Richard J. Paul

A role for Smad6 in development and homeostasis of the cardiovascular system

Smad proteins are intracellular mediators of signalling initiated by Tgf-βsuperfamily ligands (Tgf-βs, activins and bone morphogenetic proteins (Bmps)). Smads 1, 2, 3, 5 and 8 are activated upon phosphorylation by specific type I receptors, and associate with the common partner Smad4 to trigger transcriptional responses. The inhibitory Smads (6 and 7) are transcriptionally induced in cultured cells treated with Tgf-β superfamily ligands, and downregulate signalling in in vitro assays. Gene disruption in mice has begun to reveal specific developmental and physiological functions of the signal-transducing Smads. Here we explore the role of an inhibitory Smad in vivo by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrated that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-β superfamily signalling pathways in vivo.

Fibroblast growth factor 2 control of vascular tone

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FCF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

The Na,K-ATPase α2 Isoform Is Expressed in Neurons, and Its Absence Disrupts Neuronal Activity in Newborn Mice

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the α2 isoform is widely expressed in neurons, unlike in the adult brain, in which α2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of α2 isoform expression in neurons is interesting in light of our examination of mice lacking the α2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-Bötzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase α2 isoform could be important in the modulation of neuronal activity in the neonate.

Antithetic regulation by β-adrenergic receptors of Gq receptor signaling via phospholipase C underlies the airway β-agonist paradox

β-adrenergic receptors (βARs) relax airway smooth muscle and bronchodilate, but chronic β-agonist treatment in asthma causes increased sensitivity to airway constriction (hyperreactivity) and is associated with exacerbations. This paradox was explored using mice with ablated βAR genes (βAR–/–) and transgenic mice overexpressing airway smooth muscle β2AR (β2AR-OE) representing two extremes: absence and persistent activity of airway βAR. Unexpectedly, βAR–/– mice, lacking these bronchodilating receptors, had markedly decreased bronchoconstrictive responses to methacholine and other Gq-coupled receptor agonists. In contrast, β2AR-OE mice had enhanced constrictive responses. Contraction to permeabilization with β-escin was unaltered by gene ablation or overexpression. Inositol phosphate accumulation by Gq-coupled M3-muscarinic, thromboxane-A2, and 5-HT2 receptors was desensitized in airway smooth muscle cells from βAR–/– mice and sensitized in cells from β2AR-OE mice. Thus, βAR antithetically regulates constrictive signals, affecting bronchomotor tone/reactivity by additional means other than direct dilatation. Studies of signaling elements in these pathways revealed the nodal point of this cross talk as phospholipase C-β1, whose expression was altered by βAR in a direction and magnitude consistent with the physiologic and cellular responses. These results establish a mechanism of the β-agonist paradox and identify a potential asthma modifier gene (phospholipase C-β1), which may also be a therapeutic target in asthma when chronic β-agonists are required.

LINKAGE OF AEROBIC GLYCOLYSIS TO SODIUM-POTASSIUM TRANSPORT IN RAT SKELETAL MUSCLE : IMPLICATIONS FOR INCREASED MUSCLE LACTATE PRODUCTION IN SEPSIS

Although a linkage between aerobic glycolysis and sodium-potassium transport has been demonstrated in diaphragm, vascular smooth muscle, and other cells, it is not known whether this linkage occurs in skeletal muscle generally. Metabolism of intact hind-leg muscles from young rats was studied in vitro under aerobic incubation conditions. When sodium influx into rat extensor digitorum longus (EDL) and soleus muscles was facilitated by the sodium ionophore monensin, muscle weight gain and production of lactate and alanine were markedly stimulated in a dose-dependent manner. Although lactate production rose in both muscles, it was more pronounced in EDL than in soleus. Monensin-induced lactate production was inhibited by ouabain or by incubation in sodium-free medium. Preincubation in potassium-free medium followed by potassium re-addition also stimulated ouabain-inhibitable lactate release. Replacement of glucose in the incubation medium with pyruvate abolished monensin-induced lactate production but exacerbated monensin-induced weight gain. Muscles from septic or endotoxin-treated rats exhibited an increased rate of lactate production in vitro that was partially inhibited by ouabain. Increases muscle lactate production in sepsis may reflect linked increases in activity of the Na+, K+-ATPase, consumption of ATP and stimulation of aerobic glycolysis.

Antibiotic treatment of preterm labor with intact membranes: A multicenter, randomized, double-blinded, placebo-controlled trial***

OBJECTIVE Although an association between subclinical intrauterine infection and preterm birth is well established, there is conflicting evidence regarding the benefits of antibiotic administration to women in preterm labor with intact membranes. We attempted to determine the effect of ampicillin-amoxicillin and erythromycin treatment on prolongation of pregnancy, the rate of preterm birth, and neonatal morbidity in patients with preterm labor and intact membranes. STUDY DESIGN A multicenter, randomized, double-blinded, placebo-controlled trial was designed and implemented by the Maternal-Fetal Medicine Units Network of the National Institute of Child Health and Human Development. Two hundred seventy-seven women with singleton pregnancies and preterm labor with intact membranes (24 to 34 weeks) were randomly allocated to receive either antibiotics or placebos. RESULTS Of the 2373 patients screened for participation in this study in six medical centers, 277 women were enrolled (n = 133 for antibiotics group vs n = 144 for placebo group). In each study group, 60% of patients completed all the study medications. The overall prevalence of microbial invasion of the amniotic cavity was 5.8% (14/239). No significant difference between the antibiotic group and the placebo group was found in maternal outcomes, including duration of randomization-to-delivery interval, frequency of preterm delivery (< 37 weeks), frequency of preterm premature rupture of membranes, clinical chorioamnionitis, endometritis, and number of subsequent admissions for preterm labor. Similarly, no significant difference in neonatal outcomes could be detected between the two groups including respiratory distress syndrome, bronchopulmonary dysplasia, intraventricular hemorrhage, sepsis, and admission and duration of newborn intensive special care unit hospitalization. CONCLUSION The results of this study do not support the routine use of antibiotic administration to women in preterm labor with intact membranes.

Predictors of pre-eclampsia in women at high risk

OBJECTIVE We assessed several variables as predictors for pre-eclampsia risk in a group of women at high risk. STUDY DESIGN We studied 2503 women with either diabetes mellitus, chronic hypertension, multifetal gestation, or pre-eclampsia in a previous pregnancy who participated in a multicenter study comparing aspirin and placebo in preventing pre-eclampsia. We evaluated multiple variables for predicting pre-eclampsia risk with use of univariate and multivariable analysis. RESULTS Parity and mean arterial pressure at randomization were most predictive of pre-eclampsia risk. The risk was 8% with a mean arterial pressure at enrollment of <75 mm Hg versus 27% with a mean arterial pressure >85 mm Hg (relative risk and 95% confidence interval 3.3 [2.4 to 4.4]). The risk of pre-eclampsia was 26% in nulliparous patients versus 17% in parous subjects (relative risk and 95% confidence interval 1.5 [1.3-1.8]). CONCLUSIONS The finding that second-trimester mean arterial pressure affects pre-eclampsia risk suggests that the pathophysiologic process of preeclampsia is initiated before that time.

Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3.

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 ± 5.6% in wild type, 35.4 ± 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.

Positively selected G6PD-Mahidol mutation reduces Plasmodium vivax density in Southeast Asians.

Ghosts of Selection Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency of humans, and it has been long suspected to exert an effect on Plasmodium falciparum malaria in Africa. Likewise, the increase in prevalence of the G6PD-Mahidol 487A allele among Karen people in Thailand, who only in the past few thousand years have migrated into malarious zones, may be the result of selection by Plasmodium vivax malaria. P. vivax has recently been implicated in more severe disease than previously suspected, providing both a direct selective effect through mortality and an indirect selective effect through morbidity and reproductive failure. Louicharoen et al. (p. 1546) link population-genetic evidence for positive selection in an 8-year family-based study of 3000 Karen individuals and reveal that there is an association between the presence of the G6PD-Mahidol 487A allele and a reduction in the density of P. vivax parasites circulating in the bloodstreams of infected individuals. The mutation appears to exert its effect on the physiology of immature red blood cells, which are the preferred niche for P. vivax but not of P. falciparum. Positive selection acts on a hemolytic anemia–causing mutation that affects the proliferation of a blood parasite in humans. Glucose-6-phosphate dehydrogenase (G6PD) deficiency—the most common known enzymopathy—is associated with neonatal jaundice and hemolytic anemia usually after exposure to certain infections, foods, or medications. Although G6PD-deficient alleles appear to confer a protective effect against malaria, the link with clinical protection from Plasmodium infection remains unclear. We investigated the effect of a common G6PD deficiency variant in Southeast Asia—the G6PD-Mahidol487A variant—on human survival related to vivax and falciparum malaria. Our results show that strong and recent positive selection has targeted the Mahidol variant over the past 1500 years. We found that the G6PD-Mahidol487A variant reduces vivax, but not falciparum, parasite density in humans, which indicates that Plasmodium vivax has been a driving force behind the strong selective advantage conferred by this mutation.

Aquaporin 5-Deficient Mouse Lungs are Hyperresponsive to Cholinergic Stimulation

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5−/−) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5+/+ mice, Aqp5−/− mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5−/− mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5−/− mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.