Cleide G. da Silva

Mechanism of Purinergic Activation of Endothelial Nitric Oxide Synthase in Endothelial Cells

Background— Decreased endothelial nitric oxide (NO) synthase (eNOS) activity and NO production are critical contributors to the endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation; however, the mechanism of this observation is not fully clarified. Methods and Results— To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells were treated with several nucleotides, including ATP, UTP, and ADP, in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2, and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation and demonstrated that this process was adenosine independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase C&dgr; inhibitor), and protein kinase C&dgr; siRNA. In contrast, blockade of AMP-activated protein kinase, calcium/calmodulin-dependent kinase II, calcium/calmodulin-dependent kinase kinase, serine/threonine protein kinase B, protein kinase A, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase did not affect nucleotide-mediated eNOS phosphorylation. Conclusions— The present study indicates that extracellular nucleotide–mediated eNOS phosphorylation is calcium and protein kinase C&dgr; dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction.

Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells Involvement of P2 Receptors and Adenosine Transporters

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5′-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5′-nucleotidase had no effect on ATP/ADP/UTP-induced phospho- rylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca2+ and by Ca2+/calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca2+ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function.

A20 promotes liver regeneration by decreasing SOCS3 expression to enhance IL‐6/STAT3 proliferative signals

Liver regeneration is of major clinical importance in the setting of liver injury, resection, and transplantation. A20, a potent antiinflammatory and nuclear factor kappa B (NF‐κB) inhibitory protein, has established pro‐proliferative properties in hepatocytes, in part through decreasing expression of the cyclin dependent kinase inhibitor, p21. Both C‐terminal (7‐zinc fingers; 7Zn) and N‐terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF‐κB. However, both independently increased hepatocyte proliferation, suggesting that additional mechanisms contributed to the pro‐proliferative function of A20 in hepatocytes. We ascribed one of A20′s pro‐proliferative mechanisms to increased and sustained interleukin (IL)‐6‐induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, as a result of decreased hepatocyte expression of the negative regulator of IL‐6 signaling, suppressor of cytokine signaling 3 (SOCS3). This novel A20 function segregates with its 7Zn not Nter domain. Conversely, total and partial loss of A20 in hepatocytes increased SOCS3 expression, hampering IL‐6‐induced STAT3 phosphorylation. Following liver resection in mice pro‐proliferative targets downstream of IL‐6/STAT3 signaling were increased by A20 overexpression and decreased by A20 knockdown. In contrast, IL‐6/STAT3 proinflammatory targets were increased in A20‐deficient livers, and decreased or unchanged in A20 overexpressing livers. Upstream of SOCS3, levels of its microRNA regulator miR203 were significantly decreased in A20‐deficient livers. Conclusion: A20 enhances IL‐6/STAT3 pro‐proliferative signals in hepatocytes by down‐regulating SOCS3, likely through a miR203‐dependent manner. This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro‐proliferative properties in hepatocytes and prompts the pursuit of A20‐based therapies to promote liver regeneration and repair. (HEPATOLOGY 2013)

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.

A20 Protects Mice from Lethal Liver Ischemia Reperfusion Injury by Increasing Peroxisome Proliferator-Activated Receptor-α Expression

The nuclear factor‐κB inhibitory protein A20 demonstrates hepatoprotective abilities through combined antiapoptotic, anti‐inflammatory, and pro‐proliferative functions. Accordingly, overexpression of A20 in the liver protects mice from toxic hepatitis and lethal radical hepatectomy, whereas A20 knockout mice die prematurely from unfettered liver inflammation. The effect of A20 on oxidative liver damage, as seen in ischemia/reperfusion injury (IRI), is unknown. In this work, we evaluated the effects of A20 upon IRI using a mouse model of total hepatic ischemia. Hepatic overexpression of A20 was achieved by recombinant adenovirus (rAd.)‐mediated gene transfer. Although only 10%‐25% of control mice injected with saline or the control rAd.β galactosidase survived IRI, the survival rate reached 67% in mice treated with rAd.A20. This significant survival advantage in rAd.A20‐treated mice was associated with improved liver function, pathology, and repair potential. A20‐treated mice had significantly lower bilirubin and aminotransferase levels, decreased hemorrhagic necrosis and steatosis, and increased hepatocyte proliferation. A20 protected against liver IRI by increasing hepatic expression of peroxisome proliferator‐activated receptor alpha (PPARα), a regulator of lipid homeostasis and of oxidative damage. A20‐mediated protection of hepatocytes from hypoxia/reoxygenation and H2O2‐mediated necrosis was reverted by pretreatment with the PPARα inhibitor MK886. In conclusion, we demonstrate that PPARα is a novel target for A20 in hepatocytes, underscoring its novel protective effect against oxidative necrosis. By combining hepatocyte protection from necrosis and promotion of proliferation, A20‐based therapies are well‐poised to protect livers from IRI, especially in the context of small‐for‐size and steatotic liver grafts. Liver Transpl 15:1613–1621, 2009.

A20 Modulates Lipid Metabolism and Energy Production to Promote Liver Regeneration

Background Liver Regeneration is clinically of major importance in the setting of liver injury, resection or transplantation. We have demonstrated that the NF-κB inhibitory protein A20 significantly improves recovery of liver function and mass following extended liver resection (LR) in mice. In this study, we explored the Systems Biology modulated by A20 following extended LR in mice. Methodology and Principal Findings We performed transcriptional profiling using Affymetrix-Mouse 430.2 arrays on liver mRNA retrieved from recombinant adenovirus A20 (rAd.A20) and rAd.βgalactosidase treated livers, before and 24 hours after 78% LR. A20 overexpression impacted 1595 genes that were enriched for biological processes related to inflammatory and immune responses, cellular proliferation, energy production, oxidoreductase activity, and lipid and fatty acid metabolism. These pathways were modulated by A20 in a manner that favored decreased inflammation, heightened proliferation, and optimized metabolic control and energy production. Promoter analysis identified several transcriptional factors that implemented the effects of A20, including NF-κB, CEBPA, OCT-1, OCT-4 and EGR1. Interactive scale-free network analysis captured the key genes that delivered the specific functions of A20. Most of these genes were affected at basal level and after resection. We validated a number of A20s target genes by real-time PCR, including p21, the mitochondrial solute carriers SLC25a10 and SLC25a13, and the fatty acid metabolism regulator, peroxisome proliferator activated receptor alpha. This resulted in greater energy production in A20-expressing livers following LR, as demonstrated by increased enzymatic activity of cytochrome c oxidase, or mitochondrial complex IV. Conclusion This Systems Biology-based analysis unravels novel mechanisms supporting the pro-regenerative function of A20 in the liver, by optimizing energy production through improved lipid/fatty acid metabolism, and down-regulated inflammation. These findings support pursuit of A20-based therapies to improve patients’ outcomes in the context of extreme liver injury and extensive LR for tumor treatment or donation.

A20-mediated Modulation of Inflammatory and Immune Responses in Aortic Allografts and Development of Transplant Arteriosclerosis

Background. Transplant arteriosclerosis (TA) is the pathognomonic feature of chronic rejection, the primary cause of allograft failure. We have shown that the NF-&kgr;B inhibitory protein A20 exerts vasculoprotective effects in endothelial and smooth muscle cells (SMC), and hence is a candidate to prevent TA. We sought direct proof for this hypothesis. Methods. Fully mismatched, C57BL/6 (H2b) into BALB/c (H2d), aorta to carotid allografts were preperfused with saline, recombinant A20 adenovirus (rAd.A20) or rAd.&bgr;-galactosidase (&bgr;-gal), implanted, harvested 4 weeks after transplantation, and analyzed by histology, immunohistochemistry, and immunofluorescence staining. We measured indoleamine 2,3-dioxygenase, interleukin-6, and transforming growth factor-&bgr; mRNA and protein levels in nontransduced, and rAd.A20 or rAd.&bgr;-gal-transduced human SMC cultures after cytokine treatment. Results. Vascular overexpression of A20 significantly reduced TA lesions. This correlated with decreased graft inflammation and increased apoptosis of neointimal SMC. Paradoxically, T-cell infiltrates increased in A20-expressing allografts, including the immunoprivileged media, which related to A20 preventing indoleamine 2,3-dioxygenase upregulation in SMC. However, infiltrating T cells were predominantly T-regulatory cells (CD25+/Forkhead Box P3 [FoxP3+]). This agrees with A20 inhibiting interleukin-6 and promoting transforming growth factor-&bgr; production by medial SMC and in SMC cultures exposed to cytokines, which favors differentiation of regulatory over pathogenic T cells. Conclusions. In summary, A20 prevents immune-mediated remodeling of vascular allografts, therefore reduces TA lesions by affecting apoptotic and inflammatory signals and modifying the local cytokine milieu to promote an immunoregulatory response within the vessel wall. This highlights a novel function for A20 in local immunosurveillance, which added to its vasculoprotective effects, supports its therapeutic promise in TA.

A20 inhibits post-angioplasty restenosis by blocking macrophage trafficking and decreasing adventitial neovascularization

OBJECTIVE Neointimal hyperplasia is an inflammatory and proliferative process that occurs as a result of injury to the vessel wall. We have shown that the homeostatic protein A20 prevents neointimal hyperplasia by affecting endothelial cell (EC) and smooth muscle cell (SMC) responses to injury. In this work, we questioned whether A20 impacts other pathogenic effectors of neointimal hyperplasia including homing of monocyte/macrophages and EC/SMC precursors to the site of vascular injury, vascular endothelial growth factor (VEGF) secretion, and adventitial neovascularization. METHODS AND RESULTS Carotid balloon angioplasty was performed on rat recipients of a bone marrow transplant from green fluorescent rats. Adenoviral delivery of A20 prevented neointimal hyperplasia and decreased macrophage infiltration. This was associated with decreased ICAM-1 and MCP-1 expression in vitro. Additionally, A20 reduced neovascularization in the adventitia of balloon injured carotid arteries, which correlated with fewer VEGF positive cells. CONCLUSIONS A20 downregulates adhesion markers, chemokine production, and adventitial angiogenesis, all of which are required for macrophage trafficking to sites of vascular injury. This, in turn, diminishes the inflammatory milieu to prevent neointimal hyperplasia.

Hepatocyte growth factor preferentially activates the anti‐inflammatory arm of NF‐κB signaling to induce A20 and protect renal proximal tubular epithelial cells from inflammation

Inflammation induces the NF‐κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF‐κB activation. We surmised that inducing A20 without engaging the pro‐inflammatory arm of NF‐κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF‐κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF‐κB dependent, pro‐inflammatory MCP‐1, VCAM‐1, and ICAM‐1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro‐inflammatory NF‐κB dependent genes. Upregulation of A20 supported the anti‐inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα‐mediated increase of ICAM‐1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT‐p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20‐based anti‐inflammatory therapies are beneficial. J. Cell. Physiol. 227: 1382–1390, 2012.

Bidirectional crosstalk between periventricular endothelial cells and neural progenitor cells promotes the formation of a neurovascular unit.

Interactions between neural progenitor cells (NPC) and endothelial cells (EC) from adult vascular beds have been well explored previously. However, the factors and signaling mechanisms that regulate neurogenesis and angiogenesis are most prevalent during embryonic development. This study aimed to determine whether embryonic brain endothelial cells from the periventricular region (PVEC) present an advantage over adult brain EC in supporting NPC growth and differentiation. PVEC were isolated from E15 mouse brains, processed, and sorted with immunomagnetic beads using antibodies against CD31/PECAM. On immunofluorescence (IF) staining, nearly all cells were positive for EC markers CD31 and CD144/VE-Cadherin. In proliferation studies, NPC proliferation was highest in transwell co-culture with PVEC, approximately 2.3 fold increase compared to baseline versus 1.4 fold increase when co-cultured with adult brain endothelial cells (ABEC). These results correlated with the PVEC mediated delay in NPC differentiation, evidenced by high expression of progenitor marker Nestin evaluated by IF staining. Upon further characterization of PVEC in an angiogenesis assay measuring cord length, PVEC exhibited a high capacity to form cords in basal conditions compared to ABEC. This was enhanced in the presence of NPC, with both cell types displaying a preferential structural alignment resembling neurovascular networks. PVEC also expressed high Vegfa levels at baseline in comparison to NPC and ABEC. Vegfa levels increased when co-cultured with NPC. We demonstrate that PVEC and NPC co-cultures act synergistically to promote the formation of a neurovascular unit through dynamic and reciprocal communication. Our results suggest that PVEC/NPC could provide promising neuro-regenerative therapies for patients suffering brain injuries.