Leptin acts in the carotid bodies to increase minute ventilation during wakefulness and sleep and augment the hypoxic ventilatory response.

KEY POINTS: Leptin is a potent respiratory stimulant. A long functional isoform of leptin receptor, LepRb , was detected in the carotid body (CB), a key peripheral hypoxia sensor. However, the effect of leptin on minute ventilation (VE ) and the hypoxic ventilatory response (HVR) has not been sufficiently studied. We report that LepRb is present in approximately 74% of the CB glomus cells. Leptin increased carotid sinus nerve activity at baseline and in response to hypoxia in vivo. Subcutaneous infusion of leptin increased VE and HVR in C57BL/6J mice and this effect was abolished by CB denervation. Expression of LepRb in the carotid bodies of LepRb deficient obese db/db mice increased VE during wakefulness and sleep and augmented the HVR. We conclude that leptin acts on LepRb in the CBs to stimulate breathing and HVR, which may protect against sleep disordered breathing in obesity. ABSTRACT: Leptin is a potent respiratory stimulant. The carotid bodies (CB) express the long functional isoform of leptin receptor, LepRb , but the role of leptin in CB has not been fully elucidated. The objectives of the current study were (1) to examine the effect of subcutaneous leptin infusion on minute ventilation (VE ) and the hypoxic ventilatory response to 10% O2 (HVR) in C57BL/6J mice before and after CB denervation; (2) to express LepRb in CB of LepRb -deficient obese db/db mice and examine its effects on breathing during sleep and wakefulness and on HVR. We found that leptin enhanced carotid sinus nerve activity at baseline and in response to 10% O2 in vivo. In C57BL/6J mice, leptin increased VE from 1.1 to 1.5 mL/min/g during normoxia (P < 0.01) and from 3.6 to 4.7 mL/min/g during hypoxia (P < 0.001), augmenting HVR from 0.23 to 0.31 mL/min/g/Δ FIO2 (P < 0.001). The effects of leptin on VE and HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB increased VE from 1.1 to 1.3 mL/min/g during normoxia (P < 0.05) and from 2.8 to 3.2 mL/min/g during hypoxia (P < 0.02), increasing HVR. Compared to control db/db mice, LepRb transfected mice showed significantly higher VE throughout non-rapid eye movement (20.1 vs. -27.7 mL/min respectively, P < 0.05) and rapid eye movement sleep (16.5 vs 23.4 mL/min, P < 0.05). We conclude that leptin acts in CB to augment VE and HVR, which may protect against sleep disordered breathing in obesity.

Silencing of Hypoglossal Motoneurons Leads to Sleep Disordered Breathing in Lean Mice.

Obstructive Sleep Apnea (OSA) is a prevalent condition and a major cause of morbidity and mortality in Western Society. The loss of motor input to the tongue and specifically to the genioglossus muscle during sleep is associated with pharyngeal collapsibility and the development of OSA. We applied a novel chemogenetic method to develop a mouse model of sleep disordered breathing Our goal was to reversibly silence neuromotor input to the genioglossal muscle using an adeno-associated viral vector carrying inhibitory designer receptors exclusively activated by designer drugs AAV5-hM4Di-mCherry (DREADD), which was delivered bilaterally to the hypoglossal nucleus in fifteen C57BL/6J mice. In the in vivo experiment, 4 weeks after the viral administration mice were injected with a DREADD ligand clozapine-N-oxide (CNO, i.p., 1mg/kg) or saline followed by a sleep study; a week later treatments were alternated and a second sleep study was performed. Inspiratory flow limitation was recognized by the presence of a plateau in mid-respiratory flow; oxyhemoglobin desaturations were defined as desaturations >4% from baseline. In the in vitro electrophysiology experiment, four males and three females of 5 days of age were used. Sixteen-nineteen days after DREADD injection brain slices of medulla were prepared and individual hypoglossal motoneurons were recorded before and after CNO application. Positive mCherry staining was detected in the hypoglossal nucleus in all mice confirming successful targeting. In sleep studies, CNO markedly increased the frequency of flow limitation n NREM sleep (from 1.9 ± 1.3% after vehicle injection to 14.2 ± 3.4% after CNO, p < 0.05) and REM sleep (from 22.3% ± 4.1% to 30.9 ± 4.6%, respectively, p < 0.05) compared to saline treatment, but there was no significant oxyhemoglobin desaturation or sleep fragmentation. Electrophysiology recording in brain slices showed that CNO inhibited firing frequency of DREADD-containing hypoglossal motoneurons. We conclude that chemogenetic approach allows to silence hypoglossal motoneurons in mice, which leads to sleep disordered breathing manifested by inspiratory flow limitation during NREM and REM sleep without oxyhemoglobin desaturation or sleep fragmentation. Other co-morbid factors, such as compromised upper airway anatomy, may be needed to achieve recurrent pharyngeal obstruction observed in OSA.

Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes.

Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ) which spontaneously develop in vitro without additional growth factors or cytokines. Gϕ are CD66b(+)/CD63(+)/MPO(+)/LC3B(+) and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕ formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished Gϕ formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phox resembled Gϕ formed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕ formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b(+)/LC3B(+) Gϕ and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕ development.

Endothelial progenitor cells in acute myocardial infarction and sleep-disordered breathing.

RATIONALE: Mobilization and functions of endothelial progenitor cells (EPCs) are increased in patients with acute myocardial infarction (AMI). Yet, sleep-disordered breathing (SDB) is highly prevalent in patients with AMI. OBJECTIVES: To compare EPC numbers and functions in patients with AMI with SDB (AMI-SDB) and without SDB (AMI-only) and to determine the effects of intermittent hypoxia (IH) in vitro on EPC proliferative and angiogenic properties. METHODS: Forty male patients with AMI underwent a whole-night sleep study using ambulatory monitoring. Nineteen had SDB (oxygen desaturation index > 5 events/h). AMI-SDB and AMI-only patients were matched by age, body mass index, blood chemistry, and comorbidities. Blood samples were analyzed by flow cytometry, endothelial cell colony-forming units (EC-CFU), paracrine measures, blood chemistry, and oxidative stress, inflammatory, and angiogenic markers. Effects of IH in vitro were studied in 12 healthy subjects. MEASUREMENTS AND MAIN RESULTS: Circulating EPCs (CD34(+)/KDR(+)), angiogenic T cells (CD3(+)/CD31(+)/CXCR4(+)), and vascular endothelial growth factor in monocytes were significantly higher in AMI-SDB patients, whereas plasma stromal cell-derived factor (SDF)-1α levels were significantly lower. Also, EC-CFU numbers and EC-CFU paracrine effects on endothelial tube formation were significantly higher in AMI-SDB compared with AMI-only patients. Similarly, in cell cultures from healthy subjects, EC-CFU numbers and their paracrine effects on endothelial tube formation were increased after exposure to IH in vitro compared with normoxia. CONCLUSIONS: Coexistent mild to moderate SDB in patients with AMI increased the mobilization, proliferative and angiogenic capacities of EPCs, angiogenic T-cell numbers, and vascular endothelial growth factor expression in monocytes compared with patients with AMI without SDB. IH in vitro had similar effects on healthy EPC functions.

Short-term fibronectin treatment induces endothelial-like and angiogenic properties in monocyte-derived immature dendritic cells: involvement of intracellular VEGF and MAPK regulation.

Fibronectin (FN) is an extracellular matrix protein promoting cell proliferation, adhesion, and survival and is localized in the intimal layer of normal and atherosclerotic blood vessels. Dendritic cells (DCs) are potent antigen-presenting cells located in healthy and diseased intima, and thus may predispose arteries to atherosclerosis. Besides their pro-atherogenic activities DCs can promote neovascularization, by releasing pro-angiogenic mediators and/or by trans-differentiating into endothelial-like cells. Here, we investigated changes in morphology, function and angiogenic properties of monocyte-derived immature DCs (Mo-iDCs) after a short-term FN treatment and some of the signaling pathways involved in these processes. The cells were analyzed by time-lapse, confocal microscopy and flow cytometry. Within 90 min of re-plating, FN induced a swift morphologic transformation of most round iDCs into spindle-shaped iDCs (sp-iDCs). This was characterized by redistribution of mitochondria into dendritic spindles, decreased CD1c, and increased thrombomodulin (CD141) expression. Functionally, sp-iDCs acquired Ulex-europaeus-agglutinin-1 lectin binding, phagocytosis was decreased and intracellular (nuclear and cytosolic) vascular endothelial growth factor (VEGF) was increased. FN also induced ERK1/2 phosphorylation in round-iDCs, and p38MAPK phosphorylation in sp-iDCs. Inhibiting p38MAPK, but not ERK1/2, restrained the FN-induced transformation into sp-iDCs. Furthermore, FN-treatment of Mo-iDCs induced a paracrine angiogenic effect on endothelial tube formation, which was abolished by inhibiting ERK1/2 or VEGF. Inhibiting p38MAPK had no effect on endothelial tube formation. By contrast, in laminin-treated Mo-iDCs, which had round-shaped morphology, CD1c and CD141 expression was similar to control untreated cells, but intracellular VEGF levels were higher, and endothelial tube formation was an individual trait. We conclude that a short-term FN treatment induced angiogenic intracrine and paracrine properties in Mo-iDCs. This may act as an immediate protective mechanism to maintain vascular homeostasis. Moreover, inducing sp-iDCs by short term FN-treatment or ERK1/2 modulation might be considered as new approaches for regulating angiogenesis through the production/inhibition of pro-angiogenic mediators. Collectively, these findings may support a role for FN and Mo-iDCs in vascular function and angiogenesis.