Neeraj Vij

Role of Cigarette Smoke–Induced Aggresome Formation in Chronic Obstructive Pulmonary Disease–Emphysema Pathogenesis

Cigarette smoke (CS) exposure is known to induce proteostasis imbalance that can initiate accumulation of ubiquitinated proteins. Therefore, the primary goal of this study was to determine if first- and secondhand CS induces localization of ubiquitinated proteins in perinuclear spaces as aggresome bodies. Furthermore, we sought to determine the mechanism by which smoke-induced aggresome formation contributes to chronic obstructive pulmonary disease (COPD)-emphysema pathogenesis. Hence, Beas2b cells were treated with CS extract (CSE) for in vitro experimental analysis of CS-induced aggresome formation by immunoblotting, microscopy, and reporter assays, whereas chronic CS-exposed murine model and human COPD-emphysema lung tissues were used for validation. In preliminary analysis, we observed a significant (P < 0.01) increase in ubiquitinated protein aggregation in the insoluble protein fraction of CSE-treated Beas2b cells. We verified that CS-induced ubiquitin aggregrates are localized in the perinuclear spaces as aggresome bodies. These CS-induced aggresomes (P < 0.001) colocalize with autophagy protein microtubule-associated protein 1 light chain-3B(+) autophagy bodies, whereas U.S. Food and Drug Administration-approved autophagy-inducing drug (carbamazepine) significantly (P < 0.01) decreases their colocalization and expression, suggesting CS-impaired autophagy. Moreover, CSE treatment significantly increases valosin-containing protein-p62 protein-protein interaction (P < 0.0005) and p62 expression (aberrant autophagy marker; P < 0.0001), verifying CS-impaired autophagy as an aggresome formation mechanism. We also found that inhibiting protein synthesis by cycloheximide does not deplete CS-induced ubiquitinated protein aggregates, suggesting the role of CS-induced protein synthesis in aggresome formation. Next, we used an emphysema murine model to verify that chronic CS significantly (P < 0.0005) induces aggresome formation. Moreover, we observed that autophagy induction by carbamazepine inhibits CS-induced aggresome formation and alveolar space enlargement (P < 0.001), confirming involvement of aggresome bodies in COPD-emphysema pathogenesis. Finally, significantly higher p62 accumulation in smokers and severe COPD-emphysema lungs (Global Initiative for Chronic Obstructive Lung Disease Stage III/IV) as compared with normal nonsmokers (Global Initiative for Chronic Obstructive Lung Disease Stage 0) substantiates the pathogenic role of autophagy impairment in aggresome formation and COPD-emphysema progression. In conclusion, CS-induced aggresome formation is a novel mechanism involved in COPD-emphysema pathogenesis.

Lactosylceramide-accumulation in lipid-rafts mediate aberrant-autophagy, inflammation and apoptosis in cigarette smoke induced emphysema

Ceramide-accumulation is known to be involved in the pathogenesis of chronic inflammatory lung diseases including cigarette smoke-induced emphysema (CS-emphysema) but the exact sphingolipid metabolite that initiates emphysema progression remains ambiguous. We evaluated here a novel role for the sphingolipid, lactosylceramide (LacCer), as a potential mechanism for pathogenesis of CS-emphysema. We assessed the expression of LacCer, and LacCer-dependent inflammatory, apoptosis and autophagy responses in lungs of mice exposed to CS, as well as peripheral lung tissues from COPD subjects followed by experimental analysis to verify the role of LacCer in CS-emphysema. We observed significantly elevated LacCer-accumulation in human COPD lungs with increasing severity of emphysema over non-emphysema controls. Moreover, increased expression of defective-autophagy marker, p62, in lung tissues of severe COPD subjects suggest that LacCer induced aberrant-autophagy may contribute to the pathogenesis of CS-emphysema. We verified that CS-extract treatment significantly induces LacCer-accumulation in both bronchial-epithelial cells (BEAS2B) and macrophages (Raw264.7) as a mechanism to initiate aberrant-autophagy (p62-accumulation) and apoptosis that was rescued by pharmacological inhibitor of LacCer-synthase. Further, we corroborated that CS exposure induces LacCer-accumulation in murine lungs that can be controlled by LacCer-synthase inhibitor. We propose LacCer-accumulation as a novel prognosticator of COPD-emphysema severity, and provide evidence on the therapeutic efficacy of LacCer-synthase inhibitor in CS induced COPD-emphysema.

Nicotine exposure induces bronchial epithelial cell apoptosis and senescence via ROS mediated autophagy-impairment

Waterpipe smoking and e-cigarette vaping, the non-combustible sources of inhaled nicotine exposure are increasingly becoming popular and marketed as safer alternative to cigarette smoking. Hence, this study was designed to investigate the impact of inhaled nicotine exposure on disease causing COPD-emphysema mechanisms. For in vitro studies, human bronchial epithelial cells (Beas2b) were treated with waterpipe smoke extract (WPSE, 5%), nicotine (5mM), and/or cysteamine (250μM, an autophagy inducer and anti-oxidant drug), for 6hrs. We observed significantly (p<0.05) increased ubiquitinated protein-accumulation in the insoluble protein fractions of Beas2b cells treated with WPSE or nicotine that could be rescued by cysteamine treatment, suggesting aggresome-formation and autophagy-impairment. Moreover, our data also demonstrate that both WPSE and nicotine exposure significantly (p<0.05) elevates Ub-LC3β co-localization to aggresome-bodies while inducing Ub-p62 co-expression/accumulation, verifying autophagy-impairment. We also found that WPSE and nicotine exposure impacts Beas2b cell viability by significantly (p<0.05) inducing cellular apoptosis/senescence via ROS-activation, as it could be controlled by cysteamine, which is known to have an anti-oxidant property. For murine studies, C57BL/6 mice were administered with inhaled nicotine (intranasal, 500μg/mouse/day for 5 days), as an experimental model of non-combustible nicotine exposure. The inhaled nicotine exposure mediated oxidative-stress induces autophagy-impairment in the murine lungs as seen by significant (p<0.05, n=4) increase in the expression levels of nitrotyrosine protein-adduct (oxidative-stress marker, soluble-fraction) and Ub/p62/VCP (impaired-autophagy marker, insoluble-fraction). Overall, our data shows that nicotine, a common component of WPS, e-cigarette vapor and cigarette smoke, induces bronchial epithelial cell apoptosis and senescence via ROS mediated autophagy-impairment as a potential mechanism for COPD-emphysema pathogenesis.

Second-Hand Cigarette Smoke Impairs Bacterial Phagocytosis in Macrophages by Modulating CFTR Dependent Lipid-Rafts

Introduction First/Second-hand cigarette-smoke (FHS/SHS) exposure weakens immune defenses inducing chronic obstructive pulmonary disease (COPD) but the underlying mechanisms are not fully understood. Hence, we evaluated if SHS induced changes in membrane/lipid-raft (m-/r)-CFTR (cystic fibrosis transmembrane conductance regulator) expression/activity is a potential mechanism for impaired bacterial phagocytosis in COPD. Methods RAW264.7 murine macrophages were exposed to freshly prepared CS-extract (CSE) containing culture media and/or Pseudomonas-aeruginosa-PA01-GFP for phagocytosis (fluorescence-microscopy), bacterial survival (colony-forming-units-CFU), and immunoblotting assays. The CFTR-expression/activity and lipid-rafts were modulated by transient-transfection or inhibitors/inducers. Next, mice were exposed to acute/sub-chronic-SHS or room-air (5-days/3-weeks) and infected with PA01-GFP, followed by quantification of bacterial survival by CFU-assay. Results We investigated the effect of CSE treatment on RAW264.7 cells infected by PA01-GFP and observed that CSE treatment significantly (p<0.01) inhibits PA01-GFP phagocytosis as compared to the controls. We also verified this in murine model, exposed to acute/sub-chronic-SHS and found significant (p<0.05, p<0.02) increase in bacterial survival in the SHS-exposed lungs as compared to the room-air controls. Next, we examined the effect of impaired CFTR ion-channel-activity on PA01-GFP infection of RAW264.7 cells using CFTR172-inhibitor and found no significant change in phagocytosis. We also similarly evaluated the effect of a CFTR corrector-potentiator compound, VRT-532, and observed no significant rescue of CSE impaired PA01-GFP phagocytosis although it significantly (p<0.05) decreases CSE induced bacterial survival. Moreover, induction of CFTR expression in macrophages significantly (p<0.03) improves CSE impaired PA01-GFP phagocytosis as compared to the control. Next, we verified the link between m-/r-CFTR expression and phagocytosis using methyl-β-cyclodextran (CD), as it is known to deplete CFTR from membrane lipid-rafts. We observed that CD treatment significantly (p<0.01) inhibits bacterial phagocytosis in RAW264.7 cells and adding CSE further impairs phagocytosis suggesting synergistic effect on CFTR dependent lipid-rafts. Conclusion Our data suggest that SHS impairs bacterial phagocytosis by modulating CFTR dependent lipid-rafts.

Neutrophil targeted nano-drug delivery system for chronic obstructive lung diseases.

The success of drug delivery to target airway cell(s) remains a significant challenge due to the limited ability of nanoparticle (NP) systems to circumvent protective airway-defense mechanisms. The size, density, surface and physical-chemical properties of nanoparticles are the key features that determine their ability to navigate across the airway-barrier. We evaluated here the efficacy of a PEGylated immuno-conjugated PLGA-nanoparticle (PINP) to overcome this challenge and selectively deliver drug to specific inflammatory cells (neutrophils). We first characterized the size, shape, surface-properties and neutrophil targeting using dynamic laser scattering, transmission electron microscopy and flow cytometry. Next, we assessed the efficacy of neutrophil-targeted PINPs in transporting through the airway followed by specific binding and release of drug to neutrophils. Finally, our results demonstrate the efficacy of PINP mediated non-steroidal anti-inflammatory drug-(ibuprofen) delivery to neutrophils in murine models of obstructive lung diseases, based on its ability to control neutrophilic-inflammation and resulting lung disease.

Nano-based rescue of dysfunctional autophagy in chronic obstructive lung diseases

ABSTRACT Introduction: ΔF508-CFTR (cystic fibrosis transmembrane conductance regulator) is a common CF-mutation that is known to induce oxidative-inflammatory stress through activation of reactive oxygen species (ROS), which induces autophagy-impairment resulting in accumulation of CFTR in aggresome-bodies. Cysteamine, the reduced form of cystamine, is a FDA-approved drug that has anti-oxidant, anti-bacterial, and mucolytic properties. This drug has been shown in a recent clinical trial to decrease lung inflammation and improve lung function in CF patients by potentially restoring autophagy and allowing CFTR to be trafficked to the cell membrane. Areas covered: The delivery of cysteamine to airway epithelia of chronic subjects prerequisite the need for a delivery system to allow rescue of dysfunctional autophagy. Expert opinion: We anticipate based on our ongoing studies that PLGA-PEG- or Dendrimer-mediated cysteamine delivery could allow sustained airway delivery over standard cysteamine tablets or delay release capsules that are currently used for systemic treatment. In addition, proposed nano-based autophagy induction strategy can also allow rescue of cigarette smoke (CS) induced acquired-CFTR dysfunction seen in chronic obstructive pulmonary disease (COPD)-emphysema subjects. The CS induced acquired-CFTR dysfunction involves CFTR-accumulation in aggresome-bodies that can be rescued by an autophagy-inducing antioxidant drug, cysteamine. Moreover, chronic CS-exposure generates ROS that induces overall protein-misfolding and aggregation of ubiquitinated-proteins as aggresome-bodies via autophagy-impairment that can be also be resolved by treatment with autophagy-inducing antioxidant drug, cysteamine.

Augmenting autophagy for prognosis based intervention of COPD-pathophysiology

Chronic obstructive pulmonary disease (COPD) is foremost among the non-reversible fatal ailments where exposure to tobacco/biomass-smoke and aging are the major risk factors for the initiation and progression of the obstructive lung disease. The role of smoke-induced inflammatory-oxidative stress, apoptosis and cellular senescence in driving the alveolar damage that mediates the emphysema progression and severe lung function decline is apparent, although the central mechanism that regulates these processes was unknown. To fill in this gap in knowledge, the central role of proteostasis and autophagy in regulating chronic lung disease causing mechanisms has been recently described. Recent studies demonstrate that cigarette/nicotine exposure induces proteostasis/autophagy-impairment that leads to perinuclear accumulation of polyubiquitinated proteins as aggresome-bodies, indicative of emphysema severity. In support of this concept, autophagy inducing FDA-approved anti-oxidant drugs control tobacco-smoke induced inflammatory-oxidative stress, apoptosis, cellular senescence and COPD-emphysema progression in variety of preclinical models. Hence, we propose that precise and early detection of aggresome-pathology can allow the timely assessment of disease severity in COPD-emphysema subjects for prognosis-based intervention. While intervention with autophagy-inducing drugs is anticipated to reduce alveolar damage and lung function decline, resulting in a decrease in the current mortality rates in COPD-emphysema subjects.

Inhibition of histone-deacetylase activity rescues inflammatory cystic fibrosis lung disease by modulating innate and adaptive immune responses

BackgroundChronic lung disease resulting from dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and NFκB-mediated neutrophilic-inflammation forms the basis of CF-related mortality. Here we aimed to evaluate if HDAC inhibition controls Pseudomonas-aeruginosa-lipopolysaccharide (Pa-LPS) induced airway inflammation and CF-lung disease.MethodsFor in vitro experiments, HEK293-cells were transfected with IL-8 or NFκB-firefly luciferase, and SV40-renilla- luciferase reporter constructs or ΔF508-CFTR-pCEP, followed by treatment with suberoylanilide hydroxamic acid (SAHA), Trichostatin-A (TSA) and/or TNFα. For murine studies, Cftr+/+ or Cftr−/− mice (n = 3) were injected/instilled with Pa-LPS and/or treated with SAHA or vehicle control. The progression of lung disease was monitored by quantifying changes in inflammatory markers (NFκB), cytokines (IL-6/IL-10), neutrophil activity (MPO, myeloperoxidase and/or NIMP-R14) and T-reg numbers.ResultsSAHA treatment significantly (p < 0.05) suppresses TNFα-induced NFκB and IL-8 reporter activities in HEK293-cells. Moreover, SAHA, Tubacin (selective HDAC6-inhibitor) or HDAC6-shRNAs controls CSE-induced ER-stress activities (p < 0.05). In addition, SAHA restores trafficking of misfolded-ΔF508-CFTR, by inducing protein levels of both B and C forms of CFTR. Murine studies using Cftr+/+ or Cftr−/− mice verified that SAHA controls Pa-LPS induced IL-6 levels, and neutrophil (MPO levels and/or NIMP-R14), NFκB-(inflammation) and Nrf2 (oxidative-stress marker) activities, while promoting FoxP3+ T-reg activity.ConclusionIn summary, SAHA-mediated HDAC inhibition modulates innate and adaptive immune responses involved in pathogenesis and progression of inflammatory CF-lung disease.

Targeted nano-based proteostasis-inhibition for controlling lung cancer pathogenesis & progression

S and natural biopolymers, such as polylactides/polyglycolides or chitosan, are increasingly replacing steel or bone cement as biomaterials for orthopedic surgery and regenerative engineering. Amongst the major issue is the dichotomy between mechanical strength and bioactivity of these biomaterials. Synthetic materials often exhibit sufficient mechanical strength, but lack bioactivity to promote tissue regeneration, while some of the natural materials, specifically chitosan, have excellent bioactive features, but lack the necessary mechanical strength to serve as useful materials in bone tissue engineering. In this presentation we will discuss the critical role that nanoparticles can play in modulating the mechanical properties and bioactivity of biodegradable biopolymers. As first examples we will describe the incorporation of hydroxyapatite (HA) nanoparticles into electrospun chitosan fibers, which renders this material mechanically suitable for non-weight bearing applications in maxillofacial orthopedic surgery. Specifically we will demonstrate that HA-containing chitosan scaffolds are both osteoinductive in vitro and osteoconductive in vivo and catalyze the de novo bone formation tissue regeneration in a mouse model of critical size calvarial defects. As a second example, we will describe the use of carbon nanodiamond (CND) particles to modulate the mechanical properties of polylactides, such as PLLA, used as biodegradable biomaterials for interference screws that are commonly employed in the surgical repair of tendons and ligaments. Inclusion of CND reinforced the mechanical properties PLLA and specifically reduced its brittleness and improved its ductile properties. Surprising, addition of CND also significantly enhanced the bioactivity of PLLA, as inferred from the acceleration and increased levels of biomineralization.