Brian Fouty

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.

Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle.

Abstract— Bone morphogenetic peptides (BMPs), a family of cytokines critical to normal development, were recently implicated in the pathogenesis of familial pulmonary arterial hypertension. The type-II receptor (BMPRII) is required for recognition of all BMPs, and targeted deletion of BMPRII in mice results in fetal lethality before gastrulation. To overcome this limitation and study the role of BMP signaling in postnatal vascular disease, we constructed a smooth muscle–specific transgenic mouse expressing a dominant-negative BMPRII under control of the tetracycline gene switch (SM22-tet-BMPRIIdelx4+ mice). When the mutation was activated after birth, mice developed increased pulmonary artery pressure, RV/LV+S ratio, and pulmonary arterial muscularization with no increase in systemic arterial pressure. Studies with SM22-tet-BMPRIIdelx4+ mice support the hypothesis that loss of BMPRII signaling in smooth muscle is sufficient to produce the pulmonary hypertensive phenotype.

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.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.

Riboflavin to treat nucleoside analogue-induced lactic acidosis

Fetal blood was obtained by transabdominal cardiac puncture. In each case, a detailed transvaginal ultrasound was done to confirm gestational age and to assess spontaneous and reflex fetal movements. Written consent was obtained from each woman after receiving complete information on the procedure. This study was approved by the University College London Hospitals Committee on the Ethics of Human Research. The fetal samples were collected between 5 and 20 min following intravenous (IV) administration of a bolus of 3 mg/kg of propofol (Diprivan 1%, Zeneca, Macclesfield, UK) to the mother. Peripheral maternal venous blood was collected simultaneously. Anaesthesia was maintained by spontaneous breathing of 70% nitrous oxide with 0·5–1% isoflurane in oxygen via a laryngeal mask. The propofol concentration was determined by high-performance liquid chromatography with fluorescence. The method has a lower limit of sensitivity of 0·1 g/mL and the intra-assay and interassay coefficients of variation were both 5%. No spontaneous fetal movements or reflux responses were observed in any case during the time interval between induction of anaesthesia and the surgical procedure or during the sampling procedure. Propofol was detected in all maternal and fetal serum samples. No propofol was found in coelomic or amniotic fluid samples at any stage of gestation. Propofol concentration decreased exponentially with time in both maternal (r=0·73, p<0·0001) and fetal (r=0·58, p<0·0001) serum. Maternal serum concentration of propofol was always higher than fetal serum concentration. In 14 matched samples, the mean propofol concentration was 1·96 g/mL (95% CI 1·49–2·42) in maternal serum and 0·90 g/mL (95% CI 0·68–1·11) in fetal serum. Over the 20 min interval between injection and sampling, little change was observed in the fetal/maternal propofol concentration ratio. This finding can be explained by the fact that propofol binds 97–98% to albumin, which is in much lower concentration in fetal serum during the first half of pregnancy. Overall, the pharmacodynamics of propofol found in pregnant women at 12–18 weeks of gestation are similar to those described at term 1–3 indicating that our data can be extrapolated to the period of gestation between 24 and 37 weeks and that our model can be used to study the placental transfer of other analgesic drugs in early pregnancy. Propofol has no known teratogenic effect in humans and our results also indicate that, during short maternal general

Low-Voltage-Activated (T-Type) Calcium Channels Control Proliferation of Human Pulmonary Artery Myocytes

While Ca2+ influx is essential for activation of the cell cycle machinery, the processes that regulate Ca2+ influx in this context have not been fully elucidated. Electrophysiological and molecular studies have identified multiple Ca2+ channel genes expressed in mammalian cells. Cav3.x gene family members, encoding low voltage-activated (LVA) or T-type channels, were first identified in the central nervous system and subsequently in non-neuronal tissue. Reports of a potential role for T-type Ca2+ channels in controlling cell proliferation conflict. The present study tested the hypothesis that T-type Ca2+ channels, encoded by Cav3.x genes, control pulmonary artery smooth muscle cell proliferation and cell cycle progression. Using quantitative RT/PCR, immunocytochemistry, and immunohistochemistry we found that Cav3.1 was the predominant Cav3.x channel expressed in early passage human pulmonary artery smooth muscle cells in vitro and in the media of human pulmonary arteries, in vivo. Selective blockade of Cav3.1 expression with small interfering RNA (siRNA) and pharmacological blockade of T-type channels completely inhibited proliferation in response to 5% serum and prevented cell cycle entry. These studies establish that T-type voltage-operated Ca2+ channels are required for cell cycle progression and proliferation of human PA SMC.

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

BackgroundThe 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.MethodMice 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.ResultsHypoxic 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.ConclusionIn 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.

Mevastatin Can Cause G1 Arrest and Induce Apoptosis in Pulmonary Artery Smooth Muscle Cells Through a p27Kip1-Independent Pathway

Abstract— Advanced pulmonary arterial hypertension is characterized by extensive vascular remodeling that is usually resistant to vasodilator therapy. Mevastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting step for cholesterol synthesis. HMG-CoA reductase inhibitors have been shown to upregulate the cyclin-dependent kinase inhibitor p27Kip1 and to block cell proliferation through cholesterol-independent pathways. The aim of this study was to determine the effect of mevastatin on DNA synthesis, cell cycle progression, and cell proliferation in rat pulmonary artery smooth muscle cells (PASMCs). We found that mevastatin induced G1 arrest and decreased DNA synthesis in rat PASMCs and did so in association with an increase in both total and cyclin E-bound p27Kip1. This caused a marked decrease in cyclin E kinase activity, which suggests an important role for p27Kip1 in the ability of mevastatin to induce G1 arrest. However, in PASMCs lacking functional p27Kip1, mevastatin still decreased cyclin E kinase activity, caused G1 arrest, and decreased DNA synthesis. In p27Kip1-deficient PASMCs, mevastatin induced a greater reduction of cyclin E protein levels (to 35% of control) than in wild-type cells (to 70% of control) and also reduced the phosphorylation of cdk2 on threonine 160. Mevastatin also caused apoptosis in both wild-type and p27Kip1-deficient PASMCs and was able to do so at a dose that did not induce cell cycle arrest. These data suggest that HMG-CoA reductase inhibitors can both inhibit cell proliferation and induce apoptosis in PASMCs through p27Kip1-independent pathways and may be important therapeutic agents in pulmonary arterial hypertension.

Enhanced ETA-receptor-mediated inhibition of Kv 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+(Kv) channels and release of intracellular Ca2+, 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 ETB to ETA 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 ETA-receptor blockers to attenuate the development of hypoxic pulmonary hypertension in vivo.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.

Diabetes and the pulmonary circulation

diabetes is an epidemic in the United States, affecting ∼8% of the population. The hallmark of diabetes is hyperglycemia due to either insulin deficiency or insulin resistance. Systemic vascular dysfunction is a central part of the pathophysiology of both type I insulin-dependent and type II non-

Minimal piggyBac vectors for chromatin integration

We describe novel transposon piggyBac vectors engineered to deliver transgenes as efficiently as currently available piggyBac systems, but with significantly less helper DNA co-delivered into the host genome. To generate these plasmids, we identified a previously unreported aspect of transposon biology, that the full-length terminal domains required for successful plasmid-to-chromatin transgene delivery can be removed from the transgene delivery cassette to other parts of the plasmid without significantly impairing transposition efficiency. This is achieved by including in the same plasmid, an additional helper piggyBac sequence that contains both long terminal domains, but is modified to prevent its transposition into the host genome. This design decreases the size of the required terminal domains within the delivered gene cassette of the piggyBac vector from about 1500 to just 98 base pairs. By removing these sequences from the delivered gene cassette, they are no longer incorporated into the host genome which may reduce the risk of target cell transformation.