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There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-β type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-β signaling. In this study, we tested the hypothesis that TGF-β drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-β receptor in mammary epithelium, an increased level of TGF-β protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFβRII(WT) control tumors with intact TGF-β signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-β signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-β signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-β signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.
Copyright © 2014 by The American Association of Immunologists, Inc.
OBJECTIVE - To study molecular mechanisms involved in hematopoietic stem cell (HSC) mobilization after liver resection and determine impacts on liver regeneration.
BACKGROUND - Extracellular nucleotide-mediated cell signaling has been shown to boost liver regeneration. Ectonucleotidases of the CD39 family are expressed by bone marrow-derived cells, and purinergic mechanisms might also impact mobilization and functions of HSC after liver injury.
METHODS - Partial hepatectomy was performed in C57BL/6 wild-type, Cd39 ectonucleotidase-null mice and in chimeric mice after transplantation of wild-type or Cd39-null bone marrow. Bone marrow-derived HSCs were purified by fluorescence-activated cell sorting and administered after hepatectomy. Chemotactic studies were performed to examine effects of purinergic receptor agonists and antagonists in vitro. Mobilization of human HSCs and expression of CD39 were examined and linked to the extent of resection and liver tests.
RESULTS - Subsets of HSCs expressing Cd39 are preferentially mobilized after partial hepatectomy. Chemotactic responses of HSCs are increased by CD39-dependent adenosine triphosphate hydrolysis and adenosine signaling via A2A receptors in vitro. Mobilized Cd39 HSCs boost liver regeneration, potentially limiting interleukin 1β signaling. In clinical studies, mobilized human HSCs also express CD39 at high levels. Mobilization of HSCs correlates directly with the restoration of liver volume and function after partial hepatectomy.
CONCLUSIONS - We demonstrate CD39 to be a novel HSC marker that defines a functionally distinct stem cell subset in mice and humans. HSCs are mobilized after liver resection, limit inflammation, and boost regeneration in a CD39-dependent manner. These observations have implications for monitoring and indicate future therapeutic avenues.
Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl(3)-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein α(IIb)/β(3), in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A(2A) or A(2B) adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.
Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
UNLABELLED - CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs.
CONTROL - 44.7 ± 5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD-1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD-1 over expression affords myocardial protection from ischemia-reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia-reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia-reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3β (GSK-3β). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A(2B) adenosine receptor antagonist, MRS 1754, but not the A(1) selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A(2B) receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-3β that prevents detrimental opening of the mitochondrial permeability transition pore.
2011 Elsevier Ltd. All rights reserved.
PURPOSE - To test the hypothesis that in experimental retinopathy of prematurity (ROP), retinal neovascularization (NV) and vessel tortuosity have distinct spatial and temporal links with receptor and postreceptor ion demand.
METHODS - Newborn rats were raised in either room air (controls) or variable oxygen (50%/10% [50/10]). After 14 days, 50/10 rats were recovered in room air until postnatal day (P) 19 or P22. Peripheral retinal NV severity and incidence and panretinal arteriole and venule tortuosity indexes (TI(a), TI(v)) were measured from ADPase-stained retinal wholemounts. Intraretinal ion demand and retinal thickness were measured from high-resolution manganese-enhanced MRI (MEMRI). In separate experiments, intraretinal manganese uptake was also measured in adult rats pretreated with diltiazem, a Ca(2+) channel antagonist.
RESULTS - In 50/10 rats, peripheral retinal NV severity was significantly greater than in controls at P19 and was decreased by P22. Panretinal TI(a) and TI(v) were increased over control values at P19, but only TI(v) decreased by P22. Unlike control retinas at P19 that had a centroperipheral total retinal thickness gradient, 50/10 retinas had similar central and peripheral total retinal thickness. The 50/10 group also demonstrated a correlation between peripheral retinal NV and TI(a) and TI(v). Peripheral intraretinal uptake of manganese was significantly supernormal at P19 and decreased by P22. Increased peripheral intraretinal retinal manganese uptake was associated with peripheral NV severity and panretinal TI(a). In contrast, ion demand of central postreceptor, but not receptor, retina was significantly associated with peripheral NV severity and panretinal TI(a). Panretinal TI(v) was not correlated with intraretinal ion demand in any case. In adult rats, diltiazem suppressed (P < 0.05) intraretinal manganese uptake.
CONCLUSIONS - The present data raise the possibility that altered retinal layer-specific ion demand causes retinal circulation abnormalities in experimental ROP.
PURPOSE - The efficacy of three matrix metalloproteinase (MMP) inhibitors with various selectivities (Ro-31-9790, AG3340, and DPC-A37668) was investigated in a rat model of retinopathy of prematurity, to examine the roles of MMP-2 and -9 in retinal neovascularization. The susceptibilities of MMP-2(-/-) and -9(-/-) mice to preretinal neovascularization were investigated in a mouse model of oxygen-induced retinopathy.
METHODS - Sprague-Dawley newborn rats were exposed to alternating episodes of 50% and 10% oxygen (variable oxygen exposure) to induce retinal neovascularization. Three MMP inhibitors with various selectivity profiles were administered to variable oxygen-exposed rats via local or systemic routes. Antineovascular efficacy was determined in drug-treated versus vehicle-treated rat pups by computerized imaging of adenosine diphosphatase (ADPase)-stained retinal flatmounts. Wild-type C57BL/6J and isogenic MMP-2(-/-) and -9(-/-) mice were exposed to 75% oxygen followed by normoxia. The mice were killed immediately before or after the normoxic exposure, and eyes were either harvested for retinal dissection and flatmounting or were paraffin embedded and sectioned. Retinal vascular area and retinal neovascularization were assessed by adenosine diphosphatase staining of retinal flatmounts and by counting preretinal nuclei of hematoxylin and eosin-stained retinal sections, respectively.
RESULTS - Ro-31-9790, AG3340, and DPC-A37668 had no effect on normal development of the rat retinal vasculature, regardless of dose or route of administration. Intravitreal injection of Ro-31-9790 (broad-spectrum) immediately after variable-oxygen exposure and 2 days after exposure resulted in 78% and 82% inhibition of retinal neovascularization, respectively. AG3340 (MMP-2- and -9-selective inhibitor) and DPC-A37668 (MMP-2-selective inhibitor) resulted in 65% and 52% inhibition, respectively, when administered by intravitreal injection immediately after variable-oxygen exposure. Intraperitoneal injection of 5, 15, and 50 mg/mL AG3340 or DPC-A37668 for 6 days after variable oxygen exposure resulted in 22% to 39% and 0% to 31% inhibition of neovascularization, respectively. AG3340 and DPC-A37668 administered by oral gavage at doses of 3, 10, or 30 mg/mL provided up to 42% and 86% inhibition of neovascularization, respectively. The average vascular areas of retinas from MMP-2(-/-) or -9(-/-) mice at postnatal day 12 were not significantly different from the wild-type control. There was a 75% (P < 0.001) and 44% (P < 0.01) reduction in preretinal neovascularization in oxygen-exposed MMP-2(-/-) and -9(-/-) mice at postnatal day 19, respectively, compared with wild-type control mice.
CONCLUSIONS - The results of this study suggest that MMP-2 plays a predominant role in retinal angiogenesis in both the mouse and rat models of oxygen-induced retinopathy. Furthermore, MMP-2 inhibition may be a viable therapeutic approach for ocular diseases characterized by retinal neovascularization.
PURPOSE - Loss of retinal capillaries is an inherent component of late stage autosomal dominant retinitis pigmentosa (ADRP). This study examined the hypothetical role of tissue hyperoxia in this vascular attenuation process and tested the potential of ambient hypoxia to reverse it.
METHODS - Transgenic mice expressing a mutant opsin gene with a 3-bp deletion of isoleucine at codon 255/256 were used. This model is characterized by early onset of a rapidly progressing retinal degeneration that by postnatal day (P)20 results in the loss of all but one row of photoreceptor nuclei. At P20 some mice were placed in 12% oxygen until they were euthanatized at P26. The remainder were maintained in normoxia and killed at the same age. Retinas were dissected, stained for ADPase, and flat-mounted.
RESULTS - Deep plexus capillary density was significantly different in normoxic normals versus transgenics at 20 days of age (P: = 0. 005). An additional 65% reduction of capillary density occurred within the deep plexus of normoxic transgenics between P20 and P26 (P: = 0.005). Ambient hypoxia between days P20 and P26 reversed this trend, causing an increase in deep capillary plexus density of nearly 100% (P: = 0.001).
CONCLUSIONS - This model of ADRP demonstrates two important features of human retinitis pigmentosa: photoreceptor cell death and subsequent retinal capillary atrophy. Low ambient oxygen was used to reverse the capillary atrophy and to stimulate new capillary growth, implying that retinal oxygen tension may link these two features of the pathology. The implications of this study hold importance for strategies designed to treat retinitis pigmentosa with retinal cell transplantation.
Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell cytokine that can be up-regulated by hypoxia. There is evidence that VEGF is a significant mediator in retinal neovascular diseases and other disorders in which hypoxia is believed to influence the pathogenesis. Here we demonstrate the spatial relationships among areas of retinal non-perfusion, VEGF protein and vascular endothelial cells throughout the retina, and relate these results to cellular distribution of VEGF in cross section. Newborn albino rats were oxygen-injured by cycles of alternating 50% and 10% oxygen for 14 days and then placed in room air. On days 16, 21 and 26, oxygen-injured and control (raised in room air) rats were sacrificed, enucleated and retinas were dissected and fixed for whole mount immunostaining for VEGF or embedding in glycol methacrylate for VEGF immunohistochemistry. Intact eyes taken on days 16 and 20 were processed similarly. Vascular endothelial cells were demonstrated by staining whole-mounted retinas for adenosine diphosphatase (ADPase) activity. Preretinal neovascular growths (i.e., abnormal vessels extending from the retina into the vitreous) were VEGF-positive. There was also a pan-retinal distribution of non-endothelial cells that were VEGF-positive in both room air and oxygen-injured rats, with stronger immunostaining in day 16 oxygen-injured retinas. In cross-section, VEGF staining was confirmed in preretinal growths, normal retinal vessels, cells in the inner nuclear layer (primarily Müller cells) and ganglion cells. Retinas which had been incubated with nonimmune IgG or absorbed anti-VEGF antibody showed little or no staining. In conclusion, we have identified cells of the inner retina which express VEGF. The production of VEGF by these cells--in particular, Müller cells--may promote preretinal neovascularization in oxygen-injured eyes. We have found, moreover, that the combination of immunohistochemistry and ADPase staining of whole mount preparations is a unique and powerful tool for evaluating relationships between presumed areas of retinal ischemia, VEGF (and other cytokines) and retinal blood vessels, within an entire retina. This approach can be used to study any proliferative retinal disorders in which VEGF is a potential component of the pathogenesis.
PURPOSE - A conventional criticism of animal models of retinopathy of prematurity (ROP) concerns the common occurrence of rapid spontaneous resolution of retinal vascular sequelae. The purpose of this study was to determine whether animals subjected to a novel variable oxygen exposure protocol would undergo the rapid spontaneous resolution of retinal vascular pathology that is typical of past models.
METHODS - Newborn rats were exposed to an oxygen environment that alternated between 50% and 10% every 24 h for 14 days and then removed to room air, or were raised from birth in room air as controls. To determine early retinal vascular growth rate, both exposed and non-exposed rats were sacrificed between 3 and 28 days of age, after which eyes were enucleated and retinas dissected and stained for adenosine diphosphatase (ADPase) activity to demonstrate the vasculature. Rats were maintained in room air for 2 to 18 weeks after the variable oxygen exposure period for assessment of long-term retinal vascular abnormalities by ADPase histochemistry.
RESULTS - The retinal vasculature of oxygen-exposed rats was significantly different from that of room air-raised rats with respect to capillary density, branching frequency, and bifurcation angle. These differences were restricted to the area that was vascularized after removal to room air (the peripheral-most 25% of the retinal area), and they persisted for the duration of the study.
CONCLUSIONS - We have developed a rat model of ROP using an exposure protocol designed to create systemic oxygen levels that approximate those of premature infants. This model does not demonstrate the complete resolution of vessel abnormalities that historically has limited animal models of ROP.