Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE

Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET_B) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

EXPERIMENTAL APPROACH

ET_B receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca²⁺, F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A₂ and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively.

KEY RESULTS

Cigarette smoke extract dose-dependently increased ET_B receptor expression in HPAECs after 24 h incubation. CSE-induced ET_B expression was attenuated by bosentan, the ET_B receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET_B receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET_B receptor expression, mimicking the effect of CSE. CSE-induced ET_B receptor overexpression caused greater cell contraction; increased intracellular Ca²⁺; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA₂ and ROS production; and decreased NO after acute ET-1 (10 nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine.

CONCLUSIONS AND IMPLICATION

Cigarette smoke extract induced ET_B receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET_B receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.     

Induction of apoptosis by the ginsenoside Rh2 by internalization of lipid rafts and caveolae and inactivation of Akt

Background and purpose:

Lipid rafts and caveolae are membrane microdomains with important roles in cell survival signalling involving the Akt pathway. Cholesterol is important for the structure and function of these microdomains. The ginsenoside Rh2 exhibits anti-tumour activity. Because Rh2 is structurally similar to cholesterol, we investigated the possibility that Rh2 exerted its anti-tumour effect by modulating rafts and caveolae.

Experimental approach:

A431 cells (human epidermoid carcinoma cell line) were treated with Rh2 and the effects on cell apoptosis, raft localization and Akt activation measured. We also examined the effects of over-expression of Akt and active-Akt on Rh2-induced cell death.

Key results:

Rh2 induced apoptosis concentration- and time-dependently. Rh2 reduced the levels of rafts and caveolae in the plasma membrane and increased their internalization. Furthermore, Akt activity was decreased and consequently, Akt-dependent phosphorylation of Bad, a pro-survival protein, was decreased whereas the pro-apoptotic proteins, Bim and Bax, were increased upon Rh2 treatment. Unlike microdomain internalization induce by cholesterol depletion, Rh2-mediated internalization of rafts and caveolae was not reversed by cholesterol addition. Also, cholesterol addition did not restore Akt activation or rescue cells from Rh2-induced cell death. Rh2-induced cell death was attenuated in MDA-MB-231 cells over-expressing either wild-type or dominant-active Akt.

Conclusions and implications:

Rh2 induced internalization of rafts and caveolae, leading to Akt inactivation, and ultimately apoptosis. Because elevated levels of membrane rafts and caveolae, and Akt activation have been correlated with cancer development, internalization of these microdomains by Rh2 could potentially be used as an anti-cancer therapy.     

The relationship between inotrope exposure, six-hour postoperative physiological variables, hospital mortality and renal dysfunction in patients undergoing cardiac surgery

Introduction  

Acute haemodynamic complications are common after cardiac surgery and optimal perioperative use of inotropic agents, typically guided by haemodynamic variables, remains controversial. The aim of this study was to examine the relationship of inotrope use to hospital mortality and renal dysfunction.     

To test or not to test: A cross-sectional survey of the psychosocial determinants of self-testing for cholesterol,glucose, and HIV

Background  

Although self-tests are increasingly available and widely used, it is not clear whether their use is beneficial to the users, and little is known concerning the determinants of self-test use. The aim of this study was to identify the determinants of self-test use for cholesterol, glucose, and HIV, and to examine whether these are similar across these tests. Self-testing was defined as using in-vitro tests on body materials, initiated by consumers with the aim of diagnosing a particular disorder, condition, or risk factor for disease.     

Pulmonary arteriovenous malformations: techniques and long-term outcome of embolotherapy

Over a 10-year period, 276 pulmonary arteriovenous malformations (PAVMs) were occluded with balloon embolotherapy in 76 patients, 67 (88%) of whom had hereditary hemorrhagic telangiectasia. Eleven patients (14%) were discovered by means of family screening with measurement of arterial blood gases and chest radiography. Epistaxis, dyspnea, hemoptysis, and hemothorax occurred in 79%, 71%, 13%, and 9% of patients, respectively. Clinical histories of strokes and transient ischemic attacks were present in 18% and 37% of patients, respectively. Computed tomographic scans of 59 patients showed stroke in 36%. Sixty-five percent of PAVMs were located in the lower lobes, which correlated with the finding of more pronounced hypoxemia in the upright position. After embolotherapy, symptomatic hypoxemia was corrected, and serial values have remained constant for 5 years. Complications were minimal, and no patient required surgery. Balloon embolotherapy is effective long-term therapy for PAVMs, and family screening should be pursued because of the possibility of a higher frequency of paradoxical embolization (stroke) than previously recognized.     

Amifostine (WR-2721) shortens the engraftment period of 4- hydroperoxycyclophosphamide-purged bone marrow in breast cancer patients receiving high-dose chemotherapy with autologous bone marrow support

4-Hydroperoxycyclophosphamide (4-HC), a commonly used marrow-purging agent, is active against many tumors, but is also toxic to normal marrow progenitors. Amifostine (WR-2721) is a sulfhydryl compound with chemoprotectant activity. Preclinical studies using suspensions of bone marrow and breast cancer cells demonstrated that ex vivo treatment with amifostine followed by 4-HC resulted in protection of marrow progenitors, with no compromise in the antitumor effect of 4-HC. This fact stimulated the development of a clinical trial. Bone marrow was harvested from 15 poor-prognosis breast cancer patients and randomly assigned to ex vivo treatment with amifostine followed by 4-HC (amifostine + 4-HC), or treatment with 4-HC alone. High-dose chemotherapy was then administered followed by infusion of the purged autologous bone marrow support (ABMS). Leukocyte engraftment, defined as a white blood cell count > or = 1 x 10(9)/L, was achieved in an average of 26 days for patients whose marrow was purged with amifostine + 4-HC versus 36 days for patients whose marrow was purged with 4-HC alone (P = .032). The average number of platelet transfusions (12 v 29; P = .017) and days of antibiotic therapy (28 v 40; P = .012) were significantly less for patients whose marrow was exposed to amifostine + 4-HC, compared with 4-HC alone. Unpurged backup marrow fractions were infused into three patients whose marrow was purged with 4-HC alone, because of inadequate marrow recovery. None of the patients who received amifostine + 4-HC-purged marrow required a backup marrow fraction. Complete remissions were achieved in 83% of patients with measurable disease, with no difference between the two cohorts. Forty- three percent of patients remained alive and progression-free at a mean of 13 months posttransplant. There was no significant difference in the rate or pattern of relapse for patients whose marrow was purged with amifostine + 4-HC compared with those whose marrow was purged with 4-HC alone. Ex vivo treatment of marrow with amifostine significantly shortens the time to marrow recovery, thereby reducing the risk of myelosuppressive complications in breast cancer patients receiving high- dose chemotherapy and 4-HC-purged ABMS. Since supportive care requirements are also significantly decreased, amifostine may reduce the cost of such therapy.     

Further delineation of the KBG syndrome caused by ANKRD11 aberrations

Loss-of-function variants in ANKRD11 were identified as the cause of KBG syndrome, an autosomal dominant syndrome with specific dental, neurobehavioural, craniofacial and skeletal anomalies. We present the largest cohort of KBG syndrome cases confirmed by ANKRD11 variants reported so far, consisting of 20 patients from 13 families. Sixteen patients were molecularly diagnosed by Sanger sequencing of ANKRD11, one familial case and three sporadic patients were diagnosed through whole-exome sequencing and one patient was identified through genomewide array analysis. All patients were evaluated by a clinical geneticist. Detailed orofacial phenotyping, including orthodontic evaluation, intra-oral photographs and orthopantomograms, was performed in 10 patients and revealed besides the hallmark feature of macrodontia of central upper incisors, several additional dental anomalies as oligodontia, talon cusps and macrodontia of other teeth. Three-dimensional (3D) stereophotogrammetry was performed in 14 patients and 3D analysis of patients compared with controls showed consistent facial dysmorphisms comprising a bulbous nasal tip, upturned nose with a broad base and a round or triangular face. Many patients exhibited neurobehavioural problems, such as autism spectrum disorder or hyperactivity. One-third of patients presented with (conductive) hearing loss. Congenital heart defects, velopharyngeal insufficiency and hip anomalies were less frequent. On the basis of our observations, we recommend cardiac assessment in children and regular hearing tests in all individuals with a molecular diagnosis of KBG syndrome. As ANKRD11 is a relatively common gene in which sequence variants have been identified in individuals with neurodevelopmental disorders, it seems an important contributor to the aetiology of both sporadic and familial cases.