ influx. However, it was mimicked by inhibition of constitutive NOS with NG-monomethyl-L-arginine and chelation of intracellular Ca2+. The endothelin type B-receptor antagonist BQ-788 prevented the increases in NOx production associated with normoxic ventilation in both isolated hypertensive lungs and intact chronically hypoxic rats. These results suggest that a reduced supply of the cosubstrate molecular O2 to NOS counteracts an endothelin type B receptor-mediated stimulation of NO synthesis in hypertensive rat lungs. Thus, despite increased NOS protein in the lungs and pulmonary arteries of chronically hypoxic rats, direct hypoxic inhibition of NO production may contribute to the development of pulmonary hypertension.

Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension.

Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3-4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6-9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 +/- 46% of control levels in hypoxic lungs (P < 0.05) but were decreased by 50 +/- 5 and 60 +/- 11%, respectively, in monocrotaline-treated and fawn-hooded lungs (P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 +/- 1.4 microM; P < 0.05) but not in either monocrotaline-treated (0.7 +/- 0.3 microM) or fawn-hooded (0.45 +/- 0.1 microM) lungs vs. normotensive lungs (0.12 +/- 0.07 microM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-L-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.

The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia.

Acute hypoxic vasoconstriction and development of hypoxic pulmonary hypertension (PHTN) are unique properties of the pulmonary circulation. The pulmonary endothelium produces vasoactive factors, including nitric oxide (NO), that modify these phenomena. We tested the hypothesis that NO produced by endothelial nitric oxide synthase (eNOS) modulates pulmonary vascular responses to hypoxia using mice with targeted disruption of the eNOS gene (eNOS-/-). Marked PHTN was found in eNOS-/- mice raised in mild hypoxia when compared with either controls or eNOS-/- mice raised in conditions simulating sea level. We found an approximate twofold increase in partially and fully muscularized distal pulmonary arteries in eNOS-/- mice compared with controls. Consistent with vasoconstriction being the primary mechanism of PHTN, however, acute inhalation of 25 ppm NO resulted in normalization of RV pressure in eNOS-/- mice. In addition to studies of eNOS-/- mice, the dose-effect of eNOS was tested using heterozygous eNOS+/- mice. Although the lungs of eNOS+/- mice had 50% of normal eNOS protein, the response to hypoxia was indistinguishable from that of eNOS-/- mice. We conclude that eNOS-derived NO is an important modulator of the pulmonary vascular response to chronic hypoxia and that more than 50% of eNOS expression is required to maintain normal pulmonary vascular tone.

Effect of K+ATP channel inhibition on total and regional vascular resistance in guinea pig pregnancy.

Decreased vascular resistance and vasoconstrictor response during pregnancy enables an increase in cardiac output and regional blood flow to the uterine circulation. We sought to determine whether inhibition of vascular smooth muscle ATP-sensitive potassium (K+ATP) channel activity during pregnancy increased systemic and/or regional vascular resistance and resistance response to ANG II. A total of 32 catheterized, awake, pregnant or nonpregnant guinea pigs were treated with either the K+ATP channel inhibitor glibenclamide (3.5 mg/kg) or vehicle (DMSO) (n = 8/group). In nonpregnant and pregnant animals, glibenclamide raised blood pressure and systemic, uterine, and coronary vascular resistance, diminishing cardiac output and organ blood flow. Glibenclamide produced a greater rise in coronary vascular resistance in the pregnant than nonpregnant groups and increased renal and cerebral vascular resistance in the pregnant animals only. ANG II infusion raised blood pressure and systemic and renal vascular resistance and lowered cardiac output and renal blood flow in vehicle-treated animals. Glibenclamide augmented ANG II-induced systemic vasoconstriction in the nonpregnant and pregnant groups and the rise in uteroplacental vascular resistance in the pregnant animals. We concluded that K+ATP channel activity likely modulates systemic, uterine, and coronary vascular resistance and opposes ANG II-induced systemic vasoconstriction in nonpregnant and pregnant guinea pigs. Pregnancy augments K+ATP channel activity in the uterine, coronary, renal, and cerebral vascular beds and the uteroplacental circulation during ANG II infusion. Thus increased K+ATP channel activity appears to influence regional control of vascular resistance during guinea pig pregnancy but cannot account for the characteristic decrease in systemic vascular resistance and ANG II-induced systemic vasoconstrictor response.