The role of nitric oxide synthases in the pathophysiology of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease is a major cause of high morbidity and mortality with a high socioeconomic burden worldwide. The contribution of vascular alterations to the pathogenesis of the disease remains controversial and there is still ongoing debate about the possible development of pulmonary hypertension in COPD. Against this background, the current thesis aimed to decipher the time course for the development of lung emphysema as well as vascular alterations to the pulmonary circulation by use of a mouse model of tobacco smokeinduced COPD. For this purpose, WT mice were exposed for up to eight months to tobacco smoke (6 h/day, 5 days/week). It was demonstrated that both vascular structural and functional alterations occurred, including loss of pulmonary vessels, narrowing of vascular lumen, an increased degree of muscularization, pulmonary hypertension as well as endothelial dysfunction. Against the background, it was hypothesized that oxidative as well nitrosative stress plays a major role to the development of COPD by the regulation of inducible as well as the endothelial NO synthases. An upregulation of the inducible nitric oxide synthase (iNOS) was found in the pulmonary vasculature concomitant with increased nitrotyrosine levels. Comparing the development of vascular alteration and emphysema in WT, iNOS–/–, and eNOS–/– mice, this study found that iNOS–/– were completely protected from these structural and functional changes. Moreover, the same effect was observed by the treatment of wild-type mice with the iNOS inhibitor L-NIL. Similar regulatory processes and structural alterations as for tobacco smoke exposed mice were found in GOLD stage IV for explanted COPD patient lungs. Thus, iNOS inhibition may be a strategy for prevention of COPD in the future.COPD ist weltweit eine der wichtigsten Ursachen für hohe Morbidität und Mortalität mit hoher sozioökonomischer Bedeutung. Der Beitrag von vaskulären Veränderungen zur Pathogenese der COPD ist derzeit umstritten und es besteht eine laufende Debatte über die mögliche Entwicklung und Bedeutung der pulmonalen Hypertonie in der COPD. Vor diesem Hintergrund, war das Ziel der vorliegenden Arbeit, den zeitlichen Verlauf der Emphysementwicklung und der vaskulären Veränderungen anhand des Mausmodells der Tabakrauch induzierten COPD zu entschlüsseln. Für diesen Zweck wurden Wildtyp-Mäuse für acht Monate Tabak-Rauch für 6 Stunden/Tag und 5 Tage/Woche ausgesetzt. Es konnte gezeigt werden, dass sowohl strukturelle als auch funktionelle Gefäßveränderungen stattfinden, einschließlich des Verlusts der Lungengefäße, der Verengung des Gefäßlumens, des erhöhten Muskularisierungsgrades von Gefäßen, pulmonaler Hypertonie und endothelialer Dysfunktion. Basierend auf diesen Beobachtungen, wurde die Hypothese aufgestellt, dass oxidativer so wie nitrosativer Stress eine wichtige Rolle bei der Entwicklung von COPD spielen, indem es zu einer Regulation von induzierbaren und endothelialen NO-Synthasen kommt. Tatsächlich konnte in der vorliegenden Arbeit im pulmonalen Gefäßsystem eine Hochregulierung der induzierbaren NO Synthase (iNOS) nachgewiesen werden, einhergehend mit erhöhtem Vorkommen von Nitrotyrosin. Durch den Vergleich der Entwicklung von Gefäßveränderungen und der Emphysementwicklung in Wildtyp-, iNOS-/-- und eNOS-/-- Mäusen, konnte diese Studie zeigen, dass iNOS–/– -Mäuse vor den genannten strukturellen und funktionellen Änderungen komplett geschützt waren. Darüber hinaus, konnte der gleiche Effekt nach Behandlung von Wildtyp-Mäusen mit dem iNOS Inhibitor L-NIL beobachtet werden. Ähnliche regulatorische Prozesse und strukturelle Veränderungen wie in den Wildtyp-Mauslungen nach Rauchexposition wurden in Resektaten von Lungen von Patienten mit COPD(GOLD stage IV) nachgewiesen. Demnach könnte die Inhibierung von iNOS in Zukunft eine Strategie zur COPD Prävention darstellen

MiR-23a Regulates Skin Langerhans Cell Phagocytosis and Inflammation-Induced Langerhans Cell Repopulation

Langerhans cells (LCs) are skin-resident macrophage that act similarly to dendritic cells for controlling adaptive immunity and immune tolerance in the skin, and they are key players in the development of numerous skin diseases. While TGF-β and related downstream signaling pathways are known to control numerous aspects of LC biology, little is known about the epigenetic signals that coordinate cell signaling during LC ontogeny, maintenance, and function. Our previous studies in a total miRNA deletion mouse model showed that miRNAs are critically involved in embryonic LC development and postnatal LC homeostasis; however, the specific miRNA(s) that regulate LCs remain unknown. miR-23a is the first member of the miR-23a-27a-24-2 cluster, a direct downstream target of PU.1 and TGF-b, which regulate the determination of myeloid versus lymphoid fates. Therefore, we used a myeloid-specific miR-23a deletion mouse model to explore whether and how miR-23a affects LC ontogeny and function in the skin. We observed the indispensable role of miR-23a in LC antigen uptake and inflammation-induced LC epidermal repopulation; however, embryonic LC development and postnatal homeostasis were not affected by cells lacking miR23a. Our results suggest that miR-23a controls LC phagocytosis by targeting molecules that regulate efferocytosis and endocytosis, whereas miR-23a promotes homeostasis in bone marrow-derived LCs that repopulate the skin after inflammatory insult by targeting Fas and Bcl-2 family proapoptotic molecules. Collectively, the context-dependent regulatory role of miR-23a in LCs represents an extra-epigenetic layer that incorporates TGF-b- and PU.1-mediated regulation during steady-state and inflammation-induced repopulation

ACE2 Deficiency Worsens Epicardial Adipose Tissue Inflammation and Cardiac Dysfunction in Response to Diet-Induced Obesity

Obesity is increasing in prevalence and is strongly associated with metabolic and cardiovascular disorders. The renin-angiotensin system (RAS) has emerged as a key pathogenic mechanism for these disorders; angiotensin (Ang)-converting enzyme 2 (ACE2) negatively regulates RAS by metabolizing Ang II into Ang 1-7. We studied the role of ACE2 in obesity-mediated cardiac dysfunction. ACE2 null (ACE2KO) and wild-type (WT) mice were fed a high-fat diet (HFD) or a control diet and studied at 6 months of age. Loss of ACE2 resulted in decreased weight gain but increased glucose intolerance, epicardial adipose tissue (EAT) inflammation, and polarization of macrophages into a proinflammatory phenotype in response to HFD. Similarly, human EAT in patients with obesity and heart failure displayed a proinflammatory macrophage phenotype. Exacerbated EAT inflammation in ACE2KO-HFD mice was associated with decreased myocardial adiponectin, decreased phosphorylation of AMPK, increased cardiac steatosis and lipotoxicity, and myocardial insulin resistance, which worsened heart function. Ang 1-7 (24 µg/kg/h) administered to ACE2KO-HFD mice resulted in ameliorated EAT inflammation and reduced cardiac steatosis and lipotoxicity, resulting in normalization of heart failure. In conclusion, ACE2 plays a novel role in heart disease associated with obesity wherein ACE2 negatively regulates obesity-induced EAT inflammation and cardiac insulin resistance

The Calcineurin-TFEB-p62 Pathway Mediates the Activation of Cardiac Macroautophagy by Proteasomal Malfunction

Rationale: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP) are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and ALP defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the two catabolic pathways will help advance cardiac pathophysiology and medicine.Objective: Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac ALP.Methods and Results: Myocardial macroautophagy, transcription factor EB (TFEB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of Psmc1 (an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediated Molcn1 silencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type (WT) mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from WT mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with WT mice.Conclusions: (1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac ALP activation during proteasome malfunction

Remote ischemic preconditioning confers late protection against myocardial ischemia-reperfusion injury in mice by upregulating interleukin-10

Abstract Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia-reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of interleukin (IL)-10. Mice were exposed to lower limb RIPC or sham ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120). These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies

Phosphatase PTEN is critically involved in post-myocardial infarction remodeling through the Akt/interleukin-10 signaling pathway

The inflammatory cytokines interleukin (IL)-10 and tumor necrosis factor (TNF)-α play an important role in left ventricular (LV) remodeling after myocardial infarction (MI). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) inactivates protein kinase Akt and promotes cell death in the heart. However, it is not known whether PTEN promotes post-MI remodeling by regulating IL-10 and TNF-α. MI was induced in wild-type (WT) mice and Pten heterozygous mutant (HET) mice. Pten adenoviruses (adPten) or empty vectors (adNull) were injected into the peri-infarct area of WT mice. LV dilation was attenuated and fractional shortening was increased in HET mice compared to WT mice. Survival rate and fractional shortening were decreased in adPten mice compared to adNull mice. Leukocyte infiltration into the peri-infarct area was attenuated in HET mice and worsened in adPten mice. PTEN expression was upregulated in the infarcted heart of WT mice. Partial inactivation of PTEN increased the production of IL-10 and decreased the expression of TNF-α and matrix metalloproteinase (MMP)-2 and -9 after MI in HET mice. PTEN overexpression caused opposite effects in the infarcted heart. Moreover in the infarcted heart of HET mice, Akt inhibition decreased Stat3 phosphorylation and IL-10 expression, and blockade of the IL-10 receptor increased TNF-α and MMP-2 expression. Both Akt inhibition and IL-10 receptor blockade abolished the attenuation of post-MI remodeling in HET mice. In conclusion, PTEN is critically involved in post-MI remodeling through the Akt/IL-10 signaling pathway. Therefore, targeting PTEN may be an effective approach to post-MI remodeling.15 page(s

Smad2/4 Signaling Pathway Is Critical for Epidermal Langerhans Cell Repopulation Under Inflammatory Condition but Not Required for Their Homeostasis at Steady State

Epidermal Langerhans cells (LCs) are skin-resident dendritic cells that are essential for the induction of skin immunity and tolerance. Transforming growth factor-β 1 (TGFβ1) is a crucial factor for LC maintenance and function. However, the underlying TGFβ1 signaling pathways remain unclear. Our previous research has shown that the TGFβ1/Smad3 signaling pathway does not impact LC homeostasis and maturation. In this study, we generated mice with conditional deletions of either individual Smad2, Smad4, or both Smad2 and Smad4 in the LC lineage or myeloid lineage, to further explore the impact of TGFβ1/Smad signaling pathways on LCs. We found that interruption of Smad2 or Smad4 individually or simultaneously in the LC lineage did not significantly impact the maintenance, maturation, antigen uptake, and migration of LCs in vivo or in vitro during steady state. However, the interruption of both Smad2 and Smad4 pathways in the myeloid lineage led to a dramatic inhibition of bone marrow-derived LCs in the inflammatory state. Overall, our data suggest that canonical TGFβ1/Smad2/4 signaling pathways are dispensable for epidermal LC homeostasis and maturation at steady state, but are critical for the long-term LC repopulation directly originating from the bone marrow in the inflammatory state