Smooth Muscle Area Research Articles

The Effect of Alcohol Administration on the Corpus Cavernosum.

PurposeWe studied the effects of alcohol administration on the corpus cavernosum (CC) using an animal model.Materials and MethodsCC sections and the aortic ring of rabbits were used in an organ bath study. After acute alcohol administration, changes in blood alcohol concentration and electrical stimulation induced intracavernosal pressure/mean arterial pressure (ICP/MAP) percentage were compared in rats. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in the CC were measured using immunoassays. After chronic alcohol administration, ICP/MAP percentage, cAMP and cGMP were compared in rats. Histological changes were examined using the Masson trichrome stain and the Sircol collagen assay. Endothelial nitric oxide synthase (eNOS) expression was examined using immunohistochemistry and Western blotting.ResultsAlcohol relaxed the CC in a dose-dependent manner, and the relaxation response was suppressed when pretreated with propranolol, indomethacin, glibenclamide, and 4-aminopyridine. In rats with acute alcohol exposure, the cAMP level in the CC was significantly greater than was observed in the control group (p<0.05). In rats with chronic alcohol exposure, however, changes in cAMP and cGMP levels were insignificant, and the CC showed markedly smaller areas of smooth muscle, greater amounts of dense collagen (p<0.05). Immunohistochemical analysis of eNOS showed a less intense response, and western blotting showed that eNOS expression was significantly lower in this group (p<0.05).ConclusionsAcute alcohol administration activated the cAMP pathway with positive effects on erectile function. In contrast, chronic alcohol administration changed the ultrastructures of the CC and suppressed eNOS expression, thereby leading to erectile dysfunction.

Abstract 11814: Pulmonary Hypertension With Altered eNOS and ALK-1 Signaling in the Autoimmuno-disease Mice

Background: Pulmonary artery hypertension is often associated with connective tissue disease and major determinant of prognosis. MRL/lpr mouse, which has hypergammaglobulinemia and products various autoimmuno antibody, develops vasculitis and nephritis spontaneously and is widely used as the model of lupus nephritis or Sjogren syndrome. In this study, we examined the right ventricular pressure overload, pathological features and the signaling molecules in the lung tissues of MRL/lpr mice. Methods: We used the female MRL/lpr mice at the age of 12 to 18 weeks. We measured right ventricular systolic pressure (RVSP) by a micro-manometer catheter and the ratio of right ventricular weight to left ventricular weight including ventricular septum (RV/LV). Lung sections were stained with α-smooth muscle actin, and the medial smooth muscle area was expressed as a percentage of the external area of the vessel. Levels of endothelial nitric oxide synthase (eNOS), survivin, endothelin-1 (ET-1), and activin receptor-like kinase-1 (ALK-1) were analyzed by Western blotting. Results: RVSP in the MRL/lpr mice was significantly higher (29.8 ± 7.2 vs. 19.2 ± 1.9 mmHg, P &lt; 0.05) than that in WT C57/Bl6 mice. RV/LV was increased in the MRL/lpr mice compared to the WT C57/Bl6 mice (0.33 ± 0.58 vs. 0.20 ± 0.67, P &lt; 0.05). The medial smooth muscle area of MRL/lpr mice were larger than that of WT C57/bl6 (56.3 ± 4.0 vs. 39.5 ± 6.1%, P &lt; 0.05). Western blot analysis revealed the markedly elevated levels of ET-1 and survivin as well as decreased ALK-1 expression and eNOS phosphorylation in the lung tissue of MRL/lpr mice. Conclusion: We demonstrated that MRL/lpr mice were complicated with pulmonary hypertension. The possible mechanisms include the vasoconstriction enhanced by increased ET-1 level and impaired vasodilatation caused by decreased eNOS phosphorylation and ALK-1 expression. In addition, upregulation of survivin promoted the medial wall thickening of pulmonary vessels. MRL/lpr mice appeared to be a useful model in considering the mechanism of pulmonary hypertension associated with connective tissue diseases.

Effectiveness of bronchial thermoplasty in patients with severe refractory asthma: Clinical and histopathologic correlations

The effectiveness of bronchial thermoplasty (BT) has been reported in patients with severe asthma, yet its effect on different bronchial structures remains unknown. We sought to examine the effect of BT on bronchial structures and to explore the association with clinical outcome in patients with severe refractory asthma. Bronchial biopsy specimens (n=300) were collected from 15 patients with severe uncontrolled asthma before and 3months after BT. Immunostained sections were assessed for airway smooth muscle (ASM) area, subepithelial basement membrane thickness, nerve fibers, and epithelial neuroendocrine cells. Histopathologic findings were correlated with clinical parameters. BT significantly improved asthma control and quality of life at both 3 and 12months and decreased the numbers of severe exacerbations and the dose of oral corticosteroids. At 3months, this clinical benefit was accompanied by a reduction in ASM area (median values before and after BT, respectively: 19.7% [25th-75th interquartile range (IQR), 15.9% to 22.4%] and 5.3% [25th-75th IQR], 3.5% to 10.1%, P<.001), subepithelial basement membrane thickening (4.4μm [25th-75th IQR, 4.0-4.7μm] and 3.9μm [25th-75th IQR, 3.7-4.6μm], P=0.02), submucosal nerves (1.0 ‰ [25th-75th IQR, 0.7-1.3 ‰] immunoreactivity and 0.3 ‰ [25th-75th IQR, 0.1-0.5 ‰] immunoreactivity, P<.001), ASM-associated nerves (452.6 [25th-75th IQR, 196.0-811.2] immunoreactive pixels per mm2 and 62.7 [25th-75th IQR, 0.0-230.3] immunoreactive pixels per mm2, P=.02), and epithelial neuroendocrine cells (4.9/mm2 [25th-75th IQR, 0-16.4/mm2] and 0.0/mm2 [25th-75th IQR, 0-0/mm2], P=.02). Histopathologic parameters were associated based on Asthma Control Test scores, numbers of exacerbations, and visits to the emergency department (all P≤.02) 3 and 12months after BT. BT is a treatment option in patients with severe therapy-refractory asthma that downregulates selectively structural abnormalities involved in airway narrowing and bronchial reactivity, particularly ASM, neuroendocrine epithelial cells, and bronchial nerve endings.

Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study.

Chronic cough involves airway remodeling associated with cough reflex hypersensitivity. Whether cough itself induces these features remains unknown. Guinea pigs were assigned to receive treatment with citric acid (CA), saline (SA), or CA+dextromethorphan (DEX). All animals were exposed to 0.5M CA on days 1 and 22. On days 4-20, the CA and CA+DEX groups were exposed to CA, and the SA group to saline thrice weekly, during which the CA+DEX group was administered DEX pretreatment to inhibit cough. The number of coughs was counted during each 10-min CA or SA exposure. Terbutaline premedication was started to prevent bronchoconstriction. Bronchoalveolar lavage and pathology were examined on day 25. Average cough number for 10 CA exposures was examined as "cough index" in the CA group, which was divided into frequent (cough index>5) and infrequent (<5) cough subgroups for lavage and pathology analysis. The number of coughs significantly increased in the CA group from day 13 onwards. In the CA+DEX and SA groups, the number of coughs did not differ between days 1 and 22, while average number of coughs during days 4-20 was significantly lower than at days 1 and 22. Bronchoalveolar cell profiles were similar among the four groups. The smooth muscle area of small airways was significantly greater in the frequent-cough subgroup than in the other groups (in which it was similar), and highly correlated with cough index in CA group. Repeated cough induces airway smooth muscle remodeling associated with cough reflex hypersensitivity.

Abstract 672: Differential effects of p53 mutations on cancer invasion and metastasis in a mouse model of colon cancer

Abstract Inactivation of the APC (adenomatous polyposis coli) tumor suppressor gene plays an important role in initiating most adenomas and colorectal cancers (CRCs). Somatic mutations in the TP53 tumor suppressor gene and KRAS oncogene are found in roughly 60% and 40% of CRCs, respectively, and contribute to tumor progression. In the vast majority of human CRCs, TP53 missense mutations lead to high levels of mutant p53 protein expression and the loss of the other wild type TP53 allele. To evaluate the role of TP53 missense mutations in CRC progression, we generated a genetically engineered mouse model of colorectal carcinoma, via combined targeting of Apc, Kras, and Trp53 alleles in mouse colon epithelium, focusing on comparing phenotypic effects of the murine equivalent of the human R273H mutation (i.e., murine R270H mutation) or a large deletion mutation of mouse Trp53. Inactivation of one allele Apc and activation of an oncogenic Kras allele in colon epithelium generated serrated and hyperplastic morphologic epithelium and adenomas. The addition of either the R270H missense mutation or the Trp53 null mutation to the Apc and Kras mutations led to markedly shortened survival of the mice, due to the development of multiple colon tumors in each mouse ranging from adenomas to late stage adenocarcinomas. Evidence of invasion into the smooth muscle and serosa area was found in both compound mice with missense or deletion mutations in Trp53, with the Trp53R270H mutant mice displaying an increased prevalence of deeply invasive tumors relative to the mice with deletion of Trp53. Furthermore, we found that the missense mutant Trp53R270H allele in combination with Apc and Kras mutations, but not the null-mutant Trp53 allele, was strongly linked to metastases to lymph nodes and lung. We have developed a useful mouse model of metastatic colon cancer that recapitulates the role of TP53 mutations in cooperating with APC and KRAS mutations in human CRC development and progression. In addition, our findings strongly suggest a powerful role for missense mutant p53 proteins compared to TP53 null mutations in promoting invasion and metastasis in CRC progression. Citation Format: Ying Feng, Naoya Sakamoto, Maranne Green, Megan Greenn, Kathleen R. Cho, Eric R. Fearon. Differential effects of p53 mutations on cancer invasion and metastasis in a mouse model of colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 672.

Innervation of the Female Levator Ani Muscle and Consequences in Pelvic Floor Dysfunctions

IntroductionThe Levator Ani Muscle (LAM) and the Levator Ani Nerve (LAN) are distended and traumatized during pregnancy and vaginal delivery. Both mechanisms may sustainably impair the pelvis statics but the topography and functions of the nerve supply to the LAM remain anatomically controversial.ObjectiveTo characterize the nature and function of the LAM nerve pathways and to perform a comprehensive 3D reconstruction of the female pelvic innervation.MethodsThe Computer‐Assisted Anatomical dissection (CAAD) protocol was applied to pelvic blocks procured in 6 female human fetuses, in accordance with the National Biomedicine Agency. Specimens were serially sectioned and immunostained for: overall (S100), somatic (PMP22), adrenergic (TH), cholinergic (VaCHT) and nitrergic (nNOS) nerve fibers and for: smooth (smooth actin) and striated (myogenin) muscle fibers. Slides were digitized for 3D reconstructions on WinSurf®. A pelvic sample of an additional fetus was examined under electronic microscopy to analyze the ultrastructure of the LAM.ResultsThree main nerve pathways to the LAM were observed: the levator ani nerve (LAN), the pudendal nerve (PuN), the inferior hypogastric plexus (IHP). The PuN was both somatic and autonomic, located below the LAM (‘infralevator pathway’) and supplying innervation to the inferior aspect of the LAM. The LAN was solely somatic, located above the LAM (‘supralevator pathway’) and supplying innervation to the superior aspect of the LAM. The IHP nerve fibers were solely autonomic, located in between the LAM and the pelvic organs (‘endolevator pathway’) and supplying innervation to the medial portion of the LAM. Smooth muscle areas were identified with labelings and electronic microscopy: they 1) were located in the puboanal sling of the LAM 2) were bulging between rectum and vagina 3) were intermingled to the anococcygeal raphe 4) were innervated by the IHP and the PuN pathways.ConclusionHaving at once striated and smooth muscle fibers and being co‐innervated by the somatic and autonomic systems, the LAM represents a crucial anatomical structure involved in the pelvic floor function and its rehabilitation.Support or Funding Informationnone

Obstructive Sleep Apnoea Modulates Airway Inflammation and Remodelling in Severe Asthma.

BackgroundObstructive sleep apnoea (OSA) is frequently observed in severe asthma but the causal link between the 2 diseases remains hypothetical. The role of OSA-related systemic and airway neutrophilic inflammation in asthma bronchial inflammation or remodelling has been rarely investigated. The aim of this study was to compare hallmarks of inflammation in induced sputum and features of airway remodelling in bronchial biopsies from adult patients with severe asthma with and without OSA.Materials and MethodsAn overnight polygraphy was performed in 55 patients referred for difficult-to-treat asthma, who complained of nocturnal respiratory symptoms, poor sleep quality or fatigue. We compared sputum analysis, reticular basement membrane (RBM) thickness, smooth muscle area, vascular density and inflammatory cell infiltration in bronchial biopsies.ResultsIn total, 27/55 patients (49%) had OSA diagnosed by overnight polygraphy. Despite a moderate increase in apnoea-hypopnoea index (AHI; 14.2±1.6 event/h [5–35]), the proportion of sputum neutrophils was higher and that of macrophages lower in OSA than non-OSA patients, with higher levels of interleukin 8 and matrix metalloproteinase 9. The RBM was significantly thinner in OSA than non-OSA patients (5.8±0.4 vs. 7.8±0.4 μm, p<0.05). RBM thickness and OSA severity assessed by the AHI were negatively correlated (rho = -0.65, p<0.05). OSA and non-OSA patients did not differ in age, sex, BMI, lung function, asthma control findings or treatment.ConclusionMild OSA in patients with severe asthma is associated with increased proportion of neutrophils in sputum and changes in airway remodelling.

Sinomenine attenuates airway inflammation and remodeling in a mouse model of asthma.

Asthma is an inflammatory disease that involves airway inflammation and remodeling. Sinomenine (SIN) has been demonstrated to have immunosuppressive and anti-inflammatory properties. The aim of the present study was to investigate the inhibitory effects of SIN on airway inflammation and remodeling in an asthma mouse model and observe the effects of SIN on the transforming growth factor-β1 (TGF-β1)/connective tissue growth factor (CTGF) pathway and oxidative stress. Female BALB/c mice were sensitized by repetitive ovalbumin (OVA) challenge for 6 weeks in order to develop a mouse model of asthma. OVA-sensitized animals received SIN (25, 50 and 75 mg/kg) or dexamethasone (2 mg/kg). A blank control group received saline only. The area of smooth muscle and collagen, levels of mucus secretion and inflammatory cell infiltration were evaluated 24 h subsequent to the final OVA challenge. mRNA and protein levels of TGF-β1 and CTGF were determined by reverse transcription-quantitative polymerase chain reaction and immunohistology, respectively. The indicators of oxidative stress were detected by spectrophotometry. SIN significantly reduced allergen-induced increases in smooth muscle thickness, mucous gland hypertrophy, goblet cell hyperplasia, collagen deposition and eosinophilic inflammation. The levels of TGF-β1 and CTGF mRNA and protein were significantly reduced in the lungs of mice treated with SIN. Additionally, the total antioxidant capacity was increased in lungs following treatment with SIN. The malondialdehyde content and myeloperoxidase activities in the lungs from OVA-sensitized mice were significantly inhibited by SIN. In conclusion, SIN may inhibit airway inflammation and remodeling in asthma mouse models, and may have therapeutic efficacy in the treatment of asthma.

The effect of asthma on the perimeter of the airway basement membrane.

When comparing the pathology of airways in individuals with and without asthma, the perimeter of the basement membrane (Pbm) is used as a marker of airway size, as it is independent of airway smooth muscle shortening or airway collapse. The extent to which the Pbm is itself altered in asthma has not been quantified. The aim of this study was to compare the Pbm from the same anatomical sites in postmortem lungs from subjects with (n = 55) and without (n = 30) asthma (nonfatal or fatal). Large and small airways were systematically sampled at equidistant "levels" from the apical segment of the left upper lobes and anterior and basal segments of the left lower lobes of lungs fixed in inflation. The length of the Pbm was estimated from cross sections of airway at each relative level. Linear mixed models were used to investigate the relationships between Pbm and sex, age, height, smoking status, airway level, and asthma group. The final model showed significant interactions between Pbm and airway level in small (<3 mm) airways, in subjects having asthma (P < 0.0001), and by sex (P < 0.0001). No significant interactions for Pbm between asthma groups were observed for larger airways (equivalent to a diameter of ∼3 mm and greater) or smoking status. Asthma is not associated with remodeling of the Pbm in large airways. In medium and small airways, the decrease in Pbm in asthma (≤20%) would not account for the published differences in wall area or area of smooth muscle observed in cases of severe asthma.

Effects of Bronchial Thermoplasty on Airway Smooth Muscle and Collagen Deposition in Asthma.

The aim of bronchial thermoplasty is to improve asthma symptoms by reducing central airway smooth muscle mass. Up to now, the reduction of smooth muscle mass has been documented for only 1 group of 10 patients who had 15% or more of their pretreatment total bronchial biopsy area occupied by smooth muscle. To evaluate the effects of bronchial thermoplasty on airway smooth muscle mass and airway collagen deposition in adult patients with asthma, regardless of pretreatment smooth muscle area. Seventeen patients with asthma underwent bronchial thermoplasty over the course of three visits. At Visit 1, bronchial biopsies were taken from the lower lobe that was not treated during this session. At Visit 2 (3-14 wk after the first visit), all 17 patients underwent biopsy of the lower lobe treated during the first procedure. At Visit 3 (7-22 wk after the first visit), nine patients agreed to undergo biopsy of the same lower lobe. Histological and immunohistochemical analyses were performed on the biopsy specimens. Bronchial thermoplasty decreased airway smooth muscle from 12.9 ± 1.2% of the total biopsy surface at Visit 1 to 4.6 ± 0.8% at Visit 2 (P < 0.0001). For the nine patients who underwent a third biopsy, mean airway smooth muscle area was 5.3 ± 1.3% at Visit 3 (P = 0.0008 compared with baseline). Bronchial thermoplasty also decreased Type I collagen deposition underneath the basement membrane from 6.8 ± 0.3 μm at Visit 1 to 4.3 ± 0.2 μm at Visit 2 (P < 0.0001) and to 4.4 ± 0.4 μm for nine patients at Visit 3 (P < 0.0001 compared with baseline). Over the course of 1 year after treatment, the doses of inhaled corticosteroid, the number of severe exacerbations, and asthma control all improved (P ≤ 0.02). For patients with severe asthma, bronchial thermoplasty reduced the smooth muscle mass of treated airway segments, regardless of the baseline level of muscle mass. Treatment also altered the deposition of collagen. At follow-up, bronchial thermoplasty improved asthma control; however, the limited number of subjects did not allow us to evaluate possible correlations between these improvements and the studied histological parameters. Further studies are needed to confirm these results and evaluate their persistence.

Sputum and BAL Clara cell secretory protein and surfactant protein D levels in asthma.

Clara cell secretory protein (CC16) is associated with Th2 modulation. Surfactant protein D (SPD) plays an important role in surfactant homeostasis and eosinophil chemotaxis. We measured CC16 and SPD in sputum supernatants of 84 asthmatic patients and 12 healthy controls. In 22 asthmatics, we additionally measured CC16 and SPD levels in BAL and assessed smooth muscle area (SMA), reticular basement membrane (RBM) thickness, and epithelial detachment (ED) in bronchial biopsies. Induced sputum CC16 and SPD were significantly higher in patients with severe asthma (SRA) compared to mild-moderate and healthy controls. BAL CC16 and SPD levels were also higher in SRA compared to mild-moderate asthma. CC16 BAL levels correlated with ED, while SPD BAL levels correlated with SMA and RBM. Severity represented a significant covariate for these associations. CC16 and SPD levels are upregulated in SRA and correlate with remodeling indices, suggesting a possible role of these biomarkers in the remodeling process.

The role of SDF-1/CXCR4 on airway inflammation and airway remodeling in a rat asthma model

To explore the roles of stromal cell-derived factor 1 (SDF-1) and C-X-C chemokine receptor 4 (CXCR4) on airway inflammation and airway remodeling in rat asthma models. Eighteen female SD rats were randomly divided into 3 groups (n = 6): control group, asthmatic 4 weeks group and asthmatic 8 weeks group. The rats were sensitized and inhaled ovalbumin (OVA). After the asthma model was successfully established, the airway pressure was measured. The methods of HE staining and Image-Pro Plus image analysis software were used to detect the changes of eosinophils (EOS), the perimeter of inner bronchial lumen, the wall area, the area of bronchial smooth muscle and the number of smooth muscle cells of airway walls. RT-PCR and Western-blot were used to detect the expression of SDF-1 and CXCR4 in lung tissues among the 3 groups.Immunohistochemistry was used to detect the expression of SDF-1 in airway walls. Compared with the control group, the airway responsiveness, the count of EOS, the area of bronchial wall, the area of bronchial smooth muscle, the number of smooth muscle cells of airway walls in the asthmatic 4 weeks and asthmatic 8 weeks were significantly increased, and significant difference between the 2 asthmatic groups was also observed in the above indexes (P < 0.01) .RT-PCR showed that compared with the control group (SDF-1 was 0.146 ± 0.003 and CXCR4 was 0.281 ± 0.002) , the expression of SDF-1 (0.583 ± 0.004 and 0.724 ± 0.008) and CXCR4 (0.467 ± 0.003 and 0.655 ± 0.002) in lung tissues in the asthmatic 4 weeks and asthmatic 8 weeks were significantly increased (P < 0.01) . In addition, compared with the asthmatic 4 weeks group, the expression of SDF-1 and CXCR4 in lung tissues in the 8 weeks asthmatic group were significantly increased (P < 0.01) . Compared with the control group (0.180 ± 0.009) , the expression of SDF-1 in airway walls in the asthmatic 4 weeks and asthmatic 8 weeks groups (0.270 ± 0.006 and 0.350 ± 0.009) were significantly increased (P < 0.01) . In addition, compared with the asthmatic 4 weeks group, the expression of SDF-1 in airway walls in the 8 weeks asthmatic group was significantly increased (P < 0.01) . The expression of SDF-1 and CXCR4 was correlated positively with the airway responsiveness, the number of EOS, the area of bronchial wall, the area of bronchial smooth muscle and the number of smooth muscle cells of airway walls (P < 0.01). SDF-1/CXCR4 axis may play a key role in airway inflammation and airway remodeling of asthma.

Antiasthmatic effects of resveratrol in ovalbumin-induced asthma model mice involved in the upregulation of PTEN.

Resveratrol, a natural polyphenolic compound known for its antioxidative and antiinflammatory effects, exerts antiasthmatic effects, although the mechanism underlying these effects remains elusive. The phosphatase and tensin homology deleted on chromosome ten gene (PTEN) is involved in the pathogenesis of asthma, and PTEN overexpression in asthmatic mice improved asthma symptoms. To investigate whether the antiasthmatic mechanisms of resveratrol correlated with the upregulation of PTEN expression, an ovalbumin (OVA)-induced murine asthma model was used to determine the effectiveness of resveratrol treatment. PTEN mRNA and protein expression was assessed with real-time polymerase chain reaction (PCR) and immunochemistry. To determine whether airway remodeling occurred, the inner airway wall, mucous layer, and smooth muscle areas were each determined using an image analysis system. The lung epithelial cell line 16HBE was used to study the regulation of PTEN expression levels by resveratrol in vitro. Our data demonstrated that resveratrol inhibited OVA-induced airway inflammation and airway remodeling in asthmatic mice. PTEN expression was decreased in the murine asthma model, although the expression of PTEN was restored following treatment with resveratrol. Correlation efficiency analysis showed that PTEN expression was associated with the degree of airway remodeling. Further in vitro studies demonstrated that the inhibition of Sirtuin 1 (SIRT1) activity by a SIRT1 inhibitor and RNA interference decreased PTEN protein expression, while resveratrol attenuated the decreases in PTEN expression induced by the SIRT1 inhibitor. These data suggest the mechanism of the antiasthmatic effects of resveratrol in an OVA-induced murine asthma model, which resulted in the upregulation of PTEN via SIRT1 activation.

Triptolide suppresses airway goblet cell hyperplasia and Muc5ac expression via NF-κB in a murine model of asthma

BackgroundWe have reported that triptolide inhibited pulmonary inflammation in patients with steroid-resistant asthma. In the present study, we investigated whether suppresses airway remodeling and goblet cell hyperplasia, studied the mechanism of triptolide on mucin5ac (Muc5ac) expression in a murine model of asthma. MethodsBALB/c mice were sensitized to intraperitoneal ovalbumin (OVA) followed by repetitive ovalbumin challenge for 6 weeks. Treatments included triptolide (40μg/kg) and dexamethasone (2mg/kg). The area of bronchial airway (WAt/Pbm), smooth muscle (WAm/Pbm) and mucus index were assessed 24h after the final OVA challenge. Levels of Muc5ac were assessed by ELISA, immunohistology and real-time PCR. Western blot was performed to analyze the phosphorylation of NF-κB p65. ResultsTriptolide and dexamethasone significantly reduced allergen-induced increases in the thickness of bronchial airway, smooth muscle and goblet cell hyperplasia. Levels of lung Muc5ac and Muc5ac mRNA were significantly reduced in mice treated with triptolide and dexamethasone. Phosphorylation of NF-κB p65 was significantly reduced in mice treated with triptolide and dexamethasone. ConclusionTriptolide may inhibit airway goblet cell hyperplasia and Muc5ac expression in asthmatic mice via NF-κB. It may be a potential drug for the treatment of patients with severe asthma.

Intrauterine growth restriction is associated with structural alterations in human umbilical cord and decreased nitric oxide-induced relaxation of umbilical vein

IntroductionIntrauterine growth restriction (IUGR) affects ∼8% of all pregnancies and is associated with major perinatal mortality and morbidity, and with an increased risk to develop cardiovascular diseases in adulthood. Despite identification of several risk factors, the mechanisms implicated in the development of IUGR remain poorly understood. In case of placental insufficiency, reduced delivery of oxygen and/or nutrients to the fetus could be associated with alterations in the umbilical circulation, contributing further to the impairment of maternal–fetal exchanges. We compared the structural and functional properties of umbilical cords from growth-restricted and appropriate for gestational age (AGA) term newborns, with particular attention to the umbilical vein (UV). MethodsHuman umbilical cords were collected at delivery. Morphological changes were investigated by histomorphometry, and UV's reactivity by pharmacological studies. ResultsGrowth-restricted newborns displayed significantly lower growth parameters, placental weight and umbilical cord diameter than AGA controls. Total cross-section and smooth muscle areas were significantly smaller in UV of growth-restricted neonates than in controls. Maximal vasoconstriction achieved in isolated UV was lower in growth-restricted boys than in controls, whereas nitric oxide-induced relaxation was significantly reduced in UV of growth-restricted girls compared to controls. ConclusionIUGR is associated with structural alterations of the UV in both genders, and with a decreased nitric oxide-induced relaxation in UV of newborn girls, whereas boys display impaired vasoconstriction. Further investigations will allow to better understand the regulation of umbilical circulation in growth-restricted neonates, which could contribute to devise potential novel therapeutic strategies to prevent or limit the development of IUGR.

Central airways remodeling in COPD patients

BackgroundThe contribution to airflow obstruction by the remodeling of the peripheral airways in chronic obstructive pulmonary disease (COPD) patients has been well documented, but less is known about the role played by the large airways. Few studies have investigated the presence of histopathological changes due to remodeling in the large airways of COPD patients.ObjectivesThe aim of this study was to verify the presence of airway remodeling in the central airways of COPD patients, quantifying the airway smooth muscle (ASM) area and the extracellular matrix (ECM) protein deposition, both in the subepithelial region and in the ASM, and to verify the possible contribution to airflow obstruction by the above mentioned histopathological changes.MethodsBiopsies of segmental bronchi spurs were performed in COPD patients and control smoker subjects and immunostained for collagen type I, versican, decorin, biglycan, and alpha-smooth muscle actin. ECM protein deposition was measured at both subepithelial, and ASM layers.ResultsThe staining for collagen I and versican was greater in the subepithelial layer of COPD patients than in control subjects. An inverse correlation was found between collagen I in the subepithelial layer and both forced expiratory volume in 1 second and ratio between forced expiratory volume in 1 second and forced vital capacity. A statistically significant increase of the ASM area was observed in the central airways of COPD patients versus controls.ConclusionThese findings indicate that airway remodeling also affects the large airways in COPD patients who have greater deposition of ECM proteins in the subepithelial layer and a larger smooth muscle area than control smoker subjects. These changes may contribute to chronic airflow obstruction in COPD patients.

AB191. The roles of polyuria and hyperglycemia in bladder dysfunction in long-term diabetic rats

Aims of studyDiabetic bladder dysfunction (DBD), a collective description of clinical symptoms including decreased sensation, increased capacity, poor emptying, and also detrusor overactivity, is among the most common and costly complications of diabetes mellitus (DM). It is estimated that DBD occurs in approximately 87% of individuals diagnosed with DM, and substantially affects quality of life. Yet, little is known about the pathogenic mechanisms of DBD. Unlike other organs, the bladder experiences not only hyperglycemia, but also an increased volume of urine in DM. To aid in our knowledge of the pathophysiology of DBD and to aid development of specific treatments, identification of individual contributions of polyuria and hyperglycemia in the DBD is essential.Materials and methodsSeventy two female Sprague-Dawley rats were divided into six groups: age-matched controls (control), sham urinary diversion (sham), urinary diversion (UD), streptozotocin-induced diabetics after sham urinary DM, streptozotocin-induced diabetics after urinary diversion (UD + DM), and 5% sucrose-induced diuretics after sham urinary diversion (DIU). UD was performed 10 days before diabetes induction by surgical disconnection of the ureters from the bladder and implantation to uterine cervix. Each group was subsequently evaluated 20 weeks after diabetes or diuresis induction. Twenty-four hour drinking and voided volumes were measured. Conscious cystometry (CMG) was examined. The bladders were harvested for histological examination and quantification of smooth muscle, urothelium, and collagen. The expressions of oxidative stress-related proteins, nitrotyrosine and manganese superoxide dismutase (MnSOD), in bladder were examined.ResultsDiabetes and diuresis caused increases in drinking volume, voided volume and bladder weight. The bladder weight decreased in the UD and UD + DM group. CMG showed increased inter-contractile intervals, voided volume and compliance in DIU and DM group, whereas decreased in the UD, and further in UD + DM group. The total tissue area, the tissue area of smooth muscle and urethelium increased in DIU and DM group, whereas decreased in UD and UD + DM group. As a percentage of the total cross sectional tissue area, collagen decreased in DIU and DM group, but increased in UD and UD + DM groups; smooth muscle and urothelium decreased in UD and UD + DM groups. The expression of nitrotyrosine and MnSOD increased in the DM and the UD + DM groups compared with other four groups.ConclusionsPolyuria and hyperglycemia contribute differently in DBD. Polyuria induced significant bladder hypertrophy, whereas chronic hyperglycemia induces oxidative stress in bladder, which may play an important role in the later stage of DBD.

Implantation of Autologous Adipose-Derived Cells Reconstructs Functional Urethral Sphincters in Rabbit Cryoinjured Urethra

We investigated the ability of autologous adipose-derived cells injected into cryoinjured rabbit urethras to improve urinary continence and explored the possible mechanisms by which it occurred. Adipose tissue was harvested from the perivesical region of nine 10-week-old female New Zealand White rabbits and cultured for 7 days. Immediately after harvesting the tissue, we injured the internal urethral orifice by spraying liquid nitrogen for 20 s. The cultured cells expressed the mesenchymal cell marker STRO1, but not muscle cell markers myoglobin or smooth muscle actin (SMA). Just before implantation, the adipose-derived cells were labeled with the PKH26 fluorescent cell linker. Autologous 2.0×10(6) adipose-derived cells (five rabbits) or a cell-free control solution (four rabbits) was injected around the cryoinjured urethras at 7 days after injury. Fourteen days later, the leak point pressure (LPP) was measured, and the urethras were harvested for immunohistochemical analyses. At 14 days after implantation, LPP of the cell-implanted group was significantly higher compared with the cell-free control group (p<0.05). In immunohistochemical examination, the reconstructed skeletal and smooth muscle areas in the cell-implanted regions were significantly more developed than those in controls (p<0.01). Implanted PKH26-labeled adipose-derived cells were immunohistochemically positive for myoglobin, SMA, and Pax7 antibodies, which are markers for skeletal muscles, smooth muscles, and myoblast progenitor cells, respectively. In addition, these implanted cells were positive for the nerve cell markers, tubulin β3, S100, and the vascular endothelial cell marker, von Willebrand factor. Furthermore, some of the implanted cells were positive for the transforming growth factor β1, nerve growth factor, and vascular endothelial growth factor. In conclusion, implantation of autologous adipose-derived cells into the cryoinjured rabbit urethras promoted the recovery of urethral function by myogenic differentiation, neuroregeneration, and neoangiogenesis of the implanted cells and/or the surrounding tissues as well as by bulking effects. Thus, treatment of human radical prostatectomy-related stress urinary incontinence by adipose-derived cell implantation could have significant therapeutic effects.

Abnormal tracheal smooth muscle function in the CF mouse

Increased airway smooth muscle (ASM) contractility is thought to underlie symptoms of airway hyperresponsiveness (AHR). In the cystic fibrosis (CF) airway, ASM anomalies have been reported, but have not been fully characterized and the underlying mechanisms are largely unknown. We examined ASM in an adult CF mouse tracheal ring preparation, and determined whether changes in contractility were associated with altered ASM morphology. We looked for inherent changes in the cellular pathways involved in contractility, and characterized trachea morphology in the adult trachea and in an embryonic lung culture model during development. Results showed that that there was a reduction in tracheal caliber in CF mice as indicated by a reduction in the number of cartilage rings; proximal cross-sectional areas of cftr−/− tracheas and luminal areas were significantly smaller, but there was no difference in the area or distribution of smooth muscle. Morphological differences observed in adult trachea were not evident in the embryonic lung at 11.5 days gestation or after 72 h in culture. Functional data showed a significant reduction in the amplitude and duration of contraction in response to carbachol (CCh) in Ca-free conditions. The reduction in contraction was agonist specific, and occurred throughout the length of the trachea. These data show that there is a loss in the contractile capacity of the CF mouse trachea due to downregulation of the pathway specific to acetylcholine (ACh) activation. This reduction in contraction is not associated with changes in the area or distribution of ASM.

CGMP kinase: past, presence and future

Signaling via cGMP-dependent protein kinase is the major pathway of the NO/cGMP cascade in vascular smooth muscle (SM), heart, CNS, and other cells. cGMP-dependent protein kinases (cGK) are serine/threonine kinases that are widely distributed in eukaryotes. Two genes – prkg1 and prkg2 - code for cGKs, namely cGKI and cGKII. In mammals, two isozymes, cGKIα and cGKIβ, are generated from the prkg1 gene. The cGKI isozymes are prominent in all types of smooth muscle, platelets and specific neuronal areas such as cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The cGKII prevails in the secretory epithelium of the small intestine, the juxta-glomerular cells, the adrenal cortex, the chondrocytes, and in the nucleus suprachiasmaticus. Both cGKs are major down- stream effectors of many, but not all signalling events of the NO/cGMP and the ANP/BNP/CNP/cGMP pathways. cGKI relaxes smooth muscle tone and prevents platelet aggregation, whereas cGKII inhibits renin secretion, chloride/water secretion in the small intestine, the resetting of the clock during early night and endochondreal bone growth. cGKs are involved in cardiovascular and non-cardiovascular processes and are modulators of cell growth and many other functions. Recent evidence suggest that vascular smooth muscle relaxation is not affected by cGK-dependent phosphorylation of smooth muscle expressed TRPC channels, but by cGK-dependent inhibition of TRPC-6 in endothelial cells and attenuation of endothelial release of smooth muscle relaxing factors. Angiotensin increases blood pressure and induces cardiac hypertrophy. Hypertrophy is reduced in sildenafil treated wild type mice but not in beta rescue mice. This suggests that the sildenafil effects are mediated by inhibition of a cGMP hydrolysing phosphodiesterase. Restricted evidence supports the notion that sildenafil affects cGMP levels in cardiomyofibroblasts and/or in cardiac endothelial cells. It will be shown that cGK are essential modulators of many regulatory processes.