Erika B. Rangel

S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells

Although nitric oxide (NO) signaling promotes differentiation and maturation of endothelial progenitor cells, its role in the differentiation of mesenchymal stem cells (MSCs) into endothelial cells remains controversial. We tested the role of NO signaling in MSCs derived from WT mice and mice homozygous for a deletion of S-nitrosoglutathione reductase (GSNOR−/−), a denitrosylase that regulates S-nitrosylation. GSNOR−/− MSCs exhibited markedly diminished capacity for vasculogenesis in an in vitro Matrigel tube–forming assay and in vivo relative to WT MSCs. This decrease was associated with down-regulation of the PDGF receptorα (PDGFRα) in GSNOR−/− MSCs, a receptor essential for VEGF-A action in MSCs. Pharmacologic inhibition of NO synthase with L-NG-nitroarginine methyl ester (L-NAME) and stimulation of growth hormone–releasing hormone receptor (GHRHR) with GHRH agonists augmented VEGF-A production and normalized tube formation in GSNOR−/− MSCs, whereas NO donors or PDGFR antagonist reduced tube formation ∼50% by murine and human MSCs. The antagonist also blocked the rescue of tube formation in GSNOR−/− MSCs by L-NAME or the GHRH agonists JI-38, MR-409, and MR-356. Therefore, GSNOR−/− MSCs have a deficient capacity for endothelial differentiation due to downregulation of PDGFRα related to NO/GSNOR imbalance. These findings unravel important aspects of modulation of MSCs by VEGF-A activation of the PDGFR and illustrate a paradoxical inhibitory role of S-nitrosylation signaling in MSC vasculogenesis. Accordingly, disease states characterized by NO deficiency may trigger MSC-mediated vasculogenesis. These findings have important implications for therapeutic application of GHRH agonists to ischemic disorders.

Efficacy and Dose-Dependent Safety of Intra-Arterial Delivery of Mesenchymal Stem Cells in a Rodent Stroke Model

Intra-arterial (IA) delivery of mesenchymal stem cells (MSCs) for acute ischemic stroke is attractive for clinical translation. However, studies using rat model of stroke have demonstrated that IA MSCs delivery can decrease middle cerebral artery (MCA) flow, which may limit its clinical translation. The goal of this study is to identify a dose of IA MSCs (maximum tolerated dose; MTD) that does not compromise MCA flow and evaluate its efficacy and optimal timing in a rat model of reversible middle cerebral artery occlusion (rMCAo). We sought to determine if there is a difference in efficacy of acute (1 h) versus sub-acute (24 h) IA MSCs treatment after rMCAo. Adult female Sprague-Dawley rats underwent rMCAo (90 min) and an hour later a single dose of MSCs (at de-escalating doses 1×106, 5×105, 2×105, 1×105 and 5×104) was given using IA route. MSCs were suspended in phosphate buffered saline (PBS) and PBS alone was used for control experiments. We measured the percent change in mean laser Doppler flow signal over the ipsilateral MCA in de-escalating doses groups to determine MTD. The results demonstrated that the lowering of IA MSC dose to 1×105 and below did not compromise MCA flow and hence an IA MSC dose of 1×105 considered as MTD. Subsequently, 1 h and 24 h after rMCAo, rats were treated with IA MSCs or PBS. The 24 h delivery of IA MSCs significantly improved neurodeficit score and reduced the mean infarct volume at one month as compared to control, but not the 1 h delivery. Overall, this study suggests that the IA delivery of MSCs can be performed safely and efficaciously at the MTD of 1×105 delivered at 24 hours in rodent model of stroke.

S-nitrosoglutathione reductase–dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis

Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.

Adult c-Kit(+) progenitor cells are necessary for maintenance and regeneration of olfactory neurons

The olfactory epithelium houses chemosensory neurons, which transmit odor information from the nose to the brain. In adult mammals, the olfactory epithelium is a uniquely robust neuroproliferative zone, with the ability to replenish its neuronal and non‐neuronal populations due to the presence of germinal basal cells. The stem and progenitor cells of these germinal layers, and their regulatory mechanisms, remain incompletely defined. Here we show that progenitor cells expressing c‐Kit, a receptor tyrosine kinase marking stem cells in a variety of embryonic tissues, are required for maintenance of the adult neuroepithelium. Mouse genetic fate‐mapping analyses show that embryonically, a c‐Kit(+) population contributes to olfactory neurogenesis. In adults under conditions of normal turnover, there is relatively sparse c‐Kit(+) progenitor cell (ckPC) activity. However, after experimentally induced neuroepithelial injury, ckPCs are activated such that they reconstitute the neuronal population. There are also occasional non‐neuronal cells found to arise from ckPCs. Moreover, the selective depletion of the ckPC population, utilizing temporally controlled targeted diphtheria toxin A expression, results in failure of neurogenesis after experimental injury. Analysis of this model indicates that most ckPCs reside among the globose basal cell populations and act downstream of horizontal basal cells, which can serve as stem cells. Identification of the requirement for olfactory c‐Kit–expressing progenitors in olfactory maintenance provides new insight into the mechanisms involved in adult olfactory neurogenesis. Additionally, we define an important and previously unrecognized site of adult c‐Kit activity. J. Comp. Neurol. 523:15–31, 2015.

Interaction Between Neuronal Nitric Oxide Synthase Signaling and Temperature Influences Sarcoplasmic Reticulum Calcium Leak

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.

Interaction Between Neuronal Nitric Oxide Synthase Signaling and Temperature Influences Sarcoplasmic Reticulum Calcium Leak Role of Nitroso–Redox Balance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.

Interaction Between Neuronal NOS Signaling and Temperature Influences SR Ca2+ Leak: Role of Nitroso-Redox Balance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.

Kidney-derived c-kit + progenitor/stem cells contribute to podocyte recovery in a model of acute proteinuria

Kidney-derived c-kit+ cells exhibit progenitor/stem cell properties and can regenerate epithelial tubular cells following ischemia-reperfusion injury in rats. We therefore investigated whether c-kit+ progenitor/stem cells contribute to podocyte repair in a rat model of acute proteinuria induced by puromycin aminonucleoside (PAN), the experimental prototype of human minimal change disease and early stages of focal and segmental glomerulosclerosis. We found that c-kit+ progenitor/stem cells accelerated kidney recovery by improving foot process effacement (foot process width was lower in c-kit group vs saline treated animals, P = 0.03). In particular, these cells engrafted in small quantity into tubules, vessels, and glomeruli, where they occasionally differentiated into podocyte-like cells. This effect was related to an up regulation of α-Actinin-4 and mTORC2-Rictor pathway. Activation of autophagy by c-kit+ progenitor/stem cells also contributed to kidney regeneration and intracellular homeostasis (autophagosomes and autophagolysosomes number and LC3A/B-I and LC3A/B-II expression were higher in the c-kit group vs saline treated animals, P = 0.0031 and P = 0.0009, respectively). Taken together, our findings suggest that kidney-derived c-kit+ progenitor/stem cells exert reparative effects on glomerular disease processes through paracrine effects, to a lesser extent differentiation into podocyte-like cells and contribution to maintenance of podocyte cytoskeleton after injury. These findings have clinical implications for cell therapy of glomerular pathobiology.

Interaction Between Neuronal Nitric Oxide Synthase Signaling and Temperature Influences Sarcoplasmic Reticulum Calcium LeakNovelty and Significance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.

Interaction Between Neuronal Nitric Oxide Synthase Signaling and Temperature Influences Sarcoplasmic Reticulum Calcium LeakNovelty and Significance: Role of Nitroso–Redox Balance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.