Mario J. Perez

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α–responsive, heparan sulfate–specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Rtp801, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema

Rtp801 (also known as Redd1, and encoded by Ddit4), a stress-related protein triggered by adverse environmental conditions, inhibits mammalian target of rapamycin (mTOR) by stabilizing the TSC1-TSC2 inhibitory complex and enhances oxidative stress–dependent cell death. We postulated that Rtp801 acts as a potential amplifying switch in the development of cigarette smoke–induced lung injury, leading to emphysema. Rtp801 mRNA and protein were overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The regulation of Rtp801 expression by cigarette smoke may rely on oxidative stress–dependent activation of the CCAAT response element in its promoter. We also found that Rtp801 was necessary and sufficient for nuclear factor-κB (NF-κB) activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF-κB activation, alveolar inflammation, oxidative stress and apoptosis of alveolar septal cells. In contrast, Rtp801 knockout mice were markedly protected against acute cigarette smoke–induced lung injury, partly via increased mTOR signaling, and, when exposed chronically to cigarette smoke, against emphysema. Our data support the notion that Rtp801 may represent a major molecular sensor and mediator of cigarette smoke–induced lung injury.

Heparanase mediates renal dysfunction during early sepsis in mice

Heparanase, a heparan sulfate‐specific glucuronidase, mediates the onset of pulmonary neutrophil adhesion and inflammatory lung injury during early sepsis. We hypothesized that glomerular heparanase is similarly activated during sepsis and contributes to septic acute kidney injury (AKI). We induced polymicrobial sepsis in mice using cecal ligation and puncture (CLP) in the presence or absence of competitive heparanase inhibitors (heparin or nonanticoagulant N‐desulfated re‐N‐acetylated heparin [NAH]). Four hours after surgery, we collected serum and urine for measurement of renal function and systemic inflammation, invasively determined systemic hemodynamics, harvested kidneys for histology/protein/mRNA, and/or measured glomerular filtration by inulin clearance. CLP‐treated mice demonstrated early activation of glomerular heparanase with coincident loss of glomerular filtration, as indicated by a >twofold increase in blood urea nitrogen (BUN) and a >50% decrease in inulin clearance (P < 0.05) in comparison to sham mice. Administration of heparanase inhibitors 2 h prior to CLP attenuated sepsis‐induced loss of glomerular filtration rate, demonstrating that heparanase activation contributes to early septic renal dysfunction. Glomerular heparanase activation was not associated with renal neutrophil influx or altered vascular permeability, in marked contrast to previously described effects of pulmonary heparanase on neutrophilic lung injury during sepsis. CLP induction of renal inflammatory gene (IL‐6, TNF‐α, IL‐1β) expression was attenuated by NAH pretreatment. While serum inflammatory indices (KC, IL‐6, TNF‐α, IL‐1β) were not impacted by NAH pretreatment, heparanase inhibition attenuated the CLP‐induced increase in serum IL‐10. These findings demonstrate that glomerular heparanase is active during sepsis and contributes to septic renal dysfunction via mechanisms disparate from heparanase‐mediated lung injury.

Severe pulmonary hypertension is associated with altered right ventricle metabolic substrate uptake

In severe pulmonary hypertension (SPH), prior studies have shown an increase in right ventricle (RV) uptake of glucose, but it is unclear whether there is a change in the relative utilization of fatty acids. We hypothesized that in the RV in SPH, as in left ventricular (LV) failure, there is altered substrate utilization, with increased glucose uptake and decreased fatty acid uptake. SPH was induced in rats by treatment with the VEGF receptor inhibitor SU5416 and 3 wk of hypoxia (10% FiO2 ), followed by an additional 4 wk of normoxia (SU-Hx group). Control rats were treated with carboxymethylcellulose vehicle and 7 wk of normoxia (CMC-Nx group). The rodents then underwent positron emission tomography with sequential administration of two radiotracers, 2-deoxy-2-[(18)F]fluoroglucose ((18)F-FDG) and 14-(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid ((18)F-FTHA), analogs of glucose and fatty acid, respectively. Five CMC-Nx and 3 SU-Hx rats completed the entire experimental protocol. In the RV, there was a mild increase in (18)F-FDG uptake (1.35-fold, P = 0.085) and a significant decrease in (18)F-FTHA uptake (-2.1-fold, P < 0.05) in the SU-Hx rats relative to the CMC-Nx rats. In the LV, SU-Hx rats had less uptake of both radiotracers compared with CMC-Nx rats. Less RV fatty acid uptake in SPH was corroborated by decreased fatty acid transporters and enzymes in the RV tissue, and specifically a decrease in lipoprotein lipase. In the RV in rats with SPH, there is a major shift in metabolic substrate preference, largely due to decreased fatty acid uptake.

Novosphingobium and Its Potential Role in Chronic Obstructive Pulmonary Diseases: Insights from Microbiome Studies

Bacterial infection of lung airways underlies some of the main complications of COPD, significantly impacting disease progression and outcome. Colonization by bacteria may further synergize, amplify, or trigger pathways of tissue damage started by cigarette smoke, contributing to the characteristic airway inflammation and alveolar destruction of COPD. We sought to elucidate the presence and types of lung bacterial populations in different stages of COPD, aimed at revealing important insights into the pathobiology of the disease. Sequencing of the bacterial small subunit ribosomal RNA gene in 55 well-characterized clinical lung samples, revealed the presence of Novosphingobium spp. (>2% abundance) in lungs of patients with GOLD 3-GOLD 4 COPD, cystic fibrosis and a subset of control individuals. Novosphingobium-specific quantitative PCR was concordant with the sequence data and high levels of Novosphingobium spp. were quantifiable in advanced COPD, but not from other disease stages. Using a mouse model of subacute lung injury due to inhalation of cigarette smoke, bronchoalveolar lavage neutrophil and macrophage counts were significantly higher in mice challenged intratracheally with N. panipatense compared to control mice (p<0.01). Frequencies of neutrophils and macrophages in lung tissue were increased in mice challenged with N. panipatense at room air compared to controls. However, we did not observe an interaction between N. panipatense and subacute cigarette smoke exposure in the mouse. In conclusion, Novosphingobium spp. are present in more severe COPD disease, and increase inflammation in a mouse model of smoke exposure.

Rtp801 suppression of epithelial mTORC1 augments endotoxin-induced lung inflammation.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular responses to environmental stress. mTOR (and its primary complex mTORC1) is, therefore, ideally positioned to regulate lung inflammatory responses to an environmental insult, a function directly relevant to disease states such as the acute respiratory distress syndrome. Our previous work in cigarette smoke-induced emphysema identified a novel protective role of pulmonary mTORC1 signaling. However, studies of the impact of mTORC1 on the development of acute lung injury are conflicting. We hypothesized that Rtp801, an endogenous inhibitor of mTORC1, which is predominantly expressed in alveolar type II epithelial cells, is activated during endotoxin-induced lung injury and functions to suppress anti-inflammatory epithelial mTORC1 responses. We administered intratracheal lipopolysaccharide to wild-type mice and observed a significant increase in lung Rtp801 mRNA. In lipopolysaccharide-treated Rtp801(-/-) mice, epithelial mTORC1 activation significantly increased and was associated with an attenuation of lung inflammation. We reversed the anti-inflammatory phenotype of Rtp801(-/-) mice with the mTORC1 inhibitor, rapamycin, reassuring against mTORC1-independent effects of Rtp801. We confirmed the proinflammatory effects of Rtp801 by generating a transgenic Rtp801 overexpressing mouse, which displayed augmented inflammatory responses to intratracheal endotoxin. These data suggest that epithelial mTORC1 activity plays a protective role against lung injury, and its inhibition by Rtp801 exacerbates alveolar injury caused by endotoxin.

RTP801 Amplifies Nicotinamide Adenine Dinucleotide Phosphate Oxidase-4–Dependent Oxidative Stress Induced by Cigarette Smoke

&NA; Tobacco smoke (TS) causes chronic obstructive pulmonary disease, including chronic bronchitis, emphysema, and asthma. Rtp801, an inhibitor of mechanistic target of rapamycin, is induced by oxidative stress triggered by TS. Its up‐regulation drives lung susceptibility to TS injury by enhancing inflammation and alveolar destruction. We postulated that Rtp801 is not only increased by reactive oxygen species (ROS) in TS but also instrumental in creating a feedforward process leading to amplification of endogenous ROS generation. We used cigarette smoke extract (CSE) to model the effect of TS in wild‐type (Wt) and knockout (KO‐Rtp801) mouse lung fibroblasts (MLF). The production of superoxide anion in KO‐Rtp801 MLF was lower than that in Rtp801 Wt cells after CSE treatment, and it was inhibited in Wt MLF by silencing nicotinamide adenine dinucleotide phosphate oxidase‐4 (Nox4) expression with small interfering Nox4 RNA. We observed a cytoplasmic location of ROS formation by real‐time redox changes using reduction‐oxidation‐sensitive green fluorescent protein profluorescent probes. Both the superoxide production and the increase in the cytoplasmic redox were inhibited by apocynin. Reduction in the activity of Sod and decreases in the expression of Sod2 and Gpx1 genes were associated with Rtp801 CSE induction. The ROS produced by Nox4 in conjunction with the decrease in cellular antioxidant enzymatic defenses may account for the observed cytoplasmic redox changes and cellular damage caused by TS.

Role of vascular endothelial growth factor signaling in Schistosoma-induced experimental pulmonary hypertension

There is significant evidence that Th2 (T helper 2)-mediated inflammation supports the pathogenesis of both human and experimental animal models of pulmonary hypertension (PH). A key immune regulator is vascular endothelial growth factor (VEGF), which is produced by Th2 inflammation and can itself contribute to Th2 pulmonary responses. In this study, we interrogated the role of VEGF signaling in a murine model of schistosomiasis-induced PH with a phenotype of significant intrapulmonary Th2 inflammation, vascular remodeling, and elevated right ventricular pressures. We found that VEGF receptor blockade partially suppressed the levels of the Th2 inflammatory cytokines interleukin (IL)-4 and IL-13 in both the lung and the liver after Schistosoma mansoni exposure and suppressed pulmonary vascular remodeling. These findings suggest that VEGF positively contributes to schistosomiasis-induced vascular inflammation and remodeling, and they also provide evidence for a VEGF-dependent signaling pathway necessary for pulmonary vascular remodeling and inflammation in this model.

Vascular Adaptation of the Right Ventricle in Experimental Pulmonary Hypertension

Abstract Optimal right ventricular (RV) function in pulmonary hypertension (PH) requires structural and functional coupling between the RV cardiomyocyte and its adjacent capillary network. Prior investigations have indicated that RV vascular rarefaction occurs in PH, which could contribute to RV failure by reduced delivery of oxygen or other metabolic substrates. However, it has not been determined if rarefaction results from relative underproliferation in the setting of tissue hypertrophy or from actual loss of vessels. It is also unknown if rarefaction results in inadequate substrate delivery to the RV tissue. In the present study, PH was induced in rats by SU5416‐hypoxia‐normoxia exposure. The vasculature in the RV free wall was assessed using stereology. Steady‐state metabolomics of the RV tissue was performed by mass spectrometry. Complementary studies were performed in hypoxia‐exposed mice and rats. Rats with severe PH had evidence of RV failure by decreased cardiac output and systemic hypotension. By stereology, there was significant RV hypertrophy and increased total vascular length in the RV free wall in close proportion, with evidence of vessel proliferation but no evidence of endothelial cell apoptosis. There was a modest increase in the radius of tissue served per vessel, with decreased arterial delivery of metabolic substrates. Metabolomics revealed major metabolic alterations and metabolic reprogramming; however, metabolic substrate delivery was functionally preserved, without evidence of either tissue hypoxia or depletion of key metabolic substrates. Hypoxia‐treated rats and mice had similar but milder alterations. There is significant homeostatic vascular adaptation in the right ventricle of rodents with PH.