Lakshmi Galam

Mir-206 Regulates Pulmonary Artery Smooth Muscle Cell Proliferation and Differentiation

Pulmonary Arterial Hypertension (PAH) is a progressive devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. MicroRNA-206 (miR-206) is known to regulate proliferation and is implicated in various types of cancers. However, the role of miR-206 in PAH has not been studied. In this study, it is hypothesized that miR-206 could play a role in the proliferation of PASMCs. In the present study, the expression patterns of miR-206 were investigated in normal and hypertensive mouse PASMCs. The effects of miR-206 in modulating cell proliferation, apoptosis and smooth muscle cell markers in human pulmonary artery smooth muscle cells (hPASMCs) were investigated in vitro. miR-206 expression in mouse PASMCs was correlated with an increase in right ventricular systolic pressure. Reduction of miR-206 levels in hPASMCs causes increased proliferation and reduced apoptosis and these effects were reversed by the overexpression of miR-206. miR-206 over expression also increased the levels of smooth muscle cell differentiation markers α-smooth muscle actin and calponin implicating its importance in the differentiation of SMCs. miR-206 overexpression down regulated Notch-3 expression, which is key a factor in PAH development. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis and differentiation of PASMCs, and that it could be used as a novel treatment strategy in PAH.

NALP-3 inflammasome silencing attenuates ceramide-induced transepithelial permeability

The hallmark of acute lung injury (ALI) is the influx of proinflammatory cytokines into lung tissue and alveolar permeability that ultimately leads to pulmonary edema. However, the mechanisms involved in inflammatory cytokine production and alveolar permeability are unclear. Recent studies suggest that excessive production of ceramide has clinical relevance as a mediator of pulmonary edema and ALI. Our earlier studies indicate that the activation of inflammasome promotes the processing and secretion of proinflammatory cytokines and causes alveolar permeability in ALI. However, the role of ceramide in inflammasome activation and the underlying mechanism in relation to alveolar permeability is not known. We hypothesized that ceramide activates the inflammasome and causes inflammatory cytokine production and alveolar epithelial permeability. To test this hypothesis, we analyzed the lung ceramide levels during hyperoxic ALI in mice. The effect of ceramide on activation of inflammasome and production of inflammatory cytokine was assessed in primary mouse alveolar macrophages and THP‐1 cells. Alveolar transepithelial permeability was determined in alveolar epithelial type‐II cells (AT‐II) and THP‐1 co‐cultures. Our results reveal that ceramide causes inflammasome activation, induction of caspase‐1, IL‐1β cleavage, and release of proinflammatory cytokines. In addition, ceramide further induces alveolar epithelial permeability. Short‐hairpin RNA silencing of inflammasome components abrogated ceramide‐induced secretion of proinflammatory cytokines in vitro. Inflammasome silencing abolishes ceramide‐induced alveolar epithelial permeability in AT‐II. Collectively, our results demonstrate for the first time that ceramide‐induced secretion of proinflammatory cytokines and alveolar epithelial permeability occurs though inflammasome activation. J. Cell. Physiol. 227: 3310–3316, 2012.

MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2

Inflammasomes are multimeric protein complexes involved in the processing of IL-1β through Caspase-1 cleavage. NLRP3 is the most widely studied inflammasome, which has been shown to respond to a large number of both endogenous and exogenous stimuli. Although studies have begun to define basic pathways for the activation of inflammasome and have been instrumental in identifying therapeutics for inflammasome related disorders; understanding the inflammasome activation at the molecular level is still incomplete. Recent functional studies indicate that microRNAs (miRs) regulate molecular pathways and can lead to diseased states when hampered or overexpressed. Mechanisms involving the miRNA regulatory network in the activation of inflammasome and IL-1β processing is yet unknown. This report investigates the involvement of miR-133a-1 in the activation of inflammasome (NLRP3) and IL-1β production. miR-133a-1 is known to target the mitochondrial uncoupling protein 2 (UCP2). The role of UCP2 in inflammasome activation has remained elusive. To understand the role of miR-133a-1 in regulating inflammasome activation, we either overexpressed or suppressed miR-133a-1 in differentiated THP1 cells that express the NLRP3 inflammasome. Levels of Caspase-1 and IL-1β were analyzed by Western blot analysis. For the first time, we showed that overexpression of miR-133a-1 increases Caspase-1 p10 and IL-1β p17 cleavage, concurrently suppressing mitochondrial uncoupling protein 2 (UCP2). Surprisingly, our results demonstrated that miR-133A-1 controls inflammasome activation without affecting the basal expression of the individual inflammasome components NLRP3 and ASC or its immediate downstream targets proIL-1β and pro-Caspase-1. To confirm the involvement of UCP2 in the regulation of inflammasome activation, Caspase-1 p10 and IL-1β p17 cleavage in UCP2 of overexpressed and silenced THP1 cells were studied. Suppression of UCP2 by siRNA enhanced the inflammasome activity stimulated by H2O2 and, conversely, overexpression of UCP2 decreased the inflammasome activation. Collectively, these studies suggest that miR-133a-1 suppresses inflammasome activation via the suppression of UCP2.

MicroRNA 16 Modulates Epithelial Sodium Channel in Human Alveolar Epithelial Cells

Acute lung injury (ALI) is a devastating disease characterized by pulmonary edema. Removal of edema from the air spaces of lung is a critical function of the epithelial sodium channel (ENaC) in ALI. The molecular mechanisms behind resolution of pulmonary edema are incompletely understood. MicroRNAs (miRNA) are crucial gene regulators and are dysregulated in various diseases including ALI. Recent studies suggest that microRNA-16 (miR-16) targets serotonin transporter (SERT) involved in the serotonin (5-HT) transmitter system. Alterations in serotonin levels have been reported in various pulmonary diseases. However, the role of miR-16 on its target SERT, and ENaC, a key ion channel involved in the resolution of pulmonary edema, have not been studied. In the present study, the expression patterns of miR-16, SERT, ENaC and serotonin were investigated in mice exposed to room air and hyperoxia. The effects of miR-16 overexpression on ENaC, SERT, TGF-β and Nedd4 in human alveolar epithelial cells were analyzed. miR-16 and ENaC were downregulated in mice exposed to hyperoxia. miR-16 downregulation in mouse lung was correlated with an increase in SERT expression and pulmonary edema. Overexpression of miR-16 in human alveolar epithelial cells (A549) suppressed SERT and increased ENaCβ levels when compared to control-vector transfected cells. In addition, miR-16 over expression suppressed TGFβ release, a critical inhibitor of ENaC. Interestingly Nedd4, a negative regulator of ENaC remained unaltered in miR-16 over expressed A549 cells when compared to controls. Taken together, our data suggests that miR-16 upregulates ENaC, a major sodium channel involved in resolution of pulmonary edema in ALI.

Deletion of P2X7 attenuates hyperoxia-induced acute lung injury via inflammasome suppression

Increasing evidence shows that hyperoxia is a serious complication of oxygen therapy in acutely ill patients that causes excessive production of free radicals leading to hyperoxia-induced acute lung injury (HALI). Our previous studies have shown that P2X7 receptor activation is required for inflammasome activation during HALI. However, the role of P2X7 in HALI is unclear. The main aim of this study was to determine the effect of P2X7 receptor gene deletion on HALI. Wild-type (WT) and P2X7 knockout (P2X7 KO) mice were exposed to 100% O2 for 72 h. P2X7 KO mice treated with hyperoxia had enhanced survival in 100% O2 compared with the WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1β, TNF-α, monocyte chemoattractant protein-1, and IL-6 levels were attenuated in P2X7 KO mice. P2X7 deletion decreased lung edema and alveolar protein content, which are associated with enhanced alveolar fluid clearance. In addition, activation of the inflammasome was suppressed in P2X7-deficient alveolar macrophages and was associated with suppression of IL-1β release. Furthermore, P2X7-deficient alveolar macrophage in type II alveolar epithelial cells (AECs) coculture model abolished protein permeability across mouse type II AEC monolayers. Deletion of P2X7 does not lead to a decrease in epithelial sodium channel expression in cocultures of alveolar macrophages and type II AECs. Taken together, these findings show that deletion of P2X7 is a protective factor and therapeutic target for the amelioration of hyperoxia-induced lung injury.

Genipin suppresses NLRP3 inflammasome activation through uncoupling protein-2

Incomplete clearance of apoptotic cells and reactive oxygen species (ROS) release are known to trigger inflammasome activation causing severe inflammation in acute lung injury and various metabolic and autoimmune diseases. Moreover, it has been reported that apoptotic cell clearance and ROS-mediated apoptosis critically depend on mitochondrial uncoupling protein-2 (UCP2). However, the relationship between UCP2 and inflammasome activation has not been studied. This report investigates the role of UCP2 in the expression and activation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in human macrophages. We found that UCP2 overexpression significantly enhanced the expression levels of NLRP3. The NLRP3 expression levels were significantly suppressed in THP1 cells treated with genipin, a UCP2 inhibitor, compared to controls. In addition, genipin altered adenosine triphosphate (ATP)- and hydrogen peroxide (H2O2)-mediated interleukin-1 beta (IL-1β) secretion and significantly suppressed caspase-1 activity in inflammasome-activated human macrophages. Taken together, our results suggest that genipin modulates NLRP3 inflammasome activation and ATP- or H2O2-mediated IL-1β release.

High-Throughput Screen of Natural Product Libraries for Hsp90 Inhibitors

Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine.

4-hydroxynonenal regulates mitochondrial function in human small airway epithelial cells.

Prolonged exposure to oxidative stress causes Acute Lung Injury (ALI) and significantly impairs pulmonary function. Previously we have demonstrated that mitochondrial dysfunction is a key pathological factor in hyperoxic ALI. While it is known that hyperoxia induces the production of stable, but toxic 4-hydroxynonenal (4-HNE) molecule, it is unknown how the reactive aldehyde disrupts mitochondrial function. Our previous in vivo study indicated that exposure to hyperoxia significantly increases 4-HNE-Protein adducts, as well as levels of MDA in total lung homogenates. Based on the in vivo studies, we explored the effects of 4-HNE in human small airway epithelial cells (SAECs). Human SAECs treated with 25 μM of 4-HNE showed a significant decrease in cellular viability and increased caspase-3 activity. Moreover, 4-HNE treated SAECs showed impaired mitochondrial function and energy production indicated by reduced ATP levels, mitochondrial membrane potential, and aconitase activity. This was followed by a significant decrease in mitochondrial oxygen consumption and depletion of the reserve capacity. The direct effect of 4-HNE on the mitochondrial respiratory chain was confirmed using Rotenone. Furthermore, SAECs treated with 25 μM 4-HNE showed a time-dependent depletion of total Thioredoxin (Trx) proteins and Trx activity. Taken together, our results indicate that 4-HNE induces cellular and mitochondrial dysfunction in human SAECs, leading to an impaired endogenous antioxidant response.

Dysregulation of CLOCK gene expression in hyperoxia-induced lung injury

Hyperoxic acute lung injury (HALI) is characterized by inflammation and epithelial cell death. CLOCK genes are master regulators of circadian rhythm also implicated in inflammation and lung diseases. However, the relationship of CLOCK genes in hyperoxia-induced lung injury has not been studied. This study will determine if HALI alters CLOCK gene expression. To test this, wild-type and NALP3(-/-) mice were exposed to room air or hyperoxia for 24, 48, or 72 h. In addition, mice were exposed to different concentrations of hyperoxia (50, 75, or 100% O2) or room air for 72 h. The mRNA and protein levels of lung CLOCK genes, based on quantitative PCR and Western blot analysis, respectively, and their target genes are significantly elevated in mice exposed to hyperoxia compared with controls. Alterations in CLOCK genes are associated with increased inflammatory markers in bronchoalveolar lavage fluid of hyperoxic mice compared with controls. Histological examination of mice lungs exposed to hyperoxia show increased inflammation and alveolar congestion compared with controls. Our results indicate sequential increase in CLOCK gene expression in lungs of mice exposed to hyperoxia compared with controls. Additionally, data suggest a dose-dependent increase in CLOCK gene expression with increased oxygen concentrations. To validate if the expression changes related to CLOCK genes are indeed associated with inflammation, NALP3(-/-) was introduced to analyze loss of function in inflammation. Western blot analysis showed significant CLOCK gene downregulation in NALP3(-/-) mice compared with wild-type controls. Together, our results demonstrate that hyperoxia-mediated lung inflammation is associated with alterations in CLOCK gene expression.

Adenovirus-mediated transfer of the SOCS-1 gene to mouse lung confers protection against hyperoxic acute lung injury

Suppressor of cytokine signaling-1 (SOCS-1) is a member of the suppressor of cytokine signaling family of proteins and an inhibitor of interleukin-6 (IL-6) signaling. SOCS-1 has been shown to protect cells from cellular damage and apoptosis induced by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interferon gamma (IL-γ). However, it is not known whether increased SOCS-1 is protective during pulmonary oxidative stress. Therefore, we hypothesized that increased SOCS-1 in the lungs of mice would be protective in the setting of hyperoxic lung injury. We administered SOCS-1 adenovirus (Ad-SOCS-1) intratracheally into the lungs and exposed the mice to 100% O2. Mice infected with GFP adenovirus (Ad-GFP) were used as controls. Mice treated with Ad-SOCS-1 had enhanced survival in 100% oxygen compared to Ad-GFP-administered mice. After 3 days of hyperoxia, Ad-GFP mice were ill and tachypnic and died after 4 days. In contrast, all Ad-SOCS-1-treated mice survived for at least 6 days in hyperoxia and 80% survived beyond 7 days. Ad-SOCS-1 transfection protected mouse lungs from injury as indicated by lower lung wet/dry weight, alveolar-capillary protein leakage, reduced infiltration of inflammatory cells, and lower content of thiobarbituric acid-reactive substances in lung homogenate. Our results also indicated that Ad-SOCS-1 significantly inhibits hyperoxia-induced ASK-1 (apoptosis signal-regulating kinase 1) expression. Taken together, these findings show that increased expression of adenovirus-mediated SOCS-1 in the lungs of mice significantly protects against hyperoxic lung injury.