Editorial for special issue-human precision cut lung slices: an Ex vivo platform for therapeutic target discovery and drug testing in lung disease.

Publications utilizing precision cut lung slices (PCLS) steadily increased from the 1970's, with a significant increase in 2010, to tripling by 2023. PCLS have been used to study a vast array of pulmonary diseases and exposures to pathogens and toxicants to understand pathogenesis of disease but also to examine basic cellular mechanisms that underly lung biology. This Special Issue will highlight new, exciting, and novel research using PCLS, while acknowledging the substantial fund of knowledge that has been gained using this platform.

Early Correlates of School Readiness Before and During the COVID-19 Pandemic Linking Health and School Data.

Importance: Many known correlates of kindergarten readiness are captured in developmental and social screenings in primary care; little is known about how primary care data predicts school readiness. Objective: To identify early Kindergarten Readiness Assessment (KRA) correlates by linking electronic health record (EHR) data with school district KRA data and to examine potential outcomes of the COVID-19 pandemic using KRA scores between 2018 and 2021. Design, Setting, and Participants: This was a retrospective cohort study linking a large primary care practice (PCP) with school assessment data. Linkage used patient name, date of birth, and address. The setting was an urban school district and PCP affiliated with an academic medical center. Students had a KRA score from fall of 2018, 2019, or 2021 (no 2020 KRA due to the COVID-19 pandemic) and at least 1 prior well-child visit at the PCP. Exposures: Exposures included year KRA administered, reported child race and ethnicity, child sex, interpreter for medical visits, child ever failed Ages & Stages Questionnaire (ASQ) 18 to 54 months, ever rarely read to, Medicaid status, food insecurity, housing insecurity, problems with benefits, and caregiver depressive symptoms. Main Outcomes and Measures: KRA score (continuous), with a possible range of 0 to 300 (passing score = 270). Results: A total of 3204 PCP patients (mean [SD] age, 67 [4] months; 1612 male [50.3%]; 2642 Black [82.5%]; 94 Hispanic [2.9%]; 244 White [7.6%]) were matched to their KRA score. Mean (SD) KRA scores were significantly lower in 2021 (mean [SD], 260.0 [13.0]; 214 of 998 [21.4%]) compared with 2019 (mean [SD], 262.7 [13.5]; 317 of 1114 [28.5%]) and 2018 (mean [SD], 263.5 [13.6]; 351 of 1092 [32.1%]), a pattern mirrored in the larger school district. In the linear regression final model (n = 2883), the following binary variables significantly lowered the child's KRA score (points lowered [95% CI]) below a mean passing score of 270.8: child ever failed ASQ after 18 months (-6.7; 95% CI, -7.7 to -5.6), Medicaid insured (-5.7; 95% CI, -9.0 to -2.3), Hispanic ethnicity (-3.8; 95% CI, -6.9 to -0.6), requires interpreter (-3.6; 95% CI, -7.1 to -0.1), 2021 year (-3.5; 95% CI, -4.7 to -2.3), male sex (-2.7; 95% CI, -3.7 to -1.8), ever rarely read to (-1.5; 95% CI, -2.6 to -0.4), and food insecurity (-1.2; 95% CI, -2.4 to -0.1). Race, caregiver depression, housing insecurity, and problems receiving benefits were not associated with KRA scores in final model. Conclusions and Relevance: Findings of this cohort study suggest a deleterious association of the COVID-19 pandemic with early learning and development. There may be potential for PCPs and school districts to collaborate to identify and mitigate risks much earlier.

Rhinovirus induces airway remodeling: what are the physiological consequences?

BACKGROUND: Rhinovirus infections commonly evoke asthma exacerbations in children and adults. Recurrent asthma exacerbations are associated with injury-repair responses in the airways that collectively contribute to airway remodeling. The physiological consequences of airway remodeling can manifest as irreversible airway obstruction and diminished responsiveness to bronchodilators. Structural cells of the airway, including epithelial cells, smooth muscle, fibroblasts, myofibroblasts, and adjacent lung vascular endothelial cells represent an understudied and emerging source of cellular and extracellular soluble mediators and matrix components that contribute to airway remodeling in a rhinovirus-evoked inflammatory environment. MAIN BODY: While mechanistic pathways associated with rhinovirus-induced airway remodeling are still not fully characterized, infected airway epithelial cells robustly produce type 2 cytokines and chemokines, as well as pro-angiogenic and fibroblast activating factors that act in a paracrine manner on neighboring airway cells to stimulate remodeling responses. Morphological transformation of structural cells in response to rhinovirus promotes remodeling phenotypes including induction of mucus hypersecretion, epithelial-to-mesenchymal transition, and fibroblast-to-myofibroblast transdifferentiation. Rhinovirus exposure elicits airway hyperresponsiveness contributing to irreversible airway obstruction. This obstruction can occur as a consequence of sub-epithelial thickening mediated by smooth muscle migration and myofibroblast activity, or through independent mechanisms mediated by modulation of the ß2 agonist receptor activation and its responsiveness to bronchodilators. Differential cellular responses emerge in response to rhinovirus infection that predispose asthmatic individuals to persistent signatures of airway remodeling, including exaggerated type 2 inflammation, enhanced extracellular matrix deposition, and robust production of pro-angiogenic mediators. CONCLUSIONS: Few therapies address symptoms of rhinovirus-induced airway remodeling, though understanding the contribution of structural cells to these processes may elucidate future translational targets to alleviate symptoms of rhinovirus-induced exacerbations.

Rhinovirus infection induces secretion of endothelin-1 from airway epithelial cells in both in vitro and in vivo models.

BACKGROUND: Rhinovirus (RV) infection of airway epithelial cells triggers asthma exacerbations, during which airway smooth muscle (ASM) excessively contracts. Due to ASM contraction, airway epithelial cells become mechanically compressed. We previously reported that compressed human bronchial epithelial (HBE) cells are a source of endothelin-1 (ET-1) that causes ASM contraction. Here, we hypothesized that epithelial sensing of RV by TLR3 and epithelial compression induce ET-1 secretion through a TGF-ß receptor (TGFßR)-dependent mechanism. METHODS: To test this, we used primary HBE cells well-differentiated in air-liquid interface culture and two mouse models (ovalbumin and house dust mite) of allergic airway disease (AAD). HBE cells were infected with RV-A16, treated with a TLR3 agonist (poly(I:C)), or exposed to compression. Thereafter, EDN1 (ET-1 protein-encoding gene) mRNA expression and secreted ET-1 protein were measured. We examined the role of TGFßR in ET-1 secretion using either a pharmacologic inhibitor of TGFßR or recombinant TGF-ß1 protein. In the AAD mouse models, allergen-sensitized and allergen-challenged mice were subsequently infected with RV. We then measured ET-1 in bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness (AHR) following methacholine challenge. RESULTS: Our data reveal that RV infection induced EDN1 expression and ET-1 secretion in HBE cells, potentially mediated by TLR3. TGFßR activation was partially required for ET-1 secretion, which was induced by RV, poly(I:C), or compression. TGFßR activation alone was sufficient to increase ET-1 secretion. In AAD mouse models, RV induced ET-1 secretion in BALF, which positively correlated with AHR. CONCLUSIONS: Our data provide evidence that RV infection increased epithelial-cell ET-1 secretion through a TGFßR-dependent mechanism, which contributes to bronchoconstriction during RV-induced asthma exacerbations.

Using Quality Improvement to Design Early Childhood Services Navigation in Primary Care.

Enrollment in high-quality early childhood education (ECE) improves educational and health outcomes and can mitigate racial and economic disparities. Pediatricians are encouraged to promote ECE yet lack the time and knowledge to assist families effectively. In 2016, our academic primary care center hired an ECE Navigator to promote ECE and help families enroll. Our SMART aims were to increase the number of children with facilitated referrals to high-quality ECE programs from 0 to 15 per month and to confirm enrollment on a subset to achieve an enrollment rate of 50% by December 31, 2020. Methods: We used the Institute for Healthcare Improvement's Model for Improvement. Interventions included system changes in partnership with ECE agencies (eg, interactive map of subsidized preschool options, streamlined enrollment forms), case management with families, and population-based approaches to understand families' needs and the program's overall impact. We plotted the number of monthly facilitated referrals and the percentage of referrals enrolled on run and control charts. We used standard probability-based rules to identify special causes. Results: Facilitated referrals increased from 0 to 29 per month and remained above 15. The percentage of enrolled referrals increased from 30% to 74% in 2018, then decreased to 27% in 2020 when childcare availability declined during the pandemic. Conclusions: Our innovative ECE partnership improved access to high-quality ECE. Interventions could be adopted in part or whole by other clinical practices or WIC offices to equitably improve early childhood experiences for low-income families and racial minorities.

Rhinovirus C15 Attenuates Relaxation and cAMP Production in Human Airways and Smooth Muscle.

Rhinoviruses (RVs) evoke as many as 85% of acute asthma exacerbations in children and 50% in adults and can induce airway hyperresponsiveness and decrease efficacy of current therapeutics to provide symptom relief. Using human precision-cut lung slices (hPCLSs), primary human air-liquid interface-differentiated airway epithelial cells (HAECs), and human airway smooth muscle (HASM) as preclinical experimental models, we demonstrated that RV-C15 attenuates agonist-induced bronchodilation. Specifically, airway relaxation to formoterol and cholera toxin, but not forskolin (Fsk), was attenuated following hPCLS exposure to RV-C15. In isolated HASM cells, exposure to conditioned media from RV-exposed HAECs decreased cellular relaxation in response to isoproterenol and prostaglandin E2, but not Fsk. Additionally, cAMP generation elicited by formoterol and isoproterenol, but not Fsk, was attenuated following HASM exposure to RV-C15-conditioned HAEC media. HASM exposure to RV-C15-conditioned HAEC media modulated expression of components of relaxation pathways, specifically GNAI1 and GRK2. Strikingly, similar to exposure to intact RV-C15, hPCLS exposed to UV-inactivated RV-C15 showed markedly attenuated airway relaxation in response to formoterol, suggesting that the mechanism(s) of RV-C15-mediated loss of bronchodilation is independent of virus replication pathways. Further studies are warranted to identify soluble factor(s) regulating the epithelial-driven smooth muscle loss of ß2-adrenergic receptor function.

A Par3/LIM Kinase/Cofilin Pathway Mediates Human Airway Smooth Muscle Relaxation by TAS2R14.

TAS2Rs (bitter taste receptors) are GPCRs (G protein-coupled receptors) expressed on human airway smooth muscle (HASM) cells; when activated by receptor agonists they evoke marked airway relaxation. In both taste and HASM cells, TAS2Rs activate a canonical Gßγ-mediated stimulation of Ca2+ release from intracellular stores by activation of PLCß (phospholipase Cß). Alone, this [Ca2+]i signaling does not readily account for relaxation, particularly since bronchoconstrictive agonists acting at Gq-coupled receptors also increase [Ca2+]i. We established that TAS2R14 activation in HASM promotes relaxation through F-actin (filamentous actin) severing. This destabilization of actin was from agonist-promoted activation (dephosphorylation) of cofilin, which was pertussis toxin sensitive. Cofilin dephosphorylation was due to TAS2R-mediated deactivation of LIM domain kinase. The link between early receptor action and the distal cofilin dephosphorylation was found to be the polarity protein partitioning defective 3 (Par3), a known binding partner with PLCß that inhibits LIM kinase. The physiologic relevance of this pathway was assessed using knock-downs of cofilin and Par3 in HASM cells and in human precision-cut lung slices. Relaxation by TAS2R14 agonists was ablated with knock-down of either protein as assessed by magnetic twisting cytometry in isolated cells or intact airways in the slices. Blocking [Ca2+]i release by TAS2R14 inhibited agonist-promoted cofilin dephosphorylation, confirming a role for [Ca2+]i in actin-modifying pathways. These results further elucidate the mechanistic basis of TAS2R-mediated HASM relaxation and point toward nodal points that may act as asthma or chronic obstructive pulmonary disease response modifiers or additional targets for novel bronchodilators.

Epinephrine evokes shortening of human airway smooth muscle cells following ß2 adrenergic receptor desensitization.

Epinephrine (EPI), an endogenous catecholamine involved in the body's fight-or-flight responses to stress, activates α1-adrenergic receptors (α1ARs) expressed on various organs to evoke a wide range of physiological functions, including vasoconstriction. In the smooth muscle of human bronchi, however, the functional role of EPI on α1ARs remains controversial. Classically, evidence suggests that EPI promotes bronchodilation by stimulating ß2-adrenergic receptors (ß2ARs). Conventionally, the selective ß2AR agonism of EPI was thought to be, in part, due to a predominance of ß2ARs and/or a sparse, or lack of α1AR activity in human airway smooth muscle (HASM) cells. Surprisingly, we find that HASM cells express a high abundance of ADRA1B (the α1AR subtype B) and identify a spontaneous "switch-like" activation of α1ARs that evokes intracellular calcium, myosin light chain phosphorylation, and HASM cell shortening. The switch-like responses, and related EPI-induced biochemical and mechanical signals, emerged upon pharmacological inhibition of ß2ARs and/or under experimental conditions that induce ß2AR tachyphylaxis. EPI-induced procontractile effects were abrogated by an α1AR antagonist, doxazosin mesylate (DM). These data collectively uncover a previously unrecognized feed-forward mechanism driving bronchospasm via two distinct classes of G protein-coupled receptors (GPCRs) and provide a basis for reexamining α1AR inhibition for the management of stress/exercise-induced asthma and/or ß2-agonist insensitivity in patients with difficult-to-control, disease subtypes.

CEBPD modulates the airway smooth muscle transcriptomic response to glucocorticoids.

BACKGROUND: CCAAT/Enhancer Binding Protein D (CEBPD), a pleiotropic glucocorticoid-responsive transcription factor, modulates inflammatory responses. Of relevance to asthma, expression of CEBPD in airway smooth muscle (ASM) increases with glucocorticoid exposure. We sought to characterize CEBPD-mediated transcriptomic responses to glucocorticoid exposure in ASM by measuring changes observed after knockdown of CEBPD and its impact on asthma-related ASM function. METHODS: Primary ASM cells derived from four donors were transfected with CEBPD or non-targeting (NT) siRNA and exposed to vehicle control, budesonide (100 nM, 18 h), TNFα (10 ng/ml, 18 h), or both budesonide and TNFα. Subsequently, RNA-Seq was used to measure gene expression levels, and pairwise differential expression results were obtained for exposures versus vehicle and knockdown versus control conditions. Weighted gene co-expression analysis was performed to identify groups of genes with similar expression patterns across the various experimental conditions (i.e., CEBPD knockdown status, exposures). RESULTS: CEBPD knockdown altered expression of 3037 genes under at least one exposure (q-value < 0.05). Co-expression analysis identified sets of 197, 152 and 290 genes that were correlated with CEBPD knockdown status, TNFα exposure status, and both, respectively. JAK-STAT signaling pathway genes, including IL6R and SOCS3, were among those influenced by both TNFα and CEBPD knockdown. Immunoblot assays revealed that budesonide-induced IL-6R protein expression and augmented IL-6-induced STAT3 phosphorylation levels were attenuated by CEBPD knockdown in ASM. CONCLUSIONS: CEBPD modulates glucocorticoid responses in ASM, in part via modulation of IL-6 receptor signaling.

Human Precision-Cut Lung Slices: Generation of and Measurement of Contractility and Relaxation of Small Airways.

Lung slices have been used since the mid-1990's to study various aspects of lung biology that include, but are not limited to, mechanisms of airway contraction and relaxation; the pulmonary immune response in the context of inflammatory diseases of the lung like asthma and chronic obstructive pulmonary disease; mast cell-mediated airway contractility and inflammation; modulation of airway cells following pathogen exposure; and consequences of environmental toxicant exposure. Here we describe the generation of human precision-cut lung slices (hPCLS) and measurement of contraction and relaxation of small airways within the slices.

Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma.

Asthma and inflammatory airway diseases restrict airflow in the lung, compromising gas exchange and lung function. Inhaled corticosteroids (ICSs) can reduce inflammation, control symptoms, and improve lung function; however, a growing number of patients with severe asthma do not benefit from ICS. Using bronchial airway epithelial brushings from patients with severe asthma or primary human cells, we delineated a corticosteroid-driven fibroblast growth factor (FGF)-dependent inflammatory axis, with FGF-responsive fibroblasts promoting downstream granulocyte colony-stimulating factor (G-CSF) production, hyaluronan secretion, and neutrophilic inflammation. Allergen challenge studies in mice demonstrate that the ICS, fluticasone propionate, inhibited type 2-driven eosinophilia but induced a concomitant increase in FGFs, G-CSF, hyaluronan, and neutrophil infiltration. We developed a model of steroid-induced neutrophilic inflammation mediated, in part, by induction of an FGF-dependent epithelial-mesenchymal axis, which may explain why some individuals do not benefit from ICS. In further proof-of-concept experiments, we found that combination therapy with pan-FGF receptor inhibitors and corticosteroids prevented both eosinophilic and steroid-induced neutrophilic inflammation. Together, these results establish FGFs as therapeutic targets for severe asthma patients who do not benefit from ICS.

Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface ß2AR (ß2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces ß2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases ß-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

Resolving Pediatric Preventive Care Gaps Through Hospital Inreach.

OBJECTIVES: Panel management processes have been used to help improve population-level care and outreach to patients outside the health care system. Opportunities to resolve gaps in preventive care are often missed when patients present outside of primary care settings but still within the larger health care system. We hypothesized that we could design a process of "inreach" capable of resolving care gaps traditionally addressed solely in primary care settings. Our aim was to identify and resolve gaps in vaccinations and screening for lead exposure for children within our primary care registry aged 2 to 66 months who were admitted to the hospital. We sought to increase care gaps closed from 12% to 50%. METHODS: We formed a multidisciplinary team composed of primary care and hospital medicine physicians, nursing leadership, and quality improvement experts within the Division of General and Community Pediatrics. The team identified a smart aim, mapped the process, predicted failure modes, and developed a key driver diagram. We identified, tested, and implemented multiple interventions related to role assignment, identification of admitted patients with care gaps, and communication with the inpatient teams. RESULTS: After increasing the reliability of our process to identify and contact the hospital medicine team caring for patients who needed action to 88%, we observed an increase in the preventive care gaps closed from 12% to 41%. CONCLUSIONS: A process to help improve preventive care for children can be successfully implemented by using quality improvement methodologies outside of the traditional domains of primary care.

Airway smooth muscle and airway hyperresponsiveness in asthma: mechanisms of airway smooth muscle dysfunction.

Airway smooth muscle plays a pivotal role in modulating bronchomotor tone. Modulation of contractile and relaxation signaling is critical to alleviate the airway hyperresponsiveness (AHR) associated with asthma. Emerging studies examining the phenotype of ASM in the context of asthma provide rich avenues to develop more effective therapeutics to attenuate the AHR associated with the disease.

TGF-ß: The missing link in obesity-associated airway diseases?

Obesity is emerging as a global public health epidemic. The co-morbidities associated with obesity significantly contribute to reduced quality of life, mortality, and global healthcare burden. Compared to other asthma comorbidities, obesity prominently engenders susceptibility to inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), contributes to greater disease severity and evokes insensitivity to current therapies. Unlike in other metabolic diseases associated with obesity, the mechanistic link between obesity and airway diseases is only poorly defined. Transforming growth factor-ß (TGF-ß) is a pleiotropic inflammatory cytokine belonging to a family of growth factors with pivotal roles in asthma. In this review, we summarize the role of TGF-ß in major obesity-associated co-morbidities to shed light on mechanisms of the diseases. Literature evidence shows that TGF-ß mechanistically links many co-morbidities with obesity through its profibrotic, remodeling, and proinflammatory functions. We posit that TGF-ß plays a similar mechanistic role in obesity-associated inflammatory airway diseases such as asthma and COPD. Concerning the role of TGF-ß on metabolic effects of obesity, we posit that TGF-ß has a similar mechanistic role in obesity-associated inflammatory airway diseases in interplay with different comorbidities such as hypertension, metabolic diseases like type 2 diabetes, and cardiomyopathies. Future studies in TGF-ß-dependent mechanisms in obesity-associated inflammatory airway diseases will advance our understanding of obesity-induced asthma and help find novel therapeutic targets for prevention and treatment.

Integration and Impact of Telemedicine in Underserved Pediatric Primary Care.

Telemedicine, more novel in provision of pediatric care, rapidly expanded due to the recent coronavirus disease 2019 pandemic. This study aimed to determine the feasibility of telemedicine for acute and chronic care provision in an underserved pediatric primary care center. Items assessed included patient demographic data, chief complaint, and alternative care locations if telemedicine was not available. In our setting, 62% of telemedicine visits were for acute concerns and 38% for chronic concerns. Of acute telemedicine visits, 16.5% of families would have sought care in the Emergency Department/Urgent Care, and 11.3% would have opted for no care had telemedicine not been offered. The most common chronic issues addressed were attention deficit hyperactivity disorder (80.3%) and asthma (16.9%). Racial disparities existed among our telemedicine visits with Black patients utilizing telemedicine services less frequently than non-Black patients. Telemedicine is feasible for pediatric acute and chronic care, but systems must be designed to mitigate widening racial disparities.

Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells.

N-methyl-D-aspartate (NMDA) receptors are widely expressed in the central nervous system. However, their presence and function at extraneuronal sites is less well characterized. In the present study, we examined the expression of NMDA receptor subunit mRNA and protein in human pulmonary artery (HPA) by quantitative polymerase chain reaction (PCR), immunohistochemistry and immunoblotting. We demonstrate that both GluN1 and GluN2 subunit mRNAs are expressed in HPA. In addition, GluN1 and GluN2 (A-D) subunit proteins are expressed by human pulmonary artery smooth muscle cells (HPASMCs) in vitro and in vivo. These subunits localize on the surface of HPASMCs and form functional ion channels as evidenced by whole-cell patch-clamp electrophysiology and reduced phenylephrine-induced contractile responsiveness of human pulmonary artery by the NMDA receptor antagonist MK801 under hypoxic condition. HPASMCs also express high levels of serine racemase and vesicular glutamate transporter 1, suggesting a potential source of endogenous agonists for NMDA receptor activation. Our findings show HPASMCs express functional NMDA receptors in line with their effect on pulmonary vasoconstriction, and thereby suggest a novel therapeutic target for pharmacological modulations in settings associated with pulmonary vascular dysfunction.