The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis.

The epididymis functions as transition zone for post-testicular sperm maturation and storage and faces contrasting immunological challenges, i.e. tolerance towards spermatozoa vs. reactivity against pathogens. Thus, normal organ function and integrity relies heavily on a tightly controlled immune balance. Previous studies described inflammation-associated tissue damage solely in the distal regions (corpus, cauda), but not in the proximal regions (initial segment, caput). To understand the observed region-specific immunity along the epididymal duct, we have used an acute bacterial epididymitis mouse model and analyzed the disease progression. Whole transcriptome analysis using RNAseq 10 days post infection showed a pro-inflammatory environment within the cauda, while the caput exhibited only minor transcriptional changes. High-dimensional flow cytometry analyses revealed drastic changes in the immune cell composition upon infection with uropathogenic Escherichia coli. A massive influx of neutrophils and monocytes was observed exclusively in distal regions and was associated with bacterial appearance and tissue alterations. In order to clarify the reasons for the region-specific differences in the intensity of immune responses, we investigated the heterogeneity of resident immune cell populations under physiological conditions by scRNASeq analysis of extravascular CD45+ cells. Twelve distinct immune cell subsets were identified, displaying substantial differences in distribution along the epididymis as further assessed by flow cytometry and immunofluorescence staining. Macrophages constituted the majority of resident immune cells and were further separated in distinct subgroups based on their transcriptional profile, tissue location and monocyte-dependence. Crucially, the proximal and distal regions showed striking differences in their immunological landscapes. These findings indicate that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for addressing the contrasting immunological challenges and thus, preserving tissue integrity and organ function.

Three tissue resident macrophage subsets coexist across organs with conserved origins and life cycles.

Resident macrophages orchestrate homeostatic, inflammatory, and reparative activities. It is appreciated that different tissues instruct specialized macrophage functions. However, individual tissues contain heterogeneous subpopulations, and how these subpopulations are related is unclear. We asked whether common transcriptional and functional elements could reveal an underlying framework across tissues. Using single-cell RNA sequencing and random forest modeling, we observed that four genes could predict three macrophage subsets that were present in murine heart, liver, lung, kidney, and brain. Parabiotic and genetic fate mapping studies revealed that these core markers predicted three unique life cycles across 17 tissues. TLF+ (expressing TIMD4 and/or LYVE1 and/or FOLR2) macrophages were maintained through self-renewal with minimal monocyte input; CCR2+ (TIMD4−LYVE1−FOLR2−) macrophages were almost entirely replaced by monocytes, and MHC-IIhi macrophages (TIMD4−LYVE1−FOLR2−CCR2−), while receiving modest monocyte contribution, were not continually replaced. Rather, monocyte-derived macrophages contributed to the resident macrophage population until they reached a defined upper limit after which they did not outcompete pre-existing resident macrophages. Developmentally, TLF+ macrophages were first to emerge in the yolk sac and early fetal organs. Fate mapping studies in the mouse and human single-cell RNA sequencing indicated that TLF+ macrophages originated from both yolk sac and fetal monocyte precursors. Furthermore, TLF+ macrophages were the most transcriptionally conserved subset across mouse tissues and between mice and humans, despite organ- and species-specific transcriptional differences. Here, we define the existence of three murine macrophage subpopulations based on common life cycle properties and core gene signatures and provide a common starting point to understand tissue macrophage heterogeneity.

Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress.

Hypertension affects one-third of the world's population, leading to cardiac dysfunction that is modulated by resident and recruited immune cells. Cardiomyocyte growth and increased cardiac mass are essential to withstand hypertensive stress; however, whether immune cells are involved in this compensatory cardioprotective process is unclear. In normotensive animals, single-cell transcriptomics of fate-mapped self-renewing cardiac resident macrophages (RMs) revealed transcriptionally diverse cell states with a core repertoire of reparative gene programs, including high expression of insulin-like growth factor-1 (Igf1). Hypertension drove selective in situ proliferation and transcriptional activation of some cardiac RM states, directly correlating with increased cardiomyocyte growth. During hypertension, inducible ablation of RMs or selective deletion of RM-derived Igf1 prevented adaptive cardiomyocyte growth, and cardiac mass failed to increase, which led to cardiac dysfunction. Single-cell transcriptomics identified a conserved IGF1-expressing macrophage subpopulation in human cardiomyopathy. Here we defined the absolute requirement of RM-produced IGF-1 in cardiac adaptation to hypertension.

Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis.

Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.

Publisher Correction: Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

In the version of this article initially published, the equal contribution of the third author was omitted. The footnote links for that author should be "Sara Nejat1,11" and the correct statement is as follows: "11These authors contributed equally: Sarah A. Dick, Jillian A. Macklin, Sara Nejat." The error has been corrected in the HTML and PDF versions of the article.

Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4+LYVE1+MHC-IIloCCR2- resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4-LYVE1-MHC-IIhiCCR2- macrophages and fully replaced TIMD4-LYVE1-MHC-IIhiCCR2+ macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4+ and TIMD4- resident macrophage abundance, whereas CCR2+ monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.

Sodium nitrite augments lung S-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats.

Deficient nitric oxide (NO) signaling plays a critical role in the pathogenesis of chronic neonatal pulmonary hypertension (PHT). Physiological NO signaling is regulated by S-nitrosothiols (SNOs), which act both as a reservoir for NO and as a reversible modulator of protein function. We have previously reported that therapy with inhaled NO (iNO) increased peroxynitrite-mediated nitration in the juvenile rat lung, although having minimal reversing effects on vascular remodeling. We hypothesized that sodium nitrite (NaNO2) would be superior to iNO in enhancing lung SNOs, thereby contributing to reversal of chronic hypoxic PHT. Rat pups were exposed to air or hypoxia (13% O2) from postnatal days 1 to 21. Dose-response prevention studies were conducted from days 1-21 to determine the optimal dose of NaNO2. Animals then received rescue therapy with daily subcutaneous NaNO2 (20 mg/kg), vehicle, or were continuously exposed to iNO (20 ppm) from days 14-21. Chronic PHT secondary to hypoxia was both prevented and reversed by treatment with NaNO2. Rescue NaNO2 increased lung NO and SNO contents to a greater extent than iNO, without causing nitration. Seven lung SNO proteins upregulated by treatment with NaNO2 were identified by multiplex tandem mass tag spectrometry, one of which was leukotriene A4 hydrolase (LTA4H). Rescue therapy with a LTA4H inhibitor, SC57461A (10 mg·kg-1·day-1 sc), partially reversed chronic hypoxic PHT. We conclude that NaNO2 was superior to iNO in increasing tissue NO and SNO generation and reversing chronic PHT, in part via upregulated SNO-LTA4H.

A CD103+ Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.

Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific T cell responses during subclinical viral myocarditis.

mTOR-Notch3 signaling mediates pulmonary hypertension in hypoxia-exposed neonatal rats independent of changes in autophagy.

BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) is a pivotal regulator of cell proliferation, survival, and autophagy. Autophagy is increased in adult experimental chronic pulmonary hypertension (PHT), but its contributory role to pulmonary vascular disease remains uncertain and has yet to be explored in the neonatal animal. Notch is a major pro-proliferative pathway activated by mTOR. A direct relationship between autophagy and Notch signaling has not been previously explored. Our aim was to examine changes in mTOR-, Notch-, and autophagy-related pathways and the therapeutic effects of autophagy modulators in experimental chronic neonatal PHT secondary to chronic hypoxia. METHODS: Rat pups were exposed to normoxia or hypoxia (13% O2 ) from postnatal days 1-21, while receiving treatment with temsirolimus (mTOR inhibitor), DAPT (Notch inhibitor), or chloroquine (inhibitor of autophagic flux). RESULTS: Exposure to hypoxia up-regulated autophagy and Notch3 signaling markers in lung, pulmonary artery (PA), and PA-derived smooth muscle cells (SMCs). Temsirolimus prevented chronic PHT and attenuated PA and SMC signaling secondary to hypoxia. These effects were replicated by DAPT. mTOR or Notch inhibition also down-regulated smooth muscle content of platelet-derived growth factor ß-receptor, a known contributor to vascular remodeling. In contrast, chloroquine had no modifying effects on markers of chronic PHT. Knockdown of Beclin-1 in SMCs had no effect on hypoxia-stimulated Notch3 signaling. CONCLUSIONS: mTOR-Notch3 signaling plays a critical role in experimental chronic neonatal PHT. Inhibition of autophagy did not suppress Notch signaling and had no effect on markers of chronic PHT.

Fingerprint of long non-coding RNA regulated by cyclic mechanical stretch in human aortic smooth muscle cells: implications for hypertension.

Emerging evidence suggests that long non-coding RNAs (lncRNAs) represent a cellular hub coordinating various cellular processes that are critical in health and disease. Mechanical stress triggers changes in vascular smooth muscle cells (VSMCs) that in turn contribute to pathophysiological changes within the vasculature. We sought to evaluate the role that lncRNAs play in mechanical stretch-induced alterations of human aortic smooth muscle cells (HASMCs). RNA (lncRNA and mRNA) samples isolated from HASMCs that had been subjected to 10 or 20% elongation (1 Hz) for 24 h were profiled with the Arraystar Human LncRNA Microarray V3.0. LncRNA expression was quantified in parallel via qRT-PCR. Of the 30,586 human lncRNAs screened, 580 were differentially expressed (DE, P < 0.05) in stretched HASMCs. Amongst the 26,109 protein-coding transcripts evaluated, 25 of those DE were associated with 25 of the aforementioned DE lncRNAs (P < 0.05). Subsequent Kyoto Encyclopedia of Genes and Genomes analysis revealed that the DE mRNAs were largely associated with the tumor necrosis factor signaling pathway and inflammation. Gene Ontology analysis indicated that the DE mRNAs were associated with cell differentiation, stress response, and response to external stimuli. We describe the first transcriptome profile of stretch-induced changes in HASMCs and provide novel insights into the regulatory switches that may be fundamental in governing aberrant VSMC remodeling.

Intermittent hypoxia during recovery from neonatal hyperoxic lung injury causes long-term impairment of alveolar development: A new rat model of BPD.

Bronchopulmonary dysplasia (BPD) is a chronic lung injury characterized by impaired alveologenesis that may persist into adulthood. Rat models of BPD using varying degrees of hyperoxia to produce injury either cause early mortality or spontaneously recover following removal of the inciting stimulus, thus limiting clinical relevance. We sought to refine an established rat model induced by exposure to 60% O2 from birth by following hyperoxia with intermittent hypoxia (IH). Rats exposed from birth to air or 60% O2 until day 14 were recovered in air with or without IH (FIO2 = 0.10 for 10 min every 6 h) until day 28 Animals exposed to 60% O2 and recovered in air had no evidence of abnormal lung morphology on day 28 or at 10-12 wk. In contrast, 60% O2-exposed animals recovered in IH had persistently increased mean chord length, more dysmorphic septal crests, and fewer peripheral arteries. Recovery in IH also increased pulmonary vascular resistance, Fulton index, and arterial wall thickness. IH-mediated abnormalities in lung structure (but not pulmonary hypertension) persisted when reexamined at 10-12 wk, accompanied by increased pulmonary vascular reactivity and decreased exercise tolerance. Increased mean chord length secondary to IH was prevented by treatment with a peroxynitrite decomposition catalyst [5,10,15,20-Tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin iron (III) chloride, 30 mg/kg/day, days 14-28], an effect accompanied by fewer inflammatory cells. We conclude that IH during recovery from hyperoxia-induced injury prevents recovery of alveologenesis and leads to changes in lung and pulmonary vascular function lasting into adulthood, thus more closely mimicking contemporary BPD.

Simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of RhoA signaling.

Chronic neonatal pulmonary hypertension (PHT) frequently results in early death. Systemically administered Rho-kinase (ROCK) inhibitors prevent and reverse chronic PHT in neonatal rats, but at the cost of severe adverse effects, including systemic hypotension and growth restriction. Simvastatin has pleiotropic inhibitory effects on isoprenoid intermediates that may limit activity of RhoA, which signals upstream of ROCK. We therefore hypothesized that statin treatment would safely limit pulmonary vascular RhoA activity and prevent and reverse experimental chronic neonatal PHT via downstream inhibitory effects on pathological ROCK activity. Sprague-Dawley rats in normoxia (room air) or moderate normobaric hypoxia (13% O2) received simvastatin (2 mg·kg-1·day-1 ip) or vehicle from postnatal days 1-14 (prevention protocol) or from days 14-21 (rescue protocol). Chronic hypoxia increased RhoA and ROCK activity in lung tissue. Simvastatin reduced lung content of the isoprenoid intermediate farnesyl pyrophosphate and decreased RhoA/ROCK signaling in the hypoxia-exposed lung. Preventive or rescue treatment of chronic hypoxia-exposed animals with simvastatin decreased pulmonary vascular resistance, right ventricular hypertrophy, and pulmonary arterial remodeling. Preventive simvastatin treatment improved weight gain, did not lower systemic blood pressure, and did not cause apparent toxic effects on skeletal muscle, liver or brain. Rescue therapy with simvastatin improved exercise capacity. We conclude that simvastatin limits RhoA/ROCK activity in the chronic hypoxia-exposed lung, thus preventing or ameliorating hemodynamic and structural markers of chronic PHT and improving long-term outcome, without causing adverse effects.

Leukotriene B4 mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury.

Systemically-administered bleomycin causes inflammation, arrested lung growth, and pulmonary hypertension (PHT) in the neonatal rat, similar to human infants with severe bronchopulmonary dysplasia (BPD). Leukotrienes (LTs) are inflammatory lipid mediators produced by multiple cell types in the lung. The major LTs, LTB4 and cysteinyl LTs, are suggested to contribute to BPD, but their specific roles remain largely unexplored in experimental models. We hypothesized that LTs are increased in bleomycin-induced BPD-like injury, and that inhibition of LT production would prevent inflammatory cell influx and thereby ameliorate lung injury. Rat pups were exposed to bleomycin (1 mg·kg(-1)·day(-1) ip) or vehicle (control) from postnatal days 1-14 and were treated with either zileuton (5-lipoxygenase inhibitor), montelukast (cysteinyl LT1 receptor antagonist), or SC57461A (LTA4 hydrolase inhibitor) 10 mg·kg(-1)·day(-1) ip. Bleomycin led to increased lung content of LTB4, but not cysteinyl LTs. Bleomycin-induced increases in tissue neutrophils and macrophages and lung contents of LTB4 and tumor necrosis factor-α were all prevented by treatment with zileuton. Treatment with zileuton or SC57461A also prevented the hemodynamic and structural markers of chronic PHT, including raised pulmonary vascular resistance, increased Fulton index, and arterial wall remodeling. However, neither treatment prevented impaired alveolarization or vascular hypoplasia secondary to bleomycin. Treatment with montelukast had no effect on macrophage influx, PHT, or on abnormal lung structure. We conclude that LTB4 plays a crucial role in lung inflammation and PHT in experimental BPD. Agents targeting LTB4 or LTB4-mediated signaling may have utility in infants at risk of developing BPD-associated PHT.

Neutrophil elastase-induced elastin degradation mediates macrophage influx and lung injury in 60% O2-exposed neonatal rats.

Neutrophil (PMNL) influx precedes lung macrophage (LM) influx into the lung following exposure of newborn pups to 60% O2. We hypothesized that PMNL were responsible for the signals leading to LM influx. This was confirmed when inhibition of PMNL influx with a CXC chemokine receptor-2 antagonist, SB-265610, also prevented the 60% O2-dependent LM influx, LM-derived nitrotyrosine formation, and pruning of small arterioles. Exposure to 60% O2 was associated with increased lung contents of neutrophil elastase and α-elastin, a marker of denatured elastin, and a decrease in elastin fiber density. This led us to speculate that neutrophil elastase-induced elastin fragments were the chemokines that led to a LM influx into the 60% O2-exposed lung. Inhibition of neutrophil elastase with sivelestat or elafin attenuated the LM influx. Sivelestat also attenuated the 60% O2-induced decrease in elastin fiber density. Daily injections of pups with an antibody to α-elastin prevented the 60% O2-dependent LM influx, impaired alveologenesis, and impaired small vessel formation. This suggests that neutrophil elastase inhibitors may protect against neonatal lung injury not only by preventing structural elastin degradation, but also by blocking elastin fragment-induced LM influx, thus preventing tissue injury from LM-derived peroxynitrite formation.

Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs.

Arginase is an enzyme that limits substrate L-arginine bioavailability for the production of nitric oxide by the nitric oxide synthases and produces L-ornithine, which is a precursor for collagen formation and tissue remodeling. We studied the pulmonary vascular effects of arginase inhibition in an established model of repeated systemic bleomycin sulfate administration in neonatal rats that results in pulmonary hypertension and lung injury mimicking the characteristics typical of bronchopulmonary dysplasia. We report that arginase expression is increased in the lungs of bleomycin-exposed neonatal rats and that treatment with the arginase inhibitor amino-2-borono-6-hexanoic acid prevented the bleomycin-induced development of pulmonary hypertension and deposition of collagen. Arginase inhibition resulted in increased L-arginine and L-arginine bioavailability and increased pulmonary nitric oxide production. Arginase inhibition also normalized the expression of inducible nitric oxide synthase, and reduced bleomycin-induced nitrative stress while having no effect on bleomycin-induced inflammation. Our data suggest that arginase is a promising target for therapeutic interventions in neonates aimed at preventing lung vascular remodeling and pulmonary hypertension.

Rho kinase mediates right ventricular systolic dysfunction in rats with chronic neonatal pulmonary hypertension.

Chronic neonatal pulmonary hypertension frequently culminates in right ventricular (RV) failure and death. In juvenile rats, RV systolic dysfunction secondary to chronic hypoxia is rescued by systemic treatment with a Rho kinase (ROCK) inhibitor. To explore the relationship between ROCK inhibitor-mediated decreases in pulmonary vascular resistance and pressure, RV hypertrophy, and systolic dysfunction, we compared the effects of systemically administered to inhaled (pulmonary-selective) ROCK inhibitor on RV systolic function. Rat pups were exposed to air or hypoxia (13% O2) from Postnatal Days 1 to 21 and received rescue treatment with aerosolized fasudil (200 mM) for 15 minutes three times daily or intraperitoneal Y27632 (15 mg/kg twice daily) from Days 14 to 21. Chronic hypoxia differentially increased RhoA and ROCK activity in the right, but not left, cardiac ventricle. Inhaled ROCK inhibitor normalized pulmonary vascular resistance and caused regression of RV hypertrophy and pulmonary arterial wall remodeling but did not improve RV systolic dysfunction (decreased stroke volume and tricuspid annular plane systolic excursion). Systemic, but not inhaled, ROCK inhibitor normalized up-regulated ROCK and phosphodiesterase 5 activities in the right ventricle. Treatment with sildenafil (100 mg/kg/d intraperitoneally from Days 14 to 21) improved RV systolic function. Collectively, these data indicate that pressure unloading and regressed arterial and cardiac remodeling did not lead to recovery of systolic function while right ventricular ROCK activity remained increased. Right ventricle-specific up-regulation of RhoA/ROCK activity is critical to hypoxia-mediated systolic dysfunction, in part by regulating the activity of phosphodiesterase 5.

Therapeutic hypercapnia prevents inhaled nitric oxide-induced right-ventricular systolic dysfunction in juvenile rats.

Chronic pulmonary hypertension in the neonate and infant frequently presents with right-ventricular (RV) failure. Current clinical management may include protracted treatment with inhaled nitric oxide (iNO), with the goal of reducing RV afterload. We have previously reported that prolonged exposure to iNO causes RV systolic dysfunction in the chronic hypoxia-exposed juvenile rat, which was prevented by a peroxynitrite decomposition catalyst. Given that inhalation of CO2 (therapeutic hypercapnia) may limit oxidative stress and upregulated cytokine expression in the lung and other organs, we hypothesized that therapeutic hypercapnia would attenuate cytokine-mediated nitric oxide synthase (NOS) upregulation, thus limiting peroxynitrite generation. Sprague-Dawley rat pups were exposed to chronic hypoxia (13% O2) from postnatal day 1 to 21, while receiving iNO (20 ppm) from day 14 to 21, with or without therapeutic hypercapnia (10% CO2). Therapeutic hypercapnia completely normalized RV systolic function, RV hypertrophy, and remodeling of pulmonary resistance arteries in animals exposed to iNO. Inhaled nitric oxide-mediated increases in RV peroxynitrite, apoptosis, and contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and NOS-2 were all attenuated by therapeutic hypercapnia. Inhibition of NOS-2 activity with 1400 W (1 mg/kg/day) prevented iNO-mediated upregulation of peroxynitrite and led to improved RV systolic function. Blockade of IL-1 receptor signaling with anakinra (500 mg/kg/day) decreased NOS-2 content and had similar effects compared to NOS-2 inhibition on iNO-mediated effects, whereas blockade of TNF-α signaling with etanercept (0.4 mg/kg on alternate days) had no effects on these parameters. We conclude that therapeutic hypercapnia prevents the adverse effects of sustained exposure to iNO on RV systolic function by limiting IL-1-mediated NOS-2 upregulation and consequent nitration. Therapeutic hypercapnia also acts synergistically with iNO in normalizing RV hypertrophy, vascular remodeling, and raised pulmonary vascular resistance secondary to chronic hypoxia.

Cyclooxygenase-2 inhibition partially protects against 60% O2 -mediated lung injury in neonatal rats.

RATIONALE: Use of the anti-inflammatory agent dexamethasone in premature infants with bronchopulmonary dysplasia has been curtailed, and no alternative anti-inflammatory agents are approved for this use. Our objective was to use a neonatal rat model of bronchopulmonary dysplasia to determine if an highly selective cyclooxygenase-2 inhibitor, 5,5-dimethyl-3-(3-fluorophenyl)4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU; 10 µg/g body weight), could prevent inflammatory cell influx and protect against lung injury. METHODS: Neonatal rats exposed to air or 60% O2 for 14 days from birth either received daily i.p. injections of (i) vehicle or DFU or (ii) vehicle or an EP(1) receptor antagonist, SC-19220. RESULTS: DFU attenuated the lung macrophage and neutrophil influx, prevented interstitial thickening and prevented the loss of peripheral blood vessels induced by 60% O2 , but did not protect against the variance in alveolar diameter induced by 60% O2 . Exposure to 60% O2 caused both an increase in lung prostaglandin E2 content and a reduction in lung mesenchymal cell mass which was reversed by DFU. Prostaglandin E2 binding to the EP(1) receptor inhibited DNA synthesis in cultures of lung fibroblasts in a dose dependent fashion. Treatment with SC-19220 attenuated the reduction in lung mesenchymal mass observed following exposure of rat pups to 60% O2 . CONCLUSIONS: An highly selective cyclooxygenase-2 inhibitor is an effective anti-inflammatory substitute for dexamethasone for preventing phagocyte influx into the neonatal lung during 60% O2 -mediated lung injury, and can modify the severity of that injury.

Rho-kinase inhibitor prevents bleomycin-induced injury in neonatal rats independent of effects on lung inflammation.

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation dominated by neutrophils and macrophages, inhibited distal airway and vascular development, and pulmonary hypertension, similar to human infants with severe bronchopulmonary dysplasia. Rho-kinase (ROCK) is known to mediate lung injury in adult animals via stimulatory effects on inflammation. We therefore hypothesized that inhibition of ROCK may ameliorate bleomycin-induced lung injury in the neonatal rat. Pups received daily intraperitoneal bleomycin or saline from Postnatal Days 1 through 14 with or without Y-27632, a ROCK inhibitor. Treatment with Y-27632 prevented bleomycin-induced pulmonary hypertension, as evidenced by normalized pulmonary vascular resistance, decreased right-ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. Bleomycin-induced changes in distal lung architecture, including septal thinning, inhibited alveolarization, and decreased numbers of peripheral arteries and capillaries, were partially or completely normalized by Y-27632. Treatment with Y-27632 or a CXCR2 antagonist, SB265610, also abrogated tissue neutrophil influx, while having no effect on macrophages. However, treatment with SB265610 did not prevent bleomycin-induced lung injury. Lung content of angiostatic thrombospondin-1 (TSP1) was increased significantly in the lungs of bleomycin-exposed animals, and was completely attenuated by treatment with Y-27632. Thrombin-stimulated TSP1 production by primary cultured rat pulmonary artery endothelial cells was also attenuated by Y-27632. Taken together, our findings suggest a preventive effect of Y-27632 on bleomycin-mediated injury by a mechanism unrelated to inflammatory cells. Our data suggest that improvements in lung morphology may have been related to indirect stimulatory effects on angiogenesis via down-regulation of TSP1.

Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle.

Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% ("physiologic") or 20% ("supraphysiologic") at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H2O2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H2O2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H2O2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.