Dustin R. Fraidenburg

Notch Activation of Ca(2+) Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.

Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension

Pulmonary vascular remodeling, mainly attributable to enhanced pulmonary arterial smooth muscle cell proliferation and migration, is a major cause for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary hypertension. The signaling cascade through Akt, comprised of three isoforms (Akt1-3) with distinct but overlapping functions, is involved in regulating cell proliferation and migration. This study aims to investigate whether the Akt/mammalian target of rapamycin (mTOR) pathway, and particularly which Akt isoform, contributes to the development and progression of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Compared with the wild-type littermates, Akt1(-/-) mice were protected against the development and progression of chronic HPH, whereas Akt2(-/-) mice did not demonstrate any significant protection against the development of HPH. Furthermore, pulmonary vascular remodeling was significantly attenuated in the Akt1(-/-) mice, with no significant effect noted in the Akt2(-/-) mice after chronic exposure to normobaric hypoxia (10% O2). Overexpression of the upstream repressor of Akt signaling, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and conditional and inducible knockout of mTOR in smooth muscle cells were also shown to attenuate the rise in right ventricular systolic pressure and the development of right ventricular hypertrophy. In conclusion, Akt isoforms appear to have a unique function within the pulmonary vasculature, with the Akt1 isoform having a dominant role in pulmonary vascular remodeling associated with HPH. The PTEN/Akt1/mTOR signaling pathway will continue to be a critical area of study in the pathogenesis of pulmonary hypertension, and specific Akt isoforms may help specify therapeutic targets for the treatment of pulmonary hypertension.

Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca(2+)]cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced [Ca(2+)]cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced [Ca(2+)]cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.

Inhaled Antibiotics for Gram-Negative Respiratory Infections

SUMMARY Gram-negative organisms comprise a large portion of the pathogens responsible for lower respiratory tract infections, especially those that are nosocomially acquired, and the rate of antibiotic resistance among these organisms continues to rise. Systemically administered antibiotics used to treat these infections often have poor penetration into the lung parenchyma and narrow therapeutic windows between efficacy and toxicity. The use of inhaled antibiotics allows for maximization of target site concentrations and optimization of pharmacokinetic/pharmacodynamic indices while minimizing systemic exposure and toxicity. This review is a comprehensive discussion of formulation and drug delivery aspects, in vitro and microbiological considerations, pharmacokinetics, and clinical outcomes with inhaled antibiotics as they apply to disease states other than cystic fibrosis. In reviewing the literature surrounding the use of inhaled antibiotics, we also highlight the complexities related to this route of administration and the shortcomings in the available evidence. The lack of novel anti-Gram-negative antibiotics in the developmental pipeline will encourage the innovative use of our existing agents, and the inhaled route is one that deserves to be further studied and adopted in the clinical arena.

Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

Thrombin‐mediated activation of Akt signaling contributes to pulmonary vascular remodeling in pulmonary hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) has been increasingly recognized as a common source of elevated pulmonary vascular resistance and pulmonary hypertension. It is clear that development of pulmonary thromboemboli is the inciting event for this process, yet it remains unclear why some patients have persistent pulmonary artery occlusion leading to distal pulmonary vascular remodeling and CTEPH. Thrombin, a serine protease, is an integral part of the common coagulation cascade, yet thrombin also has direct cellular effects through interaction with the family of PAR membrane receptors. This study is designed to determine the effects of thrombin on Akt signaling in pulmonary artery smooth muscle cells (PASMC) from normal humans and pulmonary hypertension patients. Thrombin treatment of PASMC resulted in a transient increase in Akt phosphorylation and had similar effects on the downstream targets of the Akt/mTOR pathway. Ca2+ is shown to be required for Akt phosphorylation as well as serum starvation, a distinct effect compared to platelet‐derived growth factor. Thrombin treatment was associated with a rise in intracellular [Ca2+] and enhanced store‐operated calcium entry (SOCE). These effects lead to enhanced proliferation, which is more dramatic in both IPAH and CTEPH PASMC. Enhanced proliferation is also shown to be attenuated by inhibition of Akt/mTOR in CTEPH PASMC. Thrombin has direct effects on PASMC increasing intracellular [Ca2+] and PASMC proliferation, an effect attributed to Akt phosphorylation. The current results implicate the effects of thrombin in the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) and CTEPH, which may potentially be a novel therapeutic target.

Pulmonary hypertension associated with thalassemia syndromes

Chronic hemolytic anemia has increasingly been identified as an important risk factor for the development of pulmonary hypertension (PH). Within the thalassemia syndromes, there are multiple mechanisms, both distinct and overlapping, by which PH develops and that differ among β‐thalassemia major or intermedia patients. PH in β‐thalassemia major correlates with the severity of hemolysis, yet in patients whose disease is well treated with chronic transfusion therapy, the development of PH can be related to cardiac dysfunction and the subsequent toxic effects of iron overload rather than hemolysis. β‐Thalassemia intermedia, on the other hand, has a higher incidence of PH owing to the low level of hemolysis that exists over years without the requirement for frequent transfusions, while splenectomy is shown to play an important role in both types. Standard therapies such as chronic transfusion have been shown to mitigate PH, and appropriate chelation therapy can avoid the toxic effects of iron overload, yet is not indicated in many patients. Limited evidence exists for the use of pulmonary vasodilators or other therapies, such as l‐carnitine, to treat PH associated with thalassemia. Here, we review the most recent findings regarding the pathogenic mechanisms, epidemiology, presentation, diagnosis, and treatment of PH in thalassemia syndromes.

Delivery of fetus death with misoprostol in a pregnant woman with H7N9 avian influenza A virus pneumonia and ARDS

No abstract

Benzodiazepine misadventure in acute alcohol withdrawal: The transition from delirium tremens to ICU delirium

To the Editor: In the May issue of the Journal of Anesthesia, Demuro et al. [1] describe a case series of patients receiving dexmedetomidine for alcohol withdrawal. The authors highlight that benzodiazepines may be associated with hemodynamic instability and respiratory depression. An additional complication, intensive care unit (ICU) delirium, may be an underdescribed complication of benzodiazepines in alcohol withdrawal. While not studied in alcohol withdrawal, analysis has shown an association of lorazepam use with the transition to delirium in mechanically ventilated patients, and a higher incidence of delirium with midazolam compared with dexmedetomidine [2, 3]. We recently encountered a case of severe alcohol withdrawal transitioning to ICU delirium following extensive benzodiazepine exposure. We are curious if the authors have any data regarding benzodiazepine requirements or ICU delirium assessment before/after dexmedetomidine initiation. A 38-year-old woman with past medical history of hepatitis C and alcohol abuse presented to the emergency department with pruritus and visual hallucinations after several days of abstaining from alcohol. During the first 8 h of admission, she received 31 mg of intravenous lorazepam with minimal improvement in symptoms. She was transferred to the ICU, where she was intubated and given escalating doses of continuous infusion benzodiazepines with intermittent propofol (Fig. 1). In spite of this intense regimen, the patient remained agitated and conscious enough to exit her ICU bed and stand unassisted with the endotracheal tube (ET) in place. On day 5, adjunctive phenobarbital and clonidine were added. On day 8, it was believed that the patient’s withdrawal syndrome had improved and she was transitioned to scheduled lorazepam bolus doses. However, over the next 3 days she was unable to be liberated from mechanical ventilation due to fluctuating mental status and agitation despite the addition of haloperidol. The patient was positive for ICU delirium as assessed by the confusion assessment method for the ICU (CAM-ICU). In an effort to manage the delirium and agitation, a dexmedetomidine infusion was started on day 11 at 0.2 lg/kg/h and titrated to a dose of 1.0 lg/kg/h. Over the next 24 h, the patient’s agitation improved and she was extubated on day 12. Dexmedetomidine was weaned off after 34 h, and she was transferred to the general medicine floor the following day without further need for any benzodiazepine doses. At the time of discharge from the ICU, the patient had received a total of 183.5 mg intravenous lorazepam, 24 mg oral lorazepam, and 1,221 mg intravenous midazolam. This case illustrates the potential complications of benzodiazepines in severe alcohol withdrawal. We witnessed a patient transition from severe alcohol withdrawal, to delirium tremens, and finally to ICU delirium following M. T. Johnson (&) College of Pharmacy, University of Illinois-Chicago, 833 S. Wood St, 164 Pharm [M/C 886], Chicago, IL 60612, USA e-mail: mtj1636@uic.edu

Hungry for More Autophagy in the Pathogenesis of Pulmonary Arterial Hypertension

Genetic alterations in pulmonary arterial hypertension (PAH) have become increasingly recognized in both known familial or heritable and sporadic or idiopathic PAH (IPAH). Unrecognized genetic alterations have now been found in up to 40% of the IPAH in which no familial predisposition is apparent.1 Bone morphogenetic protein (BMP) receptor II (BMPR-II) is the most common gene implicated in this hereditary form of PAH; furthermore, it is implicated in the pathogenesis of nonhereditary forms of PAH with a significant reduction in the expression of BMPR-II in both IPAH and experimental animal models of pulmonary hypertension (PH).2 BMPs represent the largest group of cytokines in the transforming growth factor-β superfamily and regulate growth, differentiation, and apoptosis in multiple cell types, whereas BMPR-II has been shown to have unique roles in differing cells. Article, see p 1159 BMPR-II is constitutively active at the cell membrane, and ligand stimulation initiates cross-linking with BMPR-I to form a receptor complex that is necessary to activate intracellular signaling. BMPR-II is most highly expressed in endothelial cells in the pulmonary vasculature, and BMPR-II activation leads to the increased proliferation and decreased apoptosis through Smad signaling.3,4 This is in contrast to pulmonary arterial smooth muscle cells, where BMP activation leads to the inhibition of proliferation and increased apoptosis through Smad signaling in large vessels, although in small pulmonary arteries, a proliferative effect is seen through activation of extracellular signal–regulated kinase and mitogen-activated protein kinase, which inhibits Smad signaling.5,6 It is these unique yet complementary functions that make BMPR-II mutations particularly damaging in the pulmonary circulation, leading to development of PAH. A dysfunctional mutation of BMPR-II, as in heritable PAH, or downregulation of protein expression, as in IPAH and animal models, can lead to endothelial dysfunction hallmarked by abnormal barrier function through …