The Effect of CFTR Modulators on Airway Infection in Cystic Fibrosis

The advent of Cystic fibrosis transmembrane receptor (CFTR) modulators in 2012 was a critical event in the history of cystic fibrosis (CF) treatment. Unlike traditional therapies that target downstream effects of CFTR dysfunction, CFTR modulators aim to correct the underlying defect at the protein level. These genotype-specific therapies are now available for an increasing number of CF patients, transforming the way we view the condition from a life-limiting disease to one that can be effectively managed. Several studies have demonstrated the vast improvement CFTR modulators have on normalization of sweat chloride, CFTR function, clinical endpoints, and frequency of pulmonary exacerbation. However, their impact on other aspects of the disease, such as pathogenic burden and airway infection, remain under explored. Frequent airway infections as a result of increased susceptibility and impaired innate immune response are a serious problem within CF, often leading to accelerated decline in lung function and disease progression. Current evidence suggests that CFTR modulators are unable to eradicate pathogenic organisms in those with already established lung disease. However, this may not be the case for those with relatively low levels of disease progression and conserved microbial diversity, such as young patients. Furthermore, it remains unknown whether the restorative effects exerted by CFTR modulators extend to immune cells, such as phagocytes, which have the potential to modulate the response of people with CF (pwCF) to infection. Throughout this review, we look at the potential impact of CFTR modulators on airway infection in CF and their ability to shape impaired pulmonary defences to pathogens.     

Chronic Inflammation as the Underlying Mechanism of the Development of Lung Diseases in Psoriasis: A Systematic Review

Psoriasis is a systemic inflammatory disease caused by dysfunctional interactions between the innate and adaptive immune responses. The systemic inflammation in psoriasis may be associated with the development of comorbidities, including lung diseases. In this review, we aimed to provide a summary of the evidence regarding the prevalence of lung diseases in patients with psoriasis and the potential underlying mechanisms. Twenty-three articles published between March 2010 and June 2021 were selected from 195 initially identified records. The findings are discussed in terms of the prevalence of asthma, chronic obstructive pulmonary disease, interstitial lung disease, obstructive sleep apnea, pulmonary hypertension, and sarcoidosis in psoriasis. A higher prevalence of lung diseases in psoriasis has been confirmed in asthma, chronic obstructive pulmonary disease, obstructive sleep apnea, and pulmonary hypertension. These conditions are important as they are previously unrecognized causes of morbidity and mortality in psoriasis. The development of lung diseases in patients with psoriasis can be explained by several mechanisms, including common risk factors, shared immune and molecular characteristics associated with chronic inflammation, as well as other mechanisms. Understanding the prevalence of lung diseases in psoriasis and their underlying mechanisms can help implement appropriate preventative and therapeutic strategies to address respiratory diseases in patients with psoriasis.     

Correctors Rescue CFTR Mutations in Nucleotide‐Binding Domain 1 (NBD1) by Modulating Proteostasis

We evaluated whether small molecule correctors could rescue four nucleotide‐binding domain 1 (NBD1) mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (A455E, S492F, ΔI507, and R560T). We first transfected Cos‐7 cells (green monkey kidney cells) with A455E, S492F, ΔI507, or R560T and created HEK‐293 (human embryonic kidney cells) cell lines stably expressing these CFTR mutations. The mutants showed lowered protein expression, instability at physiological temperature, and rapid degradation. After treatment with correctors CFFT‐002, CFFT‐003, C3, C4, and/or C18, the combination of C18+C4 showed the most correction and resulted in increased CFTR residing in the plasma membrane. We found a profound decrease in binding of CFTR to histone deacetylases (HDAC) 6 and 7 and heat shock proteins (Hsps) 27 and 40. Silencing Hsp27 or 40 rescued the mutants, but no additional amount of CFTR was rescued when both proteins were knocked down simultaneously. Thus, CFTR mutations in NBD1 can be rescued by a combination of correctors, and the treatment alters the interaction between mutated CFTR and the endoplasmic reticulum machinery.     

Intra-Peritoneal Administration of Mitochondrial DNA Provokes Acute Lung Injury and Systemic Inflammation via Toll-Like Receptor 9

The pathogenesis of sepsis is complex. Mitochondrial dysfunction, which is responsible for energy metabolism, intrinsic apoptotic pathway, oxidative stress, and systemic inflammatory responses, is closely related with severe sepsis induced death. Mitochondria DNA (mtDNA) contain un-methylated cytosine phosphate guanine (CpG) motifs, which exhibit immune stimulatory capacities. The aim of this study was to investigate the role and mechanism of mtDNA release on lipopolysaccharide (LPS) induced acute lung injury (ALI) and systemic inflammation. Following LPS injection, plasma mtDNA copies peak at 8 h. Compared with wild-type (WT) mice, mtDNA in toll like receptor 4 knockout (TLR4 KO) mice were significantly decreased. MtDNA intra-peritoneal administration causes apparent ALI as demonstrated by increased lung injury score, bronchoalveolar lavage fluid (BALF) total protein and wet/dry (W/D) ratio; mtDNA injection also directly provokes systemic inflammation, as demonstrated by increased IL-1β, IL-6, high-mobility group protein B1 (HMGB1) level; while nuclear DNA (nDNA) could not induce apparent ALI and systemic inflammation. However, compared with WT mice, TLR4 KO could not protect from mtDNA induced ALI and systemic inflammation. Specific TLR9 inhibitor, ODN 2088 pretreatment can significantly attenuate mtDNA induced ALI and systemic inflammation, as demonstrated by improved lung injury score, decreased lung wet/dry ratio, BALF total protein concentration, and decreased systemic level of IL-1β, IL-6 and HMGB1. MtDNA administration activates the expression of p-P38 mitogen-activated protein kinases (MAPK) in lung tissue and specific TLR9 inhibitor pretreatment can attenuate this activation. Thus, LPS-induced mtDNA release occurs in a TLR4-dependent manner, and mtDNA causes acute lung injury and systemic inflammation in a TLR9-dependent and TLR4-independent manner.     

Targeting S100A9 Reduces Neutrophil Recruitment,Inflammation and Lung Damage in Abdominal Sepsis

S100A9, a pro-inflammatory alarmin, is up-regulated in inflamed tissues. However, the role of S100A9 in regulating neutrophil activation, inflammation and lung damage in sepsis is not known. Herein, we hypothesized that blocking S100A9 function may attenuate neutrophil recruitment in septic lung injury. Male C57BL/6 mice were pretreated with the S100A9 inhibitor ABR-238901 (10 mg/kg), prior to cercal ligation and puncture (CLP). Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested for analysis of neutrophil infiltration as well as edema and CXC chemokine production. Blood was collected for analysis of membrane-activated complex-1 (Mac-1) expression on neutrophils as well as CXC chemokines and IL-6 in plasma. Induction of CLP markedly increased plasma levels of S100A9. ABR-238901 decreased CLP-induced neutrophil infiltration and edema formation in the lung. In addition, inhibition of S100A9 decreased the CLP-induced up-regulation of Mac-1 on neutrophils. Administration of ABR-238901 also inhibited the CLP-induced increase of CXCL-1, CXCL-2 and IL-6 in plasma and lungs. Our results suggest that S100A9 promotes neutrophil activation and pulmonary accumulation in sepsis. Targeting S100A9 function decreased formation of CXC chemokines in circulation and lungs and attenuated sepsis-induced lung damage. These novel findings suggest that S100A9 plays an important pro-inflammatory role in sepsis and could be a useful target to protect against the excessive inflammation and lung damage associated with the disease.     

Endothelial Semaphorin 7A Promotes Inflammation in Seawater Aspiration-Induced Acute Lung Injury

Inflammation is involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have shown that Semaphorin 7A (SEMA7A) promotes inflammation, there are limited reports regarding immunological function of SEMA7A in seawater aspiration-induced ALI. Therefore, we investigated the role of SEMA7A during seawater aspiration-induced ALI. Male Sprague–Dawley rats were underwent seawater instillation. Then, lung samples were collected at an indicated time for analysis. In addition, rat pulmonary microvascular endothelial cells (RPMVECs) were cultured and then stimulated with 25% seawater for indicated time point. After these treatments, cells samples were collected for analysis. In vivo, seawater instillation induced lung histopathologic changes, pro-inflammation cytokines release and increased expression of SEMA7A. In vitro, seawater stimulation led to pro-inflammation cytokine release, cytoskeleton remodeling and increased monolayer permeability in pulmonary microvascular endothelial cells. In addition, knockdown of hypoxia-inducible factor (HIF)-1α inhibited the seawater induced increase expression of SEMA7A. Meanwhile, knockdown of SEMA7A by specific siRNA inhibited the seawater induced aberrant inflammation, endothelial cytoskeleton remodeling and endothelial permeability. These results suggest that SEMA7A is critical in the development of lung inflammation and pulmonary edema in seawater aspiration-induced ALI, and may be a therapeutic target for this disease.     

Lung and Gut Microbiota Changes Associated with Pseudomonas aeruginosa Infection in Mouse Models of Cystic Fibrosis

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr ^tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa. P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice. However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.     

Exogenous Sphingosine 1-Phosphate Protects Murine Splenocytes Against Hypoxia-Induced Injury

Sphingosine-1-phosphate (S1P), a biologically active pleiotropic lipid, is involved in several physiological processes especially in the area of vascular biology and immunology encompassing cell survival, angiogenesis, vascular tone, immune response etc. by interacting with specific cell surface receptors. Hypoxia, a condition common to innumerable pathologies, is known to lethally affect cell survival by throwing off balance global gene expression, redox homeostasis, bioenergetics etc. Several molecular events of cellular adaptations to hypoxia have been closely linked to stabilization of hypoxia inducible factor-1α (HIF-1α). Signalling functions of S1P in physiological events central to hypoxia-induced pathologies led us to investigate efficacy of exogenous S1P in preconditioning murine splenocytes to sustain during cellular stress associated with sub-optimal oxygen. The present study recapitulated the pro-survival benefits of exogenous S1P under normobaric hypoxia. Results indicate a direct effect of S1P supplementation on boosting cellular adaptive responses via HIF-1α stabilization and, activation of pro-survival mediators ERK and Akt. Overwhelming anti-oxidative and anti-inflammatory benefits of S1P preconditioning could also be captured in the present study, as indicated by improved redox homeostasis, reduced oxidative damage, balanced anti/pro-inflammatory cytokine profiles and temporal regulation of nitric oxide secretion and intra-cellular calcium release. Hypoxia induced cell death and the associated stress in cellular milieu in terms of oxidative damage and inflammation could be alleviated with exogenous S1P preconditioning.     

Suppression of PTPN6 exacerbates aluminum oxide nanoparticle-induced COPD-like lesions in mice through activation of STAT pathway

Background

Inhaled nanoparticles can deposit in the deep lung where they interact with pulmonary cells. Despite numerous studies on pulmonary nanotoxicity, detailed molecular mechanisms of specific nanomaterial-induced lung injury have yet to be identified.

Results

Using whole-body dynamic inhalation model, we studied the interactions between aluminum oxide nanoparticles (Al₂O₃ NPs) and the pulmonary system in vivo. We found that seven-day-exposure to Al₂O₃ NPs resulted in emphysema and small airway remodeling in murine lungs, accompanied by enhanced inflammation and apoptosis. Al₂O₃ NPs exposure led to suppression of PTPN6 and phosphorylation of STAT3, culminating in increased expression of the apoptotic marker PDCD4. Rescue of PTPN6 expression or application of a STAT3 inhibitor, effectively protected murine lungs from inflammation and apoptosis, as well as, in part, from the induction of chronic obstructive pulmonary disease (COPD)-like effects.

Conclusion

In summary, our studies show that inhibition of PTPN6 plays a critical role in Al₂O₃ NPs-induced COPD-like lesions.

     

Dysfunctional Inflammation in Cystic Fibrosis Airways: From Mechanisms to Novel Therapeutic Approaches

Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated chloride channel expressed on the apical surface of airway epithelial cells. CFTR absence/dysfunction results in defective ion transport and subsequent airway surface liquid dehydration that severely compromise the airway microenvironment. Noxious agents and pathogens are entrapped inside the abnormally thick mucus layer and establish a highly inflammatory environment, ultimately leading to lung damage. Since chronic airway inflammation plays a crucial role in CF pathophysiology, several studies have investigated the mechanisms responsible for the altered inflammatory/immune response that, in turn, exacerbates the epithelial dysfunction and infection susceptibility in CF patients. In this review, we address the evidence for a critical role of dysfunctional inflammation in lung damage in CF and discuss current therapeutic approaches targeting this condition, as well as potential new treatments that have been developed recently. Traditional therapeutic strategies have shown several limitations and limited clinical benefits. Therefore, many efforts have been made to develop alternative treatments and novel therapeutic approaches, and recent findings have identified new molecules as potential anti-inflammatory agents that may exert beneficial effects in CF patients. Furthermore, the potential anti-inflammatory properties of CFTR modulators, a class of drugs that directly target the molecular defect of CF, also will be critically reviewed. Finally, we also will discuss the possible impact of SARS-CoV-2 infection on CF patients, with a major focus on the consequences that the viral infection could have on the persistent inflammation in these patients.     

Differential Modulation of S1PR(1–5) and Specific Activities of SphK and nSMase in Pulmonary and Cerebral Tissues of Rats Exposed to Hypobaric Hypoxia

Recent preclinical and clinical studies have unfolded the potential of pharmacological modulation of activities of sphingosine‐1‐phosphate (S1P) receptors and S1P metabolizing enzymes for the development of therapeutic interventions against a variety of pathologies. An understanding of differential and temporal effects of hypoxia exposure on the key components of S1P signalling would certainly aid in designing improved drug development strategies in this direction. In view of this, the aim of the present study was to assess the effect of progressive hypobaric hypoxia exposure on expression of S1P receptors (S1PR_1–5) and specific activities of S1P synthesizing enzymes—neutral sphingomyelinase (nSMase) and sphingosine kinase (Sphk) in pulmonary and cerebral tissues of rats exposed to simulated altitude of 21,000 feet in an animal decompression chamber. Along with this, development of cerebral and pulmonary edema and markers of inflammation were studied at 12, 24, and 48 h to validate our study model of hypobaric hypoxia‐induced stress. The protein expression of S1PR_1–5 and activities of Sphk and nSMase enzymes were observed to be dramatically affected by simulated hypobaric hypoxia exposure, concurrent with deterioration of pathology, with 12 h of exposure appearing to be the most critical of the various time points studied.     

Reconstitution of Monocyte Subsets and PD-L1 Expression but Not T Cell PD-1 Expression in Obstructive Sleep Apnea Patients upon PAP Therapy

Obstructive sleep apnea (OSA) is characterized by nocturnal breathing intermissions resulting in oxidative stress and eventually, a low-grade systemic inflammation. The study aimed to investigate the impact of positive airway pressure (PAP) therapy on the inflammatory milieu as measured by monocyte and T cell phenotypic alterations. Participants were assessed for their OSA severity before PAP therapy and about six months later, including patient-reported outcome and therapy usage by telemetry readout. The distributions of the CD14/CD16-characterized monocyte subsets as well as the CD4/CD8-characterized effector T cell subsets with regard to their PD-1 and PD-L1 expression were analyzed by flow cytometry from blood samples. Data of 25 patients revealed a significant reconstitution of the monocyte subset distribution and a decrease in PD-L1 expression on pan-monocytes and CD8⁺ T cells without an association to initial AHI and overweight. The PD-1 expression was still increased on T cell subsets, especially on CD4⁺ TH17/22 cells. We conclude that PAP therapy might have a rapid effect on the monocyte phenotype and overall PD-L1 expression levels. However, T cell immune alterations especially on TH17/22 cells persist longer, indicating an ongoing disturbance of the adaptive immune system.     

Increased Elongase 6 and Δ9-Desaturase Activity are Associated with n-7 and n-9 Fatty Acid Changes in Cystic Fibrosis

Patients with cystic fibrosis, caused by mutations in CFTR, exhibit specific and consistent alterations in the levels of particular unsaturated fatty acids compared with healthy controls. Evidence suggests that these changes may play a role in the pathogenesis of this disease. Among these abnormalities are increases in the levels of n-7 and n-9 fatty acids, particularly palmitoleate (16:1n-7), oleate (18:1n-9), and eicosatrienoate or mead acid (20:3n-9). The underlying mechanisms of these particular changes are unknown, but similar changes in the n-3 and n-6 fatty acid families have been correlated with increased expression of fatty acid metabolic enzymes. This study demonstrated that cystic fibrosis cells in culture exhibit increased metabolism along the metabolic pathways leading to 16:1n-7, 18:1n-9, and 20:3n-9 compared with wild-type cells. Furthermore, these changes are accompanied by increased expression of the enzymes that produce these fatty acids, namely Δ5, Δ6, and Δ9 desaturases and elongases 5 and 6. Taken together, these findings suggest that fatty acid abnormalities of the n-7 and n-9 series in cystic fibrosis are as a result, at least in part, of increased expression and activity of these metabolic enzymes in CFTR-mutated cells.     

Anti-Inflammatory Influences of Cystic Fibrosis Transmembrane Conductance Regulator Drugs on Lung Inflammation in Cystic Fibrosis

Cystic fibrosis (CF) is caused by a defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) which instigates a myriad of respiratory complications including increased vulnerability to lung infections and lung inflammation. The extensive influx of pro-inflammatory cells and production of mediators into the CF lung leading to lung tissue damage and increased susceptibility to microbial infections, creates a highly inflammatory environment. The CF inflammation is particularly driven by neutrophil infiltration, through the IL-23/17 pathway, and function, through NE, NETosis, and NLRP3-inflammasome formation. Better understanding of these pathways may uncover untapped therapeutic targets, potentially reducing disease burden experienced by CF patients. This review outlines the dysregulated lung inflammatory response in CF, explores the current understanding of CFTR modulators on lung inflammation, and provides context for their potential use as therapeutics for CF. Finally, we discuss the determinants that need to be taken into consideration to understand the exaggerated inflammatory response in the CF lung.     

Anti-Inflammatory,Antioxidant, and Antifibrotic Effects of Gingival-Derived MSCs on Bleomycin-Induced Pulmonary Fibrosis in Mice

Background: Mesenchymal stem cell (MSC) intervention has been associated with lung protection. We attempted to determine whether mouse gingival-derived mesenchymal stem cells (GMSCs) could protect against bleomycin-induced pulmonary fibrosis. Methods: Mice were divided into three groups: control (Con), bleomycin (Bl), and bleomycin + MSCs (Bl + MSCs). Mice were treated with 5 mg/kg bleomycin via transtracheal instillation to induce pulmonary fibrosis. We assessed the following parameters: histopathological severity of injury in the lung, liver, kidney, and aortic tissues; the degree of pulmonary fibrosis; pulmonary inflammation; pulmonary oedema; profibrotic factor levels in bronchoalveolar lavage fluid (BALF) and lung tissue; oxidative stress-related indicators and apoptotic index in lung tissue; and gene expression levels of IL-1β, IL-8, TNF-α, lysophosphatidic acid (LPA), lysophosphatidic acid receptor 1 (LPA1), TGF-β, matrix metalloproteinase 9 (MMP-9), neutrophil elastase (NE), MPO, and IL-10 in lung tissue. Results: GMSC intervention attenuated bleomycin-induced pulmonary fibrosis, pulmonary inflammation, pulmonary oedema, and apoptosis. Bleomycin instillation notably increased expression levels of the IL-1β, IL-8, TNF-α, LPA, LPA1, TGF-β, MMP-9, NE, and MPO genes and attenuated expression levels of the IL-10 gene in lung tissue, and these effects were reversed by GMSC intervention. Bleomycin instillation notably upregulated MDA and MPO levels and downregulated GSH and SOD levels in lung tissue, and these effects were reversed by GMSC intervention. GMSC intervention prevented upregulation of neutrophil content in the lung, liver, and kidney tissues and the apoptotic index in lung tissue. Conclusions: GMSC intervention exhibits anti-inflammatory and antioxidant capacities. Deleterious accumulation of neutrophils, which is reduced by GMSC intervention, is a key component of bleomycin-induced pulmonary fibrosis. GMSC intervention impairs bleomycin-induced NE, MMP-9, LPA, APL1, and TGF-β release.     

SUL-151 Decreases Airway Neutrophilia as a Prophylactic and Therapeutic Treatment in Mice after Cigarette Smoke Exposure

Chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) is featured by oxidative stress and chronic inflammation. Due to the poor efficacy of standard glucocorticoid therapy, new treatments are required. Here, we investigated whether the novel compound SUL-151 with mitoprotective properties can be used as a prophylactic and therapeutic treatment in a murine CS-induced inflammation model. SUL-151 (4 mg/kg), budesonide (500 μg/kg), or vehicle were administered via oropharyngeal instillation in this prophylactic and therapeutic treatment setting. The number of immune cells was determined in the bronchoalveolar lavage fluid (BALF). Oxidative stress response, mitochondrial adenosine triphosphate (ATP) production, and mitophagy-related proteins were measured in lung homogenates. SUL-151 significantly decreased more than 70% and 50% of CS-induced neutrophils in BALF after prophylactic and therapeutic administration, while budesonide showed no significant reduction in neutrophils. Moreover, SUL-151 prevented the CS-induced decrease in ATP and mitochondrial mtDNA and an increase in putative protein kinase 1 expression in the lung homogenates. The concentration of SUL-151 was significantly correlated with malondialdehyde level and radical scavenging activity in the lungs. SUL-151 inhibited the increased pulmonary inflammation and mitochondrial dysfunction in this CS-induced inflammation model, which implied that SUL-151 might be a promising candidate for COPD treatment.     

Nanoparticle inhalation augments particle-dependent systemic microvascular dysfunction

Background  

We have shown that pulmonary exposure to fine particulate matter (PM) impairs endothelium dependent dilation in systemic arterioles. Ultrafine PM has been suggested to be inherently more toxic by virtue of its increased surface area. The purpose of this study was to determine if ultrafine PM (or nanoparticle) inhalation produces greater microvascular dysfunction than fine PM. Rats were exposed to fine or ultrafine TiO₂ aerosols (primary particle diameters of ~1 μm and ~21 nm, respectively) at concentrations which do not alter bronchoalveolar lavage markers of pulmonary inflammation or lung damage.     

Impairment of Coronary Arteriolar Endothelium-Dependent Dilation after Multi-Walled Carbon Nanotube Inhalation: A Time-Course Study

Engineered nanomaterials have been developed for widespread applications due to many highly unique and desirable characteristics. The purpose of this study was to assess pulmonary inflammation and subepicardial arteriolar reactivity in response to multi-walled carbon nanotube (MWCNT) inhalation and evaluate the time course of vascular alterations. Rats were exposed to MWCNT aerosols producing pulmonary deposition. Pulmonary inflammation via bronchoalveolar lavage and MWCNT translocation from the lungs to systemic organs was evident 24 h post-inhalation. Coronary arterioles were evaluated 24–168 h post-exposure to determine microvascular response to changes in transmural pressure, endothelium-dependent and -independent reactivity. Myogenic responsiveness, vascular smooth muscle reactivity to nitric oxide, and α-adrenergic responses all remained intact. However, a severe impact on endothelium-dependent dilation was observed within 24 h after MWCNT inhalation, a condition which improved, but did not fully return to control after 168 h. In conclusion, results indicate that MWCNT inhalation not only leads to pulmonary inflammation and cytotoxicity at low lung burdens, but also a low level of particle translocation to systemic organs. MWCNT inhalation also leads to impairments of endothelium-dependent dilation in the coronary microcirculation within 24 h, a condition which does not fully dissipate within 168 h. The innovations within the field of nanotechnology, while exciting and novel, can only reach their full potential if toxicity is first properly assessed.