Septal Capillaries Research Articles

Prematurity and Hyperoxia Have Different Effects on Alveolar and Microvascular Lung Development in the Rabbit.

Bronchopulmonary dysplasia (BPD) is a developmental disorder of infants born prematurely, characterized by disrupted alveolarization and microvascular maturation. However, the sequence of alveolar and vascular alterations is currently not fully understood. Therefore, we used a rabbit model to evaluate alveolar and vascular development under preterm birth and hyperoxia, respectively. Pups were born by cesarean section 3 days before term and exposed for 7 days to hyperoxia (95% O2) or normoxia (21% O2). In addition, term-born rabbits were exposed to normoxia for 4 days. Rabbit lungs were fixed by vascular perfusion and prepared for stereological analysis. Normoxic preterm rabbits had a significantly lower number of alveoli than term rabbits. The number of septal capillaries was lower in preterm rabbits but less pronounced than the alveolar reduction. In hyperoxic preterm rabbits, the number of alveoli was similar to that in normoxic preterm animals; however, hyperoxia had a severe additional negative effect on the capillary number. In conclusion, preterm birth had a strong effect on alveolar development, and hyperoxia had a more pronounced effect on capillary development. The data provide a complex picture of the vascular hypothesis of BPD which rather seems to reflect the ambient oxygen concentration than the effect of premature birth.

Acute lethal leptospirosis in a red fox (Vulpesvulpes)

Leptospirosis, caused by pathogenic spirochaetes belonging to the genus Leptospira, is a globally distributed zoonosis that can affect many species of domestic and wild animals, and humans. The red fox (Vulpes vulpes) is a native species in Croatia and, due to constant food availability and lack of interspecies competition, is becoming more abundant in urban and suburban areas. Although antibodies for Leptospira spp have been detected in red foxes, lethal disease has not been reported. We necropsied a young, male red fox that had jaundice, multifocal haemorrhages in the heart, lungs and urinary bladder, hepatomegaly, non-congestive splenomegaly and slight yellow discolouration of the renal cortex and medulla. Histology revealed multifocal haemorrhages in parenchymal organs, thrombi within lung septal capillaries and other blood vessels, interstitial lymphocytic and plasmacytic nephritis and erosions of the gastric mucosa. A microscopic agglutination test on the post-mortem cardiac blood clot revealed a high titre to Leptospira interrogans serovar Icterohaemorrhagiae, which implies contact with rats, which are natural reservoirs of this serovar in Croatia. The gross and histological findings in this fox were similar to those in dogs with leptospirosis, indicating that fatal leptospirosis can occur in foxes and suggesting that this species can act as a source of infection for other animals and humans.

Differential temporal development of alveoli and the alveolar capillary network in the postnatal rat lung.

Quantitative data about the internal lung structure are needed to better understand normal and pathological lung development. Aberrant lung development causes deficits in alveolar and microvascular development; however, the normal temporal relationship between these processes is still not fully understood. We hypothesized that alveolar and capillary development show a differential time pattern. Lungs of rats aged 3, 7, 14, 21 days (d) or 3 mo (n = 8-10 each) were fixed by vascular perfusion and processed for light microscopy. Using design-based stereology number, the surface area and volume of alveoli, septal capillaries, and alveolar septa were quantified. The total number and the total volume of alveoli increased progressively during postnatal development. Interestingly, the numerical density of capillary loops was significantly higher in 14- and 21-d-old rats than before or after this age, causing a duplication of the total number of capillary loops between 1 and 2 wk of age. The mean thickness of alveolar septa started to decline slightly at the age of 14d and more pronounced at later stages. Although the septal epithelial surface area increased in proportion to alveolar number during the first 3 wk, the capillary endothelial surface area grew only slightly compared with the number of capillaries. In conclusion, the number of elements composing the alveolar capillary network expands massively during the first two postnatal weeks and exceeds the formation of alveoli. The thinning of the alveolar septa during further development suggests a reduction of the capillary network during alveolarization.

Autopsy findings of pediatric COVID-19: a systematic review

BackgroundLittle is known how COVID-19 is affecting children. Autopsies help gain an understanding of the pathophysiology of new and developing diseases. Numerous post-mortem studies had been conducted in adults with COVID-19, but few in children. Thereby, this systematic review aims to investigate the autopsy findings from pediatric COVID-19 patients.ResultsThere were a total of 15 patients from eight studies. COVID-19 mainly affects the heart and lungs. Pathology findings from the heart of COVID-19 pediatric patients include diffuse inflammatory infiltrate, myocarditis, cardiomyocyte necrosis, pericarditis, and interstitial edema. Histopathology abnormalities observed in the lungs are diffuse alveolar damage, cytopathic changes, thrombi in arterioles and septal capillaries, lung congestion, focal acute hemorrhage and edema, focal exudative changes, and mild pneumocyte hyperplasia. In addition, pathological findings from other organs, such as the liver, kidney, brain, bone marrow, lymph node, skin, spleen, muscle, colon, parotid gland, and adrenal of COVID-19 pediatric patients are also included in this review.ConclusionCardiomyocyte necrosis, interstitial edema, lung congestion, and diffuse alveolar damage are the most significant pathologic findings of the heart and lung in pediatric COVID-19 patients. More studies are needed to elucidate the pathophysiology of SARS-CoV-2 in autopsy findings and to determine the exact cause of death since it could be related to COVID-19 or other comorbidities.

DIFFUSE ALVEOLAR HEMORRHAGE RESULTING FROM RIVAROXABAN USE: A RARE COMPLICATION IN A PATIENT WITHOUT PREDISPOSING CONDITIONS

TOPIC: Diffuse Lung Disease TYPE: Fellow Case Reports INTRODUCTION: Diffuse alveolar hemorrhage (DAH) is a rare complication of direct oral anticoagulant (DOAC) use with few reported cases in patients with predisposing conditions or underlying lung disease. DAH results from disruption of the alveolar-capillary interface. Infectious, autoimmune, and drug mediated conditions may lead to DAH. Rivaroxaban is a DOAC used to treat venous thromboembolism (VTE) and non-valvular atrial fibrillation (NVAF). We present a case of a patient who developed DAH one month after starting rivaroxaban. CASE PRESENTATION: A 43-year-old female with a past medical history of hyperlipidemia and tobacco use disorder presented with frank hemoptysis and shortness of breath. She was diagnosed with lower extremity deep vein thrombosis (DVT) and was started on rivaroxaban. Vitals were stable without hypoxia. Hemoglobin dropped from 14 to 7 g/dL (normal 12.3-15.3 g/dL) requiring 2 units of blood. Prothrombin Complex Concentrate was given. Computed tomography angiogram of the chest showed bilateral lung fields with patchy ground-glass opacities with peripheral sparing, without significant intra-lobular septal thickening and no pulmonary embolus. Infectious work up was negative. Rheumatological workup with anti-glomerular basement antibodies, anti-double stranded DNA antibodies, lupus anticoagulant, antinuclear antibody, rheumatoid factor, anti-neutrophilic cytoplasmic antibody, proteinase-3 antibody, myeloperoxidase antibody, C-reactive protein and erythrocyte sedimentation rate was negative. Bronchoscopy confirmed the diagnosis of DAH. Hemoptysis resolved after discontinuing rivaroxaban, repeat lower extremity dopplers were negative for DVT. She was discharged home without anticoagulation. DISCUSSION: DAH occurs from direct injury or inflammation of the pulmonary arterioles, venules, or alveolar septal capillaries causing bleeding into the lung. It presents with non specific symptoms including cough, chest pain, hemoptysis, and dyspnea. It is life threatening with fast clinical deterioration leading to respiratory failure. The underlying vascular injury of DAH is based on histological appearance and is divided into pulmonary capillaritis, bland pulmonary hemorrhage, and diffuse alveolar damage. The diagnosis is usually made clinically and supported with imaging and bronchoscopic findings. There are few cases of rivaroxaban induced DAH in the literature. The majority of the cases report patients with an underlying medical conditions such as autoimmune disease, lung cancer, interstitial lung disease and pulmonary hypertension, predisposing them to DAH. Our patient had no predisposing factors that put her at risk of developing DAH. CONCLUSIONS: Oral anticoagulants are a under-recognized cause of DAH with limited literature on the incidence of DAH with the use of these medications. This case illustrates that patients with no underlying risk factors can develop DAH with the use of rivaroxaban. REFERENCE #1: Collard HR, Schwarz MI. Diffuse alveolar hemorrhage. Clinics in Chest Medicine 2004; 25(3): 583-92. REFERENCE #2: Park MS. Diffuse alveolar hemorrhage. Tuberc Respir Dis (Seoul) 2013; 74(4): 151-62. REFERENCE #3: Chen A, Stecker E, Warden BA. Direct Oral Anticoagulant Use: A Practical Guide to Common Clinical Challenges. Journal of the American Heart Association 2020; 9(13): e017559 DISCLOSURES: No relevant relationships by Ahmad Alhajhusain, source=Web Response No relevant relationships by Stephanie Baltaji, source=Web Response No relevant relationships by Helene Rovnan, source=Web Response

Regional and organ-level responses to local lung irradiation in sheep

Lung is a dose-limiting organ in radiotherapy. This may limit tumour control when effort is made in planning to limit the likelihood of radiation-induced lung injury (RILI). Understanding the factors that dictate susceptibility to radiation-induced pulmonary fibrosis will aid in the prevention and management of RILI, and may lead to more effective personalized radiotherapy treatment. As the interaction of regional and organ-level responses may shape the chronic consequences of RILI, we sought to characterise both aspects of the response in an ovine model. A defined volume of left pulmonary parenchyma was prescribed 5 fractions of 6 Gy within 14 days while the contralateral lung dose was constrained. Radiographic changes via computed tomography (CT) were documented to define differences in radio-exposed lung relative to non-exposed lung at d21, d63 and d171 (n = 2), and at d21, d147 and d227 (n = 2). Gross and histologic lung changes were evaluated in samples derived at necropsy examination to define the chronic pulmonary response to radiation. Irradiated lung demonstrated reduced radio-density and increased homogeneity as evidenced from texture based radiomic feature analysis, relative to the control lung. At necropsy, the radiation field was readily defined by pallor on the pleural surface, which was also evident on the cut surface of fixed lung specimens. The degree and homogeneity of pallor reflected the sparse presence of erythrocytes in alveolar septal capillaries of radiation-exposed lung. These changes contrasted with dilated and congested microvasculature in the contralateral control lung. Referencing data to measurements made in control lung volumes of sheep experiencing acute RILI indicated that interstitial collagen continues to deposit in the radio-exposed lung field. Overall lung vascularity increased during the chronic response, as evidenced by increased expression of endothelial cell marker (CD31); however, vascularity was consistently decreased in irradiated lung and was negatively correlated with lung collagen. Other organ-level responses included increased expression of alpha smooth muscle actin (ASMA), increased numbers of proliferating cells (Ki67 positive), and cells expressing the dendritic cell-lysosomal associated membrane protein (DC-LAMP) antigen. The chronic response to RILI in this model is effected at both the whole organ and local lung level. Whilst the long-term consequences of exposure to radiation involved the continued deposition of collagen in the radiation field, organ-level responses also included increased vascularization and increased expression of ASMA, Ki67 and DC-LAMP. Interrupting the interplay between these aspects may influence susceptibility to pulmonary fibrosis after radiotherapy. We advocate for the importance of large animal model systems in pursuing these opportunities to target local, organ-level and systemic mechanisms in parallel within the same subject over time.

Severe COVID-19: A multifaceted viral vasculopathy syndrome

The objective of this study was to elucidate the pathophysiology that underlies severe COVID-19 by assessing the histopathology and the in situ detection of infectious SARS-CoV-2 and viral capsid proteins along with the cellular target(s) and host response from twelve autopsies. There were three key findings: 1) high copy infectious virus was limited mostly to the alveolar macrophages and endothelial cells of the septal capillaries; 2) viral spike protein without viral RNA localized to ACE2+ endothelial cells in microvessels that were most abundant in the subcutaneous fat and brain; 3) although both infectious virus and docked viral spike protein was associated with complement activation, only the endocytosed pseudovirions induced a marked up-regulation of the key COVID-19 associated proteins IL6, TNF alpha, IL1 beta, p38, IL8, and caspase 3 in endothelium. Importantly, this microvasculitis was associated with characteristic findings on hematoxylin and eosin examination that included endothelial degeneration and resultant basement membrane zone disruption and reduplication. It is concluded that serious COVID-19 infection has two distinct mechanisms: 1) a microangiopathy of pulmonary capillaries associated with a high infectious viral load where endothelial cell death releases pseudovirions into the circulation, and 2) the pseudovirions dock on ACE2+ endothelial cells most prevalent in the skin/subcutaneous fat and brain that activates the complement pathway/coagulation cascade resulting in a systemic procoagulant state as well as endothelial expression of cytokines that produce the cytokine storm. The data predicts a favorable response to therapies based on either removal of circulating viral proteins and/or blunting of the endothelial-induced response.

Potential role of GcMAF in suppressing the severity of COVID-19-induced immune responses: Lesson learned from HIV

Over the last six months, there have been increasing numbers of reports that struggle to understand the pathogenesis of the coronavirus disease 2019 (COVID-19) pandemic. As of August 16, 2020, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for more than 21 294 000 infections and about 760 000 deaths worldwide [1], but the mechanisms of virus-induced host damage remain a mystery. Understanding the pathways behind viral pathogenicity just like cellular and tissue tropism, counteracting host defence processes and immunological responses are crucial to find new therapeutic strategies. Based on clinical reports, it is noteworthy that COVID-19 causes various degree of illness ranging from asymptomatic or milder symptomatic cases to severe lung injury or even multi-organ dysfunction with liver and kidney impairment [2], [3], [4]. Even in not severe patients, the heterogeneity of symptoms is consistent with the increasing evidence that SARS-CoV-2 shows a broad tissue tropism, being able to attack almost anything in the body [2], [3], [4], [5]. To date, the most commonly investigated hypothesis about the underlying mechanisms of multi-organ failure may be summarized into three main targets: microcirculation dysfunction, overwhelming inflammation and abnormal coagulation [7]. Radiologic and laboratory findings as well as preliminary autopsy studies seem to support this hypothesis. The most common patterns seen on chest CT were ground-glass opacity, interlobular septal thickening, air bronchogram, bilateral patchy shadowing, crazy-paving pattern, and thickening of the adjacent pleura, resembling an interstitial involvement in viral pneumonia [2], [3], [7], [8]. Under the light of microscope, the lungs revealed diffuse alveolar damage with formation of numerous hyaline membranes, very patchy and sparse interstitial chronic inflammation composed mainly of lymphocytes, thrombi within a few small pulmonary artery branches, congestion of alveolar septal capillaries, focal edema fluid, and macrophage infiltration within the airspaces [9], [10], [11], [12]. The more significant laboratory abnormalities were metabolic acidosis, lymphocytopenia, leukopenia, thrombocytopenia, elevated levels of C-reactive protein (CRP), interleukin-6 (IL-6), lactate dehydrogenase (LDH) and D-dimer [2], [3], [4], [5], [6], [7]. As firstly suggested by Huang C et al. [4], the systemic cytokine storm could play a key role in the virus-induced tissue damage. However, the question “what the link for the overproduction of pro-inflammatory mediators and the immune suppression, on the hand, and microvascular injury and thromboembolism, on the other, is”, remains unclear. Being the knowledge of this issue very scarce, lessons learned from other human pathogenic viruses, with specific reference to human immunodeficiency virus (HIV), could be diriment.

Positive pressure ventilation compresses pulmonary acinar microvessels but not their supply vessels

Pulmonary alveolar septal capillaries receive their perfusion from a web of larger surrounding acinar vessels. Using 4 μm diam. Latex particles, we showed that positive pressure ventilation narrowed the acinar vessels as evidenced by venous 4 μm particle concentrations and perfusate flows <50% of particle concentrations in negative pressure ventilated lungs. We aimed to understand the effects of positive and negative pressure ventilation on flows of larger particles through the lung. Isolated, ventilated rat lungs (air, transpulmonary pressures of 15/5 cm H2O, 25 breaths/min) were perfused with a hetastarch solution at Ppulm art/PLA pressures of 10/0 cm H2O. Red latex 7 μm (2.5 mg, 4.8 × 106) or 15 μm (2.5 mg, 5.5 × 105) particles were infused into each lung during positive or negative pressure ventilation. An equal number of green particles was infused 20 min later. Flows through lungs infused with 7 μm and 15 μm particles were not different from flows through lungs infused with 4 μm particles (p = 0.811). Venous particle concentrations of 7 μm particles relative to infused particles were lower in positive pressure lungs (0.03 ± 0.03%) compared to negative pressure lungs (0.17 ± 0.12%) (p = 0.041). Venous particle concentrations of 15 μm particles were not different between positive (2.3 ± 1.9%) and negative (3.3 ± 1.8%) pressure ventilation (p = 0.406). Together with our previous study, we conclude that 4 μm and 7 μm particles both enter acinar vessels but that the 7 μm particles are too large to flow through those vessels. In contrast, we conclude that 15 μm particles bypass the acinar vessels, flowing instead through larger intrapulmonary vessels to enter the venous outflow. These findings suggest that intrapulmonary vessels are organized as a web that allows bypass of the acinar vessels by large particles and, that these bypass vessels are not compressed by positive pressure ventilation.

Expression of von Willebrand factor, pulmonary intravascular macrophages, and Toll-like receptors in lungs of septic foals.

Sepsis causes significant mortality in neonatal foals; however, there is little data describing the cellular and molecular pathways of lung inflammation in septic foals. This study was conducted to characterize lung inflammation in septic foals. Lung tissue sections from control (n = 6) and septic (n = 17) foals were compared using histology and immunohistology. Blinded pathologic scoring of hematoxylin and eosin stained samples revealed increased features of lung inflammation such as thickened alveolar septa and sequestered inflammatory cells in septic foals. Septic foal lungs showed increased expression of von Willebrand factor in blood vessels, demonstrating vascular inflammation. Use of MAC387 antibody to detect calprotectin as a reflection of mononuclear cell infiltration revealed a significant increase in their numbers in alveolar septa of lungs from septic foals compared to those from control foals. The mononuclear cells appeared to be mature macrophages and were located in the septal capillaries, suggesting they were pulmonary intravascular macrophages (PIMs). Finally, lungs from septic foals showed increased expression of Toll-like receptor 4 and 9 in mononuclear cells relative to the control. Taken together, this study is the first to show the expression of inflammatory molecules and an increase in PIMs in lungs from foals that died from sepsis.

Lung Inflammation and Pulmonary Intravascular Macrophage Recruitment in L‐Arginine‐Induced Acute Necrotizing Pancreatitis in Mouse

Human patients suffering from severe acute pancreatitis are at higher risk of developing acute lung injury with high morbidity and mortality rates. Although there are many studies on the mechanisms leading to acute lung injury associated with acute pancreatitis in experimental models, there has not been any investigation on the induction of pulmonary intravascular macrophages (PIMs), a pro‐inflammatory cell normally present in cattle, pig, and horses, during this pathological condition in animal models. Therefore, we tested the hypothesis that lungs from mice with L‐arginine‐induced acute necrotizing pancreatitis (ANP) will be inflamed along with the recruitment of PIMs, compared to lungs of control mice. ANP was induced in C57BL/6 mice by administering two intraperitoneal injections, one hour apart, of a sterile L‐arginine monohydrochloride 9% solution at the dose of 4.5g/kg per injection, and mice were euthanized at 24h (n=7), 72h (n=7) and 120h (n=7) post‐injections. Control mice (n=9) received the same injections but with physiological sterile saline. ANP was confirmed by a pathologist using a histological grading system for pancreatic necrosis, infiltration of inflammatory cells and edema, which generated total ANP severity scores as follows: median (with range) of 0 for controls, 1 (0–3) for the 24h group, 8 (6–10) for the 72h group and 4 (2–10) for the 120h group. Serum amylase was significantly increased in the 24h group (5528±1210 U/L) and the 72h group (6547±1275 U/L), but not in the 120h group (1399±62 U/L), compared to control (1483±48 U/L), p&lt;0.001. The lung histological grading revealed infiltration of mononuclear phagocytes in the lungs of mice in the 72h group only, compared to the control lungs (p&lt;0.001). Immunohistochemistry with anti‐macrophage CD‐68 antibody revealed an increase of monocytes/macrophages present in alveolar septa of the lungs of all mice with ANP, suggesting PIMs induction, compared with control mice (p&lt;0.01), with cell counts (median; range) as follows: control mice (81; 55–147), 24h group (150; 115–190), 72h group (190; 118–200) and 120h group (135; 97–194). Bioplex assay on lung homogenates identified increased levels of IL‐6 (p &lt; 0,0001), IL‐10 (p &lt; 0,01), and MCP‐1 (p &lt; 0,05) in the 24h group only, compared to control mice. Immunohistochemistry for vWF revealed granular staining in the alveolar septal capillaries of some ANP mice, but not in control mice, suggesting activation of the lung microvasculature endothelium during ANP. The data show that lung inflammation in mice with L‐arginine‐induced ANP is initiated as early as 24h and is characterised by the infiltration of septal mononuclear phagocytes, most probably PIMs, as well as by increased lung concentrations of IL‐6, IL‐10 and MCP‐1. Because of the established role of PIMs in lung inflammation, the mouse model of ANP will be a useful tool to investigate the biology of PIMs in ANP‐associated lung inflammation.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada

Fipronil induces lung inflammation in vivo and cell death in vitro.

BackgroundFipronil is an insecticide that acts at the gamma-aminobutyric acid receptor and glutamate-gated chloride channels in the central nervous systems of target organisms. The use of fipronil is increasing across the globe. Presently, very little data exist on the potential impact of exposure to fipronil on the lungs.MethodsWe studied effects of intranasal (N = 8) and oral (N = 8) treatment with fipronil (10 mg/kg) on lungs of mice. Control mice were given groundnut oil orally (N = 7) or ethanol intranasally (N = 7) as these were the vehicles for respective treatments.ResultsHematoxylin-eosin stained lung sections showed normal histology in the control lungs compared to the thickened alveolar septa, disruption of the airways epithelium and damage to vascular endothelium in the intranasal and the oral groups. Mice exposed to fipronil either orally or intranasally showed increased von Willebrand factor staining in the endothelium and septal capillaries. Compared to the control mice, TLR4 expression in airway epithelium was increased in mice treated intranasally but not orally with fipronil. Oral fipronil reduced TLR9 staining in the airway epithelium but intranasal exposure caused intense staining in the alveolar septa and airway epithelium. There were higher numbers of TLR4 positive cells in alveolar septa in lungs of mice treated intranasally (P = 0.010) compared to the respective control and orally treated mice but no significant differences between treatments for TLR9 positive stained cells (P = 0.226). The U937 macrophage cells exposed to fipronil at concentrations of 0.29 μm to 5.72 μm/ml over 3- or 24-hour showed significant increase in cell death at higher concentrations of fipronil (P < 0.0001). Western blots revealed no effect of fipronil on TLR4 (P = 0.49) or TLR9 (P = 0.94) expression on macrophage cell line.ConclusionWhile both oral or intranasal fipronil treatments induced signs of lung inflammation, the number TLR4-positive septal cells was increased only following intranasal treatment. Fipronil causes macrophage cell death without altering TLR4 and TLR9 expression in vitro.

Lung inflammation associated with acute necrotizing pancreatitis in dogs (LB513)

There is evidence that dogs suffering from acute necrotizing pancreatitis (ANP) can develop respiratory disease. However, there are no data describing the lung histopathology in clinical cases of ANP in dogs. Therefore, we hypothesized that lungs of dogs that died due to ANP (n=21) will be inflamed along with recruitment of PIMs, a pro‐inflammatory cell normally present in ruminants and equines, compared to lungs of control dogs (n=6). Histologic sections of the pancreas graded independently by two blinded pathologists showed the presence of necrosis and infiltration of inflammatory cells (scores 3‐10) whereas control dogs received a score of 0. Lungs from ANP dogs showed no (score of 0/6) to moderate (score 3/6) inflammation, but there was no statistical difference between the lung histological score of the control (median ± SEM: 0.5±0.3) and the ANP (median ± SEM: 1.5±0.2) dogs. However, immunohistochemistry revealed an increase in the numbers of septal macrophages/monocytes in lungs of ANP dogs (median ± SEM: 138±49) compared to control dogs (median ± SEM: 1.5±0,2; p&lt;0,01). There was a moderate correlation (r=0.46) between the recruitment of macrophages/monocytes in the lungs and the total inflammatory cells score in the pancreas of ANP dogs. Only ANP dogs showed expression of vWF in alveolar septal capillaries and septal inflammatory cells. iNOS was expressed only in alveolar macrophages of ANP dogs. TLR4 and IL6 were variably expressed and localized in alveolar macrophages and septal inflammatory cells in lungs from both ANP and control dogs. We conclude that dogs suffering from ANP have pronounced lung inflammation along with recruitment of PIMs and increased expression of vWF. The PIMs may increase the susceptibility of the host to lung inflammation in response to circulating endotoxins.Grant Funding Source: Supported by Companion Animal Health Fund

Ca2+ entry via α1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca(2+) influx in response to diverse environmental stimuli. However, it is not clear whether Ca(2+) influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the alpha(1G) T-type Ca(2+) channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca(2+), immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K(+) perfusate induces Ca(2+) entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca(2+) entry in septal endothelium evoked by the selective TRPV4 activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of alpha(1G) abolishes depolarization-induced Ca(2+) entry and surface expression of P-selectin but does not prevent 4alpha-PDD-activated Ca(2+) entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4alpha-PDD-activated Ca(2+) entry and the increase in permeability, while not impacting depolarization-induced Ca(2+) entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca(2+) entry through alpha(1G) and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

Mitochondria determine TNFα receptor distribution in lung microvessels

Circulating tumor necrosis factor alpha (TNFα) ligates endothelial (EC) TNFα receptor 1 (TNFR1) to promote inflammation. Although mitochondria are capable of proinflammatory signaling in lung EC (Ichimura, JCI 2003), the mitochondrial role in TNFα‐mediated inflammation remains unknown. To determine mitochondria‐TNFR1 relationships, we viewed ECs of single microvessels in isolated, blood‐perfused rat lungs by live confocal microscopy. To determine spatial expressions of EC mitochondria and TNFR1 by fluorescence quantification, we infused microvessels respectively, with the mitochondria‐specific fluorophore, mitotracker green (MTG), or a fluorophore‐conjugated, anti‐TNFR1 mAb. Buffer microinjections removed unbound dye. Microvascular expressions for both mitochondria and TNFR1 were 3‐fold higher at venular branch points than at septal capillaries (p&lt;0.05, n=4). Fluorescence determinations along the microvascular length indicated that mitochondria and TNFR1 fluorescence intensities correlated at venular branch points (p&lt;0.05 by linear regression), but not in septal capillaries. These findings are the first evidence that in lung microvessels, TNFR1 localizes to mitochondria‐rich regions. Mitochondrial mechanisms may regulate TNFR1‐induced inflammatory responses.

Fat Embolism Secondary to Yellow Fat Disease in an Appaloosa Horse

A 4-year-old female Appaloosa horse was referred to the Department of Comparative Pathology, Veterinary Faculty, with a history of colic and acute respiratory distress. At necropsy, gross lesions consisted of extensive firm, multinodular, hemorrhagic foci in fat tissues with yellow-brown discoloration. The most affected areas were peritoneal fat and perirenal, epicardial, and subcutaneous adipose tissues. Other findings were hepatic lipidosis and multiple 1-1.5 cm hemorrhagic foci scattered in both lungs. Histopathological examination revealed severe degeneration and necrosis of adipose tissue with dystrophic calcification. The necrotic fat was infiltrated by abundant foamy macrophages and multinucleated giant cells. Based on these histopathological changes, yellow fat disease, also called nutritional panniculitis, was diagnosed. In addition, the microscopic examination of lung and kidney sections stained with osmium tetroxide and oil red O revealed numerous lipid droplets within glomerular and alveolar septal capillaries. To the authors' knowledge, this is the first description of the development of fat embolism in horses affected by yellow fat disease.

α 1G T‐type Ca 2+ channel controls plasma membrane P‐selectin expression in pulmonary microvascular endothelial cells

The objective of this study was to determine the in vivo relevance of our previous in vitro observation, that CaV3.1 (α1G) T-type Ca2+ channel controls the thrombin-stimulated exocytosis of Weibel-Palade bodies in pulmonary microvascular endothelial cells. We utilized confocal fluorescence microscopy and isolated perfused lung to characterize the surface expression of P-selectin, a Weibel-Palade body membrane protein, at the apical plasma membrane of lung endothelial cells following thrombin stimulation in α1G knockout mice and their wild-type littermate mice. We found that in wild-type mice, thrombin stimulation induced membrane P-selectin expression in lung endothelial cells in both septal capillaries and extra-alveolar vessels. Pharmacological T-type Ca2+ channel blockade using mibefradil prevented thrombin stimulated-membrane P-selectin expression in septal but not extra-alveolar endothelia. In α1G knockout mice, thrombin stimulated-membrane P-selectin expression was attenuated in septal capillary endothelial cells, but not extra-alveolar vessel endothelial cells, compared to those of wild-type mice. Taken together, these results suggest that α1G T-type Ca2+ channel importantly contributes to agonist-induced lung microvascular endothelial cell procoagulant phenotype. Supported by HL-66299.

Activation of TRPV4 increases endothelial permeability in lung septal capillaries

Activation of store-operated Ca2+ channels disrupts endothelial cell (EC) junctions in lung extra-alveolar vessels, while the channel responsible for Ca2+ entry-dependent injury in septal capillaries is unknown. Since 14,15-epoxyeicosatrienoic acid (14,15-EET) causes EC injury in rat lung septal capillaries and EETs activate Ca2+ entry via the vanilloid transient receptor potential channel TRPV4 in aortic EC, we tested whether activation of TRPV4 increases septal EC permeability. Lungs from anesthetized wild type mice (TRPV4+/+) and null littermates (TRPV4−/−) (22.2±0.9 g, mean±SE) were perfused at 0.055±0.002 mL/min/g body wt with 4% albumin/physiologic buffer (37 °C). EC permeability was measured via the filtration coefficient (Kf) at baseline and 45 min after treatment with 4α-phorbol-12,13-didecanoate (4αPDD, 10 μM) to activate TRPV4, thapsigargin (TG, 150 nM) to activate store-operated Ca2+ channels, or vehicle (DMSO, 50 μL). TG increased permeability by 2.8- and 3.2-fold in wild type and null groups, respectively (*p<0.05, paired t-test). In contrast, 4αPDD increased permeability by 2.9-fold in lungs from TRPV4+/+ mice, but had no impact in TRPV4−/− mice. Transmissionelectron microscopy revealed that 4αPDD resulted in blebbing of septal EC in wild type mice while extra-alveolar vessel EC junctions were intact, a pattern similar to that with 14,15-EET in rat lung. We conclude that TRPV4 constitutes a novel Ca2+ entry pathway involved in regulation of lung EC permeability in septal capillaries. Supported by HL081851 and SE AHA 0315049B.

Ca 2+ communication through connexin 43 in lung capillaries

The extent to which endothelial (EC) cells support gap junctional intercellular communication (GJIC) remains unknown. To determine GJIC, by fluorescence microscopy we viewed septal capillaries of isolated, blood-perfused lungs from wild type (WT) mice, and from mice with targeted deletion of the gap junctional protein, connexin 43 (Cx43) in EC (Cx43-/-) (Liao, et al., PNAS, 2001). Lung artery, left atrial and airway pressures were held at 10, 3 and 5 cm H2O, respectively. We co-loaded vessels with the Ca2+ cage, NP-EGTA and the cytosolic Ca2+ (Ca2+cyt) indicator, fluo 4. At baseline, EC Ca 2+cyt was 88±17 gray levels. To increase Ca2+cyt focally, we targeted high-intensity UV light to a single EC of a septal capillary, thereby uncaging Ca2+ from NP-EGTA. In WT mice, uncaging increased EC Ca2+cyt locally as well as at a site 100 μm distant by 33±2 and 29±2 gray levels, respectively (mean±SE, n=3, P<0.05). By contrast, in Cx43-/- mice uncaging increased Ca2+cyt locally (n=3, P<0.05), but not at a distant site. Thus, the uncaging-induced Ca2+ signal was conducted 100 μm from the uncaging site in WT, but not in Cx43-/- mice. We conclude, EC of lung capillaries rapidly communicate Ca2+ signals through Cx43-containing gap junctions. Such spatial communication may amplify Ca2+-dependent proinflammatory responses in the lung capillary bed (supported by HL75503, HL57556).

Loss of PECAM-1 Function Impairs Alveolarization

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.