Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.     

Mitochondrial dynamics and autophagy aid in removal of persistent mitochondrial DNA damage in Caenorhabditis elegans

Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mitochondrial DNA (mtDNA) by important environmental genotoxins and endogenous metabolites. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. We report that removal of such mtDNA damage is detectable by 48 h in Caenorhabditis elegans, and requires mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the in vivo relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.     

Common variants for atrial fibrillation: results from genome-wide association studies

Atrial fibrillation (AF) affects more than 5 million people worldwide; however, none of the anti-arrhythmic drugs available now are entirely optimal in terms of efficacy and safety. A better understanding of the molecular mechanism of AF will facilitate the process of finding new strategies to prevent AF. As the non-familial AF is the major form of AF, identifying common variants for AF in these populations by genome-wide association studies will definitely accelerate this process. This review summarizes the recently identified common AF variants on 4q25, 16q22, and 1q21 and discusses their implications for the clinic.     

Potent antitumor effects of combination therapy with IFNs and monocytes in mouse models of established human ovarian and melanoma tumors

Interferon-activated monocytes are known to exert cytocidal activity against tumor cells in vitro. Here, we have examined whether a combination of IFN-α2a and IFN-γ and human monocytes mediate significant antitumor effects against human ovarian and melanoma tumor xenografts in mouse models. OVCAR-3 tumors were treated i.t. with monocytes alone, IFN-α2a and IFN-γ alone or combination of all three on day 0, 15 or 30 post-tumor implantation. Mice receiving combination therapy beginning day 15 showed significantly reduced tumor growth and prolonged survival including complete regression in 40% mice. Tumor volumes measured on day 80 in mice receiving combination therapy (206 mm(3)) were significantly smaller than those of mice receiving the IFNs alone (1,041 mm(3)), monocytes alone (1,111 mm(3)) or untreated controls (1,728 mm(3)). Similarly, combination therapy with monocytes and IFNs of much larger tumor also inhibited OVCAR-3 tumor growth. Immunohistochemistry studies showed a large number of activated macrophages (CD31(+)/CD68(+)) infiltrating into OVCAR-3 tumors and higher densities of IL-12, IP10 and NOS2, markers of M1 (classical) macrophages in tumors treated with combination therapy compared to the controls. Interestingly, IFNs-activated macrophages induced apoptosis of OVCAR-3 tumor cells as monocytes alone or IFNs alone did not mediate significant apoptosis. Similar antitumor activity was observed in the LOX melanoma mouse model, but not as profound as seen with the OVCAR-3 tumors. Administration of either mixture of monocytes and IFN-α2a or monocytes and IFN-γ did not inhibit Lox melanoma growth; however, a significant inhibition was observed when tumors were treated with a mixture of monocytes, IFN-α2a and IFN-γ. These results indicate that monocytes and both IFN-α2a and IFN-γ may be required to mediate profound antitumor effect against human ovarian and melanoma tumors in mouse models.     

Timing of ischemic insult alters fetal growth trajectory, maternal angiogenic balance, and markers of renal oxidative stress in the pregnant rat

Increased uterine artery resistance and angiogenic imbalance characterized by increased soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased free vascular endothelial growth factor (VEGF) are often associated with placental insufficiency and preeclampsia but not synonymous with hypertension. We hypothesized chronic reductions in utero-placental perfusion (RUPP) for 5 days (d) during either mid- (d12-d17) or late (d14-d19) gestation would have disparate effects on plasma sFlt-1 and VEGF levels and blood pressure. Five days of chronic RUPP was achieved by placement of silver clips on the abdominal aorta and ovarian arteries on either gestational d12 or d14. Arterial pressure was increased (P < 0.05) in RUPP vs. normal pregnant (NP) in both d17 (10%) and d19 (25%) groups, respectively. Circulating free VEGF was decreased (P < 0.05) and sFlt-1:VEGF ratio increased (P < 0.05) after 5 days of RUPP ending on d19 but not d17 compared with NP controls. Angiogenic imbalance, measured by an endothelial tube formation assay, was present in the d19 RUPP but not the d17 RUPP compared with age-matched NP rats. Five days of RUPP from days 14 to 19 decreased fetal and placental weights 10% (P < 0.01) compared with d19 NP controls. After 5 days of RUPP, from days 12 to 17 of pregnancy, fetal weights were 21% lighter (P < 0.01) compared with d17 NP controls, but placental weight was unchanged. These findings suggest that the timing during which placental insufficiency occurs may play an important role in determining the extent of alterations in angiogenic balance, fetal growth restriction, and the severity of placental ischemia-induced hypertension.     

Placental and vascular adaptations to exercise training before and during pregnancy in the rat

Although exercise during pregnancy is generally recommended and thought to be beneficial to mother and fetus, the nature of the adaptations to exercise during pregnancy and how they may be beneficial remain poorly understood. Recent studies suggest that exercise may stimulate expression of several cytoprotective and pro-angiogenic molecules such as heat shock proteins (HSP) and vascular endothelial growth factors (VEGF). We hypothesized that exercise training during pregnancy improves angiogenic balance, increases HSP expression, and improves endothelial function. Female rats were given access to an exercise wheel for 6 wk before and during pregnancy. On day 19 of pregnancy tissues were collected and snap frozen for later analysis. Western blots were performed in skeletal muscle and placenta. HSP 27 (3.7 ± 0.36 vs. 2.2 ± 0.38; P < 0.05), HSP 60 (2.2 ± 0.73 vs. 0.49 ± 0.08; P < 0.05), and HSP 90 (0.33 ± 0.09 vs. 0.11 ± 0.02; P < 0.05) were increased in the placentas of exercise-trained rats compared with sedentary controls. In addition, exercise training increased (P < 0.05) plasma free VEGF and augmented (P < 0.05) endothelium-dependent vascular relaxation compared with nonexercise control rats. The present data indicates chronic exercise training stimulates HSP expression in the placenta and that regular exercise training increases circulating VEGF in pregnant but not in nonpregnant rats. Although the present findings suggest that exercise before and during pregnancy may promote the expression of molecules that could attenuate placental and vascular dysfunction in complicated pregnancies, further studies are needed to determine the safety and effectiveness of exercise training as a therapeutic modality in pregnancy.     

Increased fat deposition in injured skeletal muscle is regulated by sex-specific hormones

Sex differences in skeletal muscle regeneration are controversial; comparisons of regenerative events between sexes have not been rigorously defined in severe injury models. We comprehensively quantified inflammation and muscle regeneration between sexes and manipulated sex-specific hormones to determine effects on regeneration. Cardiotoxin injury was induced in intact, castrated and ovariectomized female and male mice; ovariectomized mice were replaced with low- or high-dose 17-β estradiol (E(2)) or progesterone (P4). Extent of injury was comparable between intact mice, but females were more efficient in removal of necrotic debris, despite similar tissue levels of inflammatory cells and chemokines. Myofiber size during regeneration was equivalent between intact mice and after castration or ovariectomy (OVX) but was decreased (P < 0.001) in ovariectomized mice with high-dose E(2) replacement. Intermuscular adipocytes were absent in uninjured muscle, whereas adipocyte area was increased among regenerated myofibers in all groups. Interestingly, intermuscular fat was greater (P = 0.03) in intact females at day 14 compared with intact males. Furthermore, castration increased (P = 0.01) and OVX decreased adipocyte accumulation. After OVX, E(2), but not P4, replacement decreased (P ≤ 0.03) fat accumulation. In conclusion, sex-dependent differences in regeneration consisted of more efficient removal of necrosis and increased fat deposition in females with similar injury, inflammation, and regenerated myofiber size; high-dose E(2) decreased myofiber size and fat deposition. Adipocyte accumulation in regenerating muscle was influenced by sex-specific hormones. Recovery following muscle injury was different between males and females, and sex-specific hormones contributed to these differences, suggesting that sex-specific treatments could be beneficial after injury.     

PTEN loss defines a TGF-β-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis

We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-β is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-β signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-β antagonism. Tubule-specific induction of TGF-β led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-β depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-β antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.