Influence of Hypoxia on the Epithelial-Pathogen Interactions in the Lung: Implications for Respiratory Disease.

Under normal physiological conditions, the lung remains an oxygen rich environment. However, prominent regions of hypoxia are a common feature of infected and inflamed tissues and many chronic inflammatory respiratory diseases are associated with mucosal and systemic hypoxia. The airway epithelium represents a key interface with the external environment and is the first line of defense against potentially harmful agents including respiratory pathogens. The protective arsenal of the airway epithelium is provided in the form of physical barriers, and the production of an array of antimicrobial host defense molecules, proinflammatory cytokines and chemokines, in response to activation by receptors. Dysregulation of the airway epithelial innate immune response is associated with a compromised immunity and chronic inflammation of the lung. An increasing body of evidence indicates a distinct role for hypoxia in the dysfunction of the airway epithelium and in the responses of both innate immunity and of respiratory pathogens. Here we review the current evidence around the role of tissue hypoxia in modulating the host-pathogen interaction at the airway epithelium. Furthermore, we highlight the work needed to delineate the role of tissue hypoxia in the pathophysiology of chronic inflammatory lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease in addition to novel respiratory diseases such as COVID-19. Elucidating the molecular mechanisms underlying the epithelial-pathogen interactions in the setting of hypoxia will enable better understanding of persistent infections and complex disease processes in chronic inflammatory lung diseases and may aid the identification of novel therapeutic targets and strategies.

The mechanism underlying hypaconitine-mediated alleviation of pancreatitis-associated lung injury through up-regulating aquaporin-1/TNF-α.

BACKGROUND/AIMS: Acute pancreatitis-associated lung injury (APALI) is one of the most common and most dangerous form of extra-pancreatic organ damage in severe acute pancreatitis (SAP). The treatment options for SAP were limited thus far; as a result, approximately 60%-80% of patients with SAP would die within a week. Hypaconitine (HC), one of the most important active ingredients in a Mongolian traditional medicine Radix Aconiti Kusnezoffii has an excellent anti-inflammatory effect. MATERIALS AND METHODS: To ascertain whether HC has a protective effect against APALI, we investigated the therapeutic effects and the underlying mechanisms in vivo and in vitro and attempted to elucidate the mechanism in detail. In this study, APALI rats and human pulmonary microvascular endothelial cells were treated with therapeutic doses of HC after establishing a model with sodium taurocholate and lipopolysaccharide, respectively. RESULTS: Serum amylase and lipase activity, lung wet/dry weight ratio, lung myeloperoxidase activity, and pancreatic and lung histopathological changes showed that HC alleviated APALI in a dose-dependent way, which can be abolished by an aquaporin-1 (AQP-1) knockdown. The results of the reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemical staining confirmed the expression of AQP-1, a kind of transmembrane protein that mainly distributed in the membranes of pulmonary cells and contributed to maintain water balance in the body by interacting with tumor necrosis factor-alpha (TNF-α), is negatively associated with APALI. On the contrary, HC treatment up-regulated AQP-1 expression and down-regulated the TNF-α expression as a consequence in APALI. CONCLUSION: These results suggest that HC has a good anti-inflammatory therapeutic effect on APALI with a possible underlying mechanism that affects the AQP-1/TNF-α pathway.

Value of pre- and intraoperative diagnostic methods in suspected glottic neoplasia.

PURPOSE: To evaluate the individual and combined ability of videostroboscopy (VS), high-speed digital imaging (HSDI), enhanced endoscopy (EE) and saline infusion (SI) to predict neoplasia, defined as glottic precursor lesion (GPL) or T1a glottic cancer, in patients suspected for glottic neoplasia. METHODS: A nationwide prospective cohort study of patients treated by cordectomy for suspected GPL or T1a glottic cancer from August 1st 2016 to October 31st 2018 was conducted in the five Danish University Departments of Head and Neck surgery. Sensitivity, specificity, negative and positive predictive values, and area under Receiver Operating Curves (AUC-ROC) were calculated with 95% confidence intervals with respect to the histological diagnosis. Logistic regression with an imputation model for missing data was applied. RESULTS: 261 patients aged 34-91 years participated; 79 (30.3%) with non-neoplasia (i.e., inflammation, papilloma, hyperkeratosis) and 182 (69.7%) neoplasia, hereof 95 (36.4%) with GPL and 87 (33.3%) with T1a glottic cancer. Data from 188 VS, 60 HSDI, 100 preoperative EE, 209 intraoperative EE, and 234 SI were analyzed. In the complete case analysis the AUC-ROC of each diagnostic test was low, but increased when the tests were combined and especially if the combination included EE. However, multinomial logistic regression with imputation showed significant association (p < 0.05) only between age, male gender, and perpendicular vasculature in intraoperative EE, and the endpoint neoplasia. CONCLUSIONS: Intraoperative EE was the most accurate diagnostic method in detecting neoplasia. The prediction ability of methods applied preoperatively was more limited, but improved when test modalities were combined.

Requirements for Successful Trachea Transplantation: A Study in the Rabbit Model.

BACKGROUND: Although creating a tracheal tube de novo might appear straightforward, the first clinical applications have shown that reconstruction of long-segment tracheal defects remains challenging. In this study, the authors aimed to refine the baseline requirements of successful trachea transplantation by means of three proof-of-concept models in the rabbit. METHODS: In each model, one characteristic of a perfect tracheal transplant was eliminated. The first model was developed to map out the immunologic response of vascularized allogenic trachea, transplanted without immunosuppression (n = 6). The second model studied (1) the need for wrapping the transplant with a highly vascularized flap and (2) the source of angiogenesis after autologous trachea transplantation (n = 18). In the third model, the authors examined the importance of an inner epithelial covering (n = 12). All models were compared to a control group of heterotopically transplanted vascularized autologous tracheae (n = 6). RESULTS: Embedded in an avascular matrix, allogenic chondrocytes were protected from an overt immune response. Orthotopic transplantation without additional external vascular wrap was successful in a well-vascularized environment. Nonetheless, an external vascular source was essential to maintain viability of the construct. Epithelial covering was necessary to prevent secondary healing. Epithelial migration from the anastomoses or graft was not sufficient to cover long-segment defects. CONCLUSIONS: These experiments provided ample evidence of the importance of baseline requirements when designing a tracheal transplant study. This study confirmed that different tracheal cell types possess different immunologic sensitivities. External revascularization, preferably in a two-stage procedure, and fast reepithelialization were both paramount to successful transplantation.

Image-enhanced bronchoscopic evaluation of bronchial mucosal microvasculature in COPD.

BACKGROUND: Bronchial vascular remodeling is an underresearched component of airway remodeling in COPD. Image-enhanced bronchoscopy may offer a less invasive method for studying bronchial microvasculature in COPD. OBJECTIVES: To evaluate endobronchial mucosal vasculature and changes in COPD by image-enhanced i-scan3 bronchoscopy and correlate them pathologically by analyzing bronchial mucosal biopsies. METHODS: This case-control study analyzed 29 COPD patients (41.4% Global initiative for chronic Obstructive Lung Disease B [GOLD B] and 58.6% GOLD D) and ten healthy controls admitted at Alexandria Main University Hospital, Egypt. Combined high-definition white light bronchoscopy (HD WLB) with i-scan3 was used to evaluate endobronchial mucosal microvasculature. The vascularity was graded according to the level of mucosal red discoloration (ie, endobronchial erythema) from decreased discoloration to normal, mild, moderate, and severe increased red discoloration (G-1, G0, G+1, G+2, and G+3, respectively) and scored by three bronchoscopists independently. Bronchial mucosal biopsies were taken for microvascular density counting using anti-CD34 antibody as angiogenesis marker. RESULTS: Different grades of endobronchial erythema were observed across/within COPD patients using combined HD WLB + i-scan3, with significant agreement among scorers (P=0.031; median score of G+1 [G-1-G+2]) being higher in GOLD D (P=0.001). Endobronchial erythema significantly correlated with COPD duration, exacerbation frequency, and body mass index (P<0.05). Angiogenesis was significantly decreased among COPD patients versus controls (10.6 [8-13.3] vs 14 [11-17.1]; P=0.02). Mucosal surface changes (including edema, atrophy, and nodules) were better visualized by the combined HD WLB + i-scan3 rather than HD WLB alone. CONCLUSION: Combined HD WLB + i-scan3 seems to be valuable in evaluating mucosal microvasculature and surface changes in COPD, which may represent vasodilatation rather than angiogenesis.

Grape seed procyanidin extract attenuates hypoxic pulmonary hypertension by inhibiting oxidative stress and pulmonary arterial smooth muscle cells proliferation.

Hypoxia-induced oxidative stress and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Grape seed procyanidin extract (GSPE) possesses antioxidant properties and has beneficial effects on the cardiovascular system. However, the effect of GSPE on HPH remains unclear. In this study, adult Sprague-Dawley rats were exposed to intermittent chronic hypoxia for 4 weeks to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio and median width of pulmonary arteries. GSPE attenuated the elevation of RVSP, RV/LV+S, and reduced the pulmonary vascular structure remodeling. GSPE also increased the levels of SOD and reduced the levels of MDA in hypoxia-induced HPH model. In addition, GSPE suppressed Nox4 mRNA levels, ROS production and PASMCs proliferation. Meanwhile, increased expression of phospho-STAT3, cyclin D1, cyclin D3 and Ki67 in PASMCs caused by hypoxia was down-regulated by GSPE. These results suggested that GSPE might potentially prevent HPH via antioxidant and antiproliferative mechanisms.

Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways.

The basic understanding of inflammatory airway diseases greatly benefits from imaging the cellular dynamics of immune cells. Current imaging approaches focus on labeling specific cells to follow their dynamics but fail to visualize the surrounding tissue. To overcome this problem, we evaluated autofluorescence multiphoton microscopy for following the motion and interaction of cells in the airways in the context of tissue morphology. Freshly isolated murine tracheae from healthy mice and mice with experimental allergic airway inflammation were examined by autofluorescence multiphoton microscopy. In addition, fluorescently labeled ovalbumin and fluorophore-labeled antibodies were applied to visualize antigen uptake and to identify specific cell populations, respectively. The trachea in living mice was imaged to verify that the ex vivo preparation reflects the in vivo situation. Autofluorescence multiphoton microscopy was also tested to examine human tissue from patients in short-term tissue culture. Using autofluorescence, the epithelium, underlying cells, and fibers of the connective tissue, as well as blood vessels, were identified in isolated tracheae. Similar structures were visualized in living mice and in the human airway tissue. In explanted murine airways, mobile cells were localized within the tissue and we could follow their migration, interactions between individual cells, and their phagocytic activity. During allergic airway inflammation, increased number of eosinophil and neutrophil granulocytes were detected that moved within the connective tissue and immediately below the epithelium without damaging the epithelial cells or connective tissues. Contacts between granulocytes were transient lasting 3 min on average. Unexpectedly, prolonged interactions between granulocytes and antigen-uptaking cells were observed lasting for an average of 13 min. Our results indicate that autofluorescence-based imaging can detect previously unknown immune cell interactions in the airways. The method also holds the potential to be used during diagnostic procedures in humans if integrated into a bronchoscope.

[Morphometric characteristics of the bronchial tree in smokers with and without chronic obstructive pulmonary disease].

AIM: To reveal the tissue, cellular, and molecular predictors leading to the development of chronic obstructive pulmonary disease (COPD) in smokers on the basis of a morphometric analysis of bronchial biopsy specimens. MATERIALS AND METHODS: A cross-sectional study was conducted in 115 smokers aged 40 to 60 years (58.3±3.24 years) with a male predominance of 90.9%. Morphological examination of bronchial biopsy specimens was first made in the smoking patients with and without COPD. RESULTS: The smokers with COPD versus those without this condition were found to have a statistically significant increase in a number of indicators: the specific volume (SV) of the surface epithelium (p=0.017), SV of basal epitheliocytes (p=0.008), the height of the epithelium (p=0.001), and the thickness of the basal membrane (p=0.006) due to impaired regeneration processes in the bronchial epithelium and to fibrosis of the lamina propria of the bronchi with a concurrent increase in the total number of fibroblasts. The group of smokers with COPD, unlike the comparison group, showed signs of vascular remodeling and microcirculatory disorders as the increased connective tissue volumetric density of the lamina propria of the bronchi with predominant perivascular localization. This was followed by reductions in the relative volume of capillaries (p=0.016), in the SV of micropinocytic vesicles (p=0.005), and the size of Weibel-Palade bodies (p=0.004) in the endotheliocytes. In the COPD patients, the total density of cell infiltrate per mm2 of the lamina propria of the bronchi was statistically significantly (p<0.001) greater than that in the comparison group at the expense of neutrophils, lowly and moderately granulated basophils, macrophages, plasma cells with a simultaneous rise in their interepithelial forms. CONCLUSION: Compensatory structural changes without signs of bronchial wall remodeling were recorded in the smokers without signs of bronchial disease. The smokers with the examined nosological entity were observed to have morphological signs of hemodynamic disorders, as well as perivascular fibrosis, atrophy, and squamous cell metaplasia of the bronchial epithelial lining.

Ex Vivo Artifacts and Histopathologic Pitfalls in the Lung.

CONTEXT: Surgical and pathologic handling of lung physically affects lung tissue. This leads to artifacts that alter the morphologic appearance of pulmonary parenchyma. OBJECTIVE: To describe and illustrate mechanisms of ex vivo artifacts that may lead to diagnostic pitfalls. DESIGN: In this study 4 mechanisms of ex vivo artifacts and corresponding diagnostic pitfalls are described and illustrated. RESULTS: The 4 patterns of artifacts are: (1) surgical collapse, due to the removal of air and blood from pulmonary resections; (2) ex vivo contraction of bronchial and bronchiolar smooth muscle; (3) clamping edema of open lung biopsies; and (4) spreading of tissue fragments and individual cells through a knife surface. Morphologic pitfalls include diagnostic patterns of adenocarcinoma, asthma, constrictive bronchiolitis, and lymphedema. CONCLUSION: Four patterns of pulmonary ex vivo artifacts are important to recognize in order to avoid morphologic misinterpretations.

Pediatric Endotracheal Tube Cuff Pressures During Aeromedical Transport.

OBJECTIVES: Cuffed endotracheal tubes (ETTs) are frequently used in children, allowing fewer air leaks and helping prevent ventilator-associated pneumonia. Tracheal mucosal perfusion is compromised at an ETT cuff pressure (ETTCP) of 30 cm H2O with blood flow completely absent above 50 cm H2O. Our objective was to compare multiple pediatric-sized ETTCPs at ground level and various altitudes during aeromedical transport. METHODS: Simulating the transport environment, 4 pediatric-sized mannequin heads were intubated with appropriately sized cuffed ETTs (3.0, 4.0, 5.0, 6.0) and transported by helicopter or nonpressurized fixed-wing aircraft 20 times each. The ETTCP was set to 10 cm H2O before transport, and the pressure was measured with a standard manometer at 1000-ft intervals until reaching peak altitude or CP greater than 60 cm H2O. Ground elevation ranged from 400-650 ft mean sea level (MSL) and peak altitude from 3500 to 5000 ft MSL. RESULTS: Increased altitude caused a significant increase in ETTCP of all ETT sizes (P < 0.001). However, there is no statistical difference in pressures between ETT sizes (P = 0.28). On average, ETTCP in 3.0, 4.0, and 6.0 ETTs surpassed 30 cm H2O at approximately 1500 ft MSL and 50 cm H2O at approximately 2800 ft MSL. In the 5.0 ETT, the CP reached 30 cm H2O at 2000 ft MSL and 50 cm H2O at 3700 ft MSL. CONCLUSIONS: The ETTCP in pediatric-sized ETTs regularly exceed recommended pressure limits at relatively low altitudes. There is no additional pressure increase related to ETT size. This has the potential to decrease mucosal blood flow, possibly increasing risk of subsequent tracheal stenosis, rupture, and other complications.

A case of massive hemoptysis related to a smoking-history: an acquired form of the Dieulafoy's disease?

The hypervascularization of the bronquial wall, secondary to chronic bronchopulmonary inflammation is a bleeding etiology in smokers, but insufficient to explain certain massive recurrent cases. We report a case of a woman with a smoking history who presented a recurrent and massive hemoptysis. A diagnostic study with laboratory tests, bronchoscopy, computed tomography and echocardiogram did not identify the etiological cause. However, bronchial arteriography showed right and left bronchial tortuous and dilated arteries and demonstrated that a bronchovascular fistula was the origin of the hemoptysis. An acquired form of the Dieulafoy's disease in this context of a smoking history might justify such findings. Bronchial arteriography as a diagnostic method should be the preferred choice rather than bronchoscopy in these cases.

The H2S-generating enzymes cystathionine ß-synthase and cystathionine γ-lyase play a role in vascular development during normal lung alveolarization.

The gasotransmitter hydrogen sulfide (H2S) is emerging as a mediator of lung physiology and disease. Recent studies revealed that H2S administration limited perturbations to lung structure in experimental animal models of bronchopulmonary dysplasia (BPD), partially restoring alveolarization, limiting pulmonary hypertension, limiting inflammation, and promoting epithelial repair. No studies have addressed roles for endogenous H2S in lung development. H2S is endogenously generated by cystathionine ß-synthase (Cbs) and cystathionine γ-lyase (Cth). We demonstrate here that the expression of Cbs and Cth in mouse lungs is dynamically regulated during lung alveolarization and that alveolarization is blunted in Cbs(-/-) and Cth(-/-) mouse pups, where a 50% reduction in the total number of alveoli was observed, without any impact on septal thickness. Laser-capture microdissection and immunofluorescence staining indicated that Cbs and Cth were expressed in the airway epithelium and lung vessels. Loss of Cbs and Cth led to a 100-500% increase in the muscularization of small- and medium-sized lung vessels, which was accompanied by increased vessel wall thickness, and an apparent decrease in lung vascular supply. Ablation of Cbs expression using small interfering RNA or pharmacological inhibition of Cth using propargylglycine in lung endothelial cells limited angiogenic capacity, causing a 30-40% decrease in tube length and a 50% decrease in number of tubes formed. In contrast, exogenous administration of H2S with GYY4137 promoted endothelial tube formation. These data confirm a key role for the H2S-generating enzymes Cbs and Cth in pulmonary vascular development and homeostasis and in lung alveolarization.

Omeprazole Attenuates Pulmonary Aryl Hydrocarbon Receptor Activation and Potentiates Hyperoxia-Induced Developmental Lung Injury in Newborn Mice.

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in human preterm infants and a similar lung phenotype characterized by alveolar simplification in newborn mice. Omeprazole (OM) is a proton pump inhibitor that is used to treat humans with gastric acid related disorders. OM-mediated aryl hydrocarbon receptor (AhR) activation attenuates acute hyperoxic lung injury (HLI) in adult mice. Whether OM activates pulmonary AhR and protects C57BL/6J newborn mice against hyperoxia-induced developmental lung (alveolar and pulmonary vascular simplification, inflammation, and oxidative stress) injury (HDLI) is unknown. Therefore, we tested the hypothesis that OM will activate pulmonary AhR and mitigate HDLI in newborn mice. Newborn mice were treated daily with i.p. injections of OM at doses of 10 (OM10) or 25 (OM25) mg/kg while being exposed to air or hyperoxia (FiO2 of 85%) for 14 days, following which their lungs were harvested to determine alveolarization, pulmonary vascularization, inflammation, oxidative stress, vascular injury, and AhR activation. To our surprise, hyperoxia-induced alveolar and pulmonary vascular simplification, inflammation, oxidative stress, and vascular injury were augmented in OM25-treated animals. These findings were associated with attenuated pulmonary vascular endothelial growth factor receptor 2 expression and decreased pulmonary AhR activation in the OM25 group. We conclude that contrary to our hypothesis, OM decreases functional activation of pulmonary AhR and potentiates HDLI in newborn mice. These observations are consistent with our previous findings, which suggest that AhR activation plays a protective role in HDLI in newborn mice.

Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

Amelioration of carbon tetrachloride-induced pulmonary toxicity with Oxalis corniculata.

This research work was planned to investigate the antioxidant potential of methanolic crude extract of Oxalis corniculata (OCME) against lung injuries initiated by carbon tetrachloride (CCl4) in rats at histological and biochemical level. A total of 42 female Sprague Dawley rats were randomly distributed in to seven groups and each group comprised of six rats. Experiment was completed in 22 days (10 doses at alternate days). Group I was not treated (control rats), while group II was administered with vehicles (olive oil and dimethyl sulfoxide), groups III, IV, and V were treated with 1 ml kg(-1) body weight (b.w.) of CCl4 (20% in olive oil). Group III received only CCl4, whereas groups IV and V were administered with 100 and 200 mg kg(-1) b.w. of OCME, respectively. Group VI was administered with OCME (200 mg kg(-1) b.w.) alone. Group VII was treated with sylimarin (50 mg kg(-1) b.w.). CCl4 enhanced the lipid peroxidation while reduced the glutathione in lung samples. Activities of antioxidant enzymes, catalase, peroxidase, superoxide dismutase, and glutathione-S-transferase decreased in lung homogenates with CCl4. Treatment of CCl4 induced deleterious changes in the microanatomy of lungs by rupturing the alveolar septa, thickening of alveolar walls, and damaging the cells with subsequent collapse of blood vessels due to the accumulation of degenerated blood cells. OCME, dose dependently, prevented the alterations in these parameters. These results suggest that OCME protected the lungs due to its intrinsic properties by scavenging of free radicals generated by CCl4.

Increased exhaled breath temperature in subjects with uncontrolled asthma.

BACKGROUND: Increased vascularity of the airway mucosa in asthma potentially increases heat loss in the airways. OBJECTIVE: To determine if the inflamed airways of subjects with uncontrolled asthma show increased exhaled breath temperature (EBT). PATIENTS AND METHODS: In 100 patients with persistent asthma and 50 healthy volunteers, we measured lung function by post-bronchodilator forced spirometry, the asthma control test (ACT) and EBT. RESULTS: Patients with asthma, of whom 49 (49%) were female, with a mean (± standard error of the mean) age of 44 (±17) years and a predicted forced expiratory volume in one second of 71% (±16), had a significantly increased EBT, particularly those with uncontrolled asthma (n = 50, ACT ≤ 19, EBT 34.9 ± 0.8°C), compared to patients with controlled asthma (n = 50, ACT ≥ 20, EBT 33.7 ± 0.8°C) and healthy volunteers (n = 50, EBT 33.2 ± 0.2°C, P < 0.001). CONCLUSION: We observed a higher temperature on exhaled breath in subjects with uncontrolled asthma than in subjects with controlled asthma and healthy controls. The increase in exhaled breath temperature may be a proxy for a raised airway inflammatory state in asthma patients.

Alveolocapillary model system to study alveolar re-epithelialization.

In the present study an in vitro bilayer model system of the pulmonary alveolocapillary barrier was established to investigate the role of the microvascular endothelium on re-epithelialization. The model system, confluent monolayer cultures on opposing sides of a porous membrane, consisted of a human microvascular endothelial cell line (HPMEC-ST1.6R) and an alveolar type II like cell line (A549), stably expressing EGFP and mCherry, respectively. These fluorescent proteins allowed the real time assessment of the integrity of the monolayers and the automated analysis of the wound healing process after a scratch injury. The HPMECs significantly attenuated the speed of re-epithelialization, which was associated with the proximity to the A549 layer. Examination of cross-sectional transmission electron micrographs of the model system revealed protrusions through the membrane pores and close contact between the A549 cells and the HPMECs. Immunohistochemical analysis showed that these close contacts consisted of heterocellular gap-, tight- and adherens-junctions. Additional analysis, using a fluorescent probe to assess gap-junctional communication, revealed that the HPMECs and A549 cells were able to exchange the fluorophore, which could be abrogated by disrupting the gap junctions using connexin mimetic peptides. These data suggest that the pulmonary microvascular endothelium may impact the re-epithelialization process.

Does the hypopharyngeal cavernous body protect the development of Zenker's diverticulum?

OBJECTIVE: The aim of this study was to examine the morphology of the cavernous body at the pharyngoesophageal segment. METHODS: In 47 cadavers the submucosal vascular plexus of the pharyngoesophageal segment and the cricopharyngeal muscle were examined. RESULTS: A vascular plexus which was macroscopically non visible or slightly identified was observed in the majority of the specimens (28 or 59.6%). Fourteen of the cadavers (29.8%) exhibited macroscopically lightly observable blood vessels which were covering a discontinuous area of the hypopharyngeal wall. In the other five specimens (10.6%) the vascular plexus was covering a large part of the dorsal wall of the pharyngoesophageal segment as a compact mass. The grade of dilation of the vascular plexus corresponded to the degree of protrusion of the cricopharyngeal muscle. CONCLUSIONS: In the case of a persistent constricted cricopharyngeal muscle, the dilation of the hypopharyngeal cavernous body may protect from developing a Zenker's diverticulum by reinforcing the Killian's dehiscence.

Relationship between immunohistochemical assessment of bronchial mucosa microvascularization and clinical stage in asthma.

Although hardly ever used in current practice, fibrobronchoscopy may provide interesting histopathological-clinical correlations in patients diagnosed with different stages of evolutive asthma. The aim of the study was to evaluate the correlation between semi-quantitative microvascularization features and the asthma severity assessed according to the GINA classification 2006. Our study group consisted in 21 patients diagnosed with asthma of different stages of severity and two-control patients investigated by fibrobronchoscopy with associated biopsy. The tissue fragments underwent standard processing procedures for the immunohistochemical exam, using CD34 as microvascularization marker. The semi-quantitative analysis was based on the "hot spot" method and on a score system that corresponds to the microvessels density. The statistical analysis of the correspondence between CD34 score and clinical parameters was performed using the SPSS 17 software, applying non-parametric correlation tests. The CD34 evaluation showed an increase in blood vessels count in all asthmatic patients in comparison to the control group and a close correlation with the asthma severity, reflected by the FEV1 values. The statistical analysis showed an inverse correlation between FEV1 [%] values and CD34 expression (r=-0.93, p<<0.01). Our data concur to other research reports, supporting the hypothesis that angiogenesis initially facilitates the edema development and later on appears to be involved in the bronchial wall thickening, as a component of the chronic inflammatory response, with concomitant distensibility reduction. The bronchial mucosa microvascularization evaluation opens new perspectives for advanced therapies, with beneficial effects for asthmatic patients' life quality.

Differential effects of inhaled methacholine on circumferential wall and vascular smooth muscle of third-generation airways in awake sheep.

Evolution and natural selection ensure that specific mechanisms exist for selective airway absorption of inhaled atmospheric molecules. Indeed, nebulized cholinoceptor agonists used in asthma-challenge tests may or may not enter the systemic circulation. We examined the hypothesis that inhaled cholinoceptor agonists have selective access. Six sheep were instrumented under general anesthesia (propofol 5 mg/kg iv, 2-3% isoflurane-oxygen), each with pulsed-Doppler blood flow transducers mounted on the single bronchial artery and sonomicrometer probes mounted on the intrapulmonary third-generation lingula lobe bronchus. Continuous measurements were made of bronchial blood flow (Q(br)), Q(br) conductance (C(br)), bronchial hemicircumference (CIRC(br)), and bronchial wall thickness (WALL TH(br)) in recovered, standing, awake sheep. Methacholine (MCh; 0.125-2.0 µg/kg iv), at the highest dose, caused a 233% rise in Q(br) (P < 0.05) and a 286% rise in C(br) (P < 0.05). CIRC(br) fell to 90% (P < 0.05); WALL TH(br) did not change. In contrast, nebulized MCh (1-32 mg/ml), inhaled through a mask at the highest dose, caused a rise in ventilation and a rise in Q(br) proportional to aortic pressure without change in C(br). CIRC(br) fell to 91% (P < 0.01), and WALL TH(br) did not change. Thus inhaled MCh has access to cholinoceptors of bronchial circumferential smooth muscle to cause airway lumen narrowing but effectively not to those of the systemic bronchovascular circulation. It is speculated that the mechanism is selective neuroparacrine inhibition of muscarinic acetylcholine receptors (M3 bronchovascular cholinoceptors) by prostanoids released by intense MCh activation of epithelial and mucosal cells lining the airway.