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Plasminogen Kringle 5 is one of the most potent cytokines identified to inhibit the proliferation and migration of vascular endothelial cells. Herein, six aptamer candidates that specifically bind to Kringle 5 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). After 10 rounds of screening against Kringle 5, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. The preferred aptamer KG-4, which demonstrated a low dissociation constant (K d ) of ∼ 432 nM and excellent selectivity for Kringle 5. A conserved "motif" of eight bases located at the stem-loop intersection, common to the aptamer, was further confirmed as the recognition element for binding with Kringle 5. The bulge formed by the motif and depression on the lysine binding site of Kringle 5 were both located at the binding interface, and the "induced fit" between their structures played a central role in the recognition process. Kringle 5 interacts KG-4 primarily through enthalpy-driven van der Waals forces and hydrogen bond. The key nucleotides A34 and C35 at motif on KG-4 and the positively charged amino acids in the loop 1 and loop 4 regions on Kringle 5 play a major role in the interaction. Furthermore, KG-4 dose-dependently reduced the proliferation inhibition of vascular endothelial cells by Kringle 5 and had a blocking effect on the function of Kringle 5 in inhibiting migration and promoting apoptosis of vascular endothelial cells in vitro. This study put a new light on protein-aptamer binding mechanism and may provide insight into the treatment of ischemic diseases by target depletion of Kringle 5., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liujiao Bian reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Overnutrition-induced endothelial inflammation plays a crucial role in high-fat diet (HFD)-induced insulin resistance in animals. Endothelial glycolysis plays a critical role in endothelial inflammation and proliferation, but its role in diet-induced endothelial inflammation and subsequent insulin resistance has not been elucidated. PFKFB3 is a critical glycolytic regulator, and its increased expression has been observed in adipose vascular endothelium of C57BL/6J mice fed with HFD in vivo, and in palmitate (PA)-treated primary human adipose microvascular endothelial cells (HAMECs) in vitro. We generated mice with Pfkfb3 deficiency selective for endothelial cells to examine the effect of endothelial Pfkfb3 in endothelial inflammation in metabolic organs and in the development of HFD-induced insulin resistance. EC Pfkfb3-deficientmice exhibited mitigated HFD-induced insulin resistance, including decreased body weight and fat mass, improved glucose clearance and insulin sensitivity, and alleviated adiposity and hepatic steatosis. Mechanistically, cultured PFKFB3 knockdown HAMECs showed decreased NF-κB activation induced by PA, and consequent suppressed adhesion molecule expression and monocyte adhesion. Taken together, these results demonstrate that increased endothelial PFKFB3 expression promotes diet-induced inflammatory responses and subsequent insulin resistance, suggesting that endothelial metabolic alteration plays an important role in the development of insulin resistance.

Acute lung injury (ALI) is one of the leading causes of death in sepsis. Endothelial inflammation and dysfunction play a prominent role in development of ALI. Glycolysis is the predominant bioenergetic pathway for endothelial cells (ECs). However, the role of EC glycolysis in ALI of sepsis remains unclear. Here we show that both the expression and activity of PFKFB3, a key glycolytic activator, were markedly increased in lipopolysaccharide (LPS)-treated human pulmonary arterial ECs (HPAECs) in vitro and in lung ECs of mice challenged with LPS in vivo. PFKFB3 knockdown significantly reduced LPS-enhanced glycolysis in HPAECs. Compared with LPS-challenged wild-type mice, endothelial-specific Pfkfb3 knockout (Pfkfb3 ΔVEC ) mice exhibited reduced endothelium permeability, lower pulmonary edema, and higher survival rate. This was accompanied by decreased expression of intracellular adhesion molecule-1 (Icam-1) and vascular cell adhesion molecule 1 (Vcam-1), as well as decreased neutrophil and macrophage infiltration to the lung. Consistently, PFKFB3 silencing or PFKFB3 inhibition in HPAECs and human pulmonary microvascular ECs (HPMVECs) significantly downregulated LPS-induced expression of ICAM-1 and VCAM-1, and monocyte adhesion to human pulmonary ECs. In contrast, adenovirus-mediated PFKFB3 overexpression upregulated ICAM-1 and VCAM-1 expression in HPAECs. Mechanistically, PFKFB3 silencing suppressed LPS-induced nuclear translocation of nuclear factor κB (NF-κB)-p65, and NF-κB inhibitors abrogated PFKFB3-induced expression of ICAM-1 and VCAM-1. Finally, administration of the PFKFB3 inhibitor 3PO also reduced the inflammatory response of vascular endothelium and protected mice from LPS-induced ALI. Overall, these findings suggest that targeting PFKFB3-mediated EC glycolysis is an efficient therapeutic strategy for ALI in sepsis., (Published by Elsevier Ltd.)

Background and Purpose: Inactivation of the gene for adenosine A 2A receptors (ADORA2A for humans and Adora2a for rodents) protects against brain injury in experimental stroke. However, the cell-specific pathogenic effects of A 2A receptors in thromboembolic stroke and the underlying mechanisms remain undefined. Here, we tested the hypothesis that inhibition of endothelial A 2A receptors after thromboembolic stroke improves post-stroke outcomes via down-regulation of inflammation., Experimental Approach: Thromboembolic stroke was induced by embolic middle cerebral artery occlusion in mice. Post-stroke outcomes were determined with neurological deficit scoring, infarct volume, inflammatory marker expression, brain leukocyte infiltration, blood-brain barrier (BBB) leakage, and oedema assessment. Anti-inflammatory effects of silencing the gene for A 2A receptors or pharmacological antagonism of these receptors were assessed in vitro., Key Results: Thromboembolic stroke induced Adora2a expression in the brain. Mice globally deficient in Adora2a (Adora2a -/- ) were resistant to stroke injury. Mice specifically deficient in endothelial Adora2a (Adora2a ΔVEC ) showed reduced leukocyte infiltration, BBB leakage, and oedema after stroke, along with attenuated downstream proinflammatory markers, both in vivo and in vitro. The A 2A receptor antagonist, KW 6002, also reduced brain injury and inflammation after stroke. Inactivation of ADORA2A inhibited endothelial inflammation via suppression of the NLRP3 inflammasome, down-regulating cleaved caspase 1 and IL-1β expression., Conclusions and Implications: Specific inactivation of endothelial A 2A receptors mitigated ischaemic brain injury and improved post-stroke outcomes, at least partly, through anti-inflammatory effects via blockade of NLRP3 inflammasome activity. Our findings may open new approaches to vascular protection after ischaemic stroke., (© 2019 The British Pharmacological Society.)

Increased aerobic glycolysis in endothelial cells of atheroprone areas of blood vessels has been hypothesized to drive increased inflammation and lesion burden but direct links remain to be established. Here we show that endothelial cells exposed to disturbed flow in vivo and in vitro exhibit increased levels of protein kinase AMP-activated (PRKA)/AMP-activated protein kinases (AMPKs). Selective deletion of endothelial Prkaa1, coding for protein kinase AMP-activated catalytic subunit alpha1, reduces glycolysis, compromises endothelial cell proliferation, and accelerates the formation of atherosclerotic lesions in hyperlipidemic mice. Rescue of the impaired glycolysis in Prkaa1-deficient endothelial cells through Slc2a1 overexpression enhances endothelial cell viability and integrity of the endothelial cell barrier, and reverses susceptibility to atherosclerosis. In human endothelial cells, PRKAA1 is upregulated by disturbed flow, and silencing PRKAA1 reduces glycolysis and endothelial viability. Collectively, these results suggest that increased glycolysis in the endothelium of atheroprone arteries is a protective mechanism.

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

Adenosine/adenosine receptor-mediated signaling has been implicated in the development of various ischemic diseases, including ischemic retinopathies. Here, we show that the adenosine A2a receptor (ADORA2A) promotes hypoxia-inducible transcription factor-1 (HIF-1)-dependent endothelial cell glycolysis, which is crucial for pathological angiogenesis in proliferative retinopathies. Adora2a expression is markedly increased in the retina of mice with oxygen-induced retinopathy (OIR). Endothelial cell-specific, but not macrophage-specific Adora2a deletion decreases key glycolytic enzymes and reduces pathological neovascularization in the OIR mice. In human primary retinal microvascular endothelial cells, hypoxia induces the expression of ADORA2A by activating HIF-2α. ADORA2A knockdown decreases hypoxia-induced glycolytic enzyme expression, glycolytic flux, and endothelial cell proliferation, sprouting and tubule formation. Mechanistically, ADORA2A activation promotes the transcriptional induction of glycolytic enzymes via ERK- and Akt-dependent translational activation of HIF-1α protein. Taken together, these findings advance translation of ADORA2A as a therapeutic target in the treatment of proliferative retinopathies and other diseases dependent on pathological angiogenesis.Pathological angiogenesis in the retina is a major cause of blindness. Here the authors show that adenosine receptor A2A drives pathological angiogenesis in the oxygen-induced retinopathy mouse model by promoting glycolysis in endothelial cells via the ERK/Akt/HIF-1α pathway, thereby suggesting new therapeutic targets for disease treatment.

The nucleoside adenosine is a potent regulator of vascular homeostasis, but it remains unclear how expression or function of the adenosine-metabolizing enzyme adenosine kinase (ADK) and the intracellular adenosine levels influence angiogenesis. We show here that hypoxia lowered the expression of ADK and increased the levels of intracellular adenosine in human endothelial cells. Knockdown (KD) of ADK elevated intracellular adenosine, promoted proliferation, migration, and angiogenic sprouting in human endothelial cells. Additionally, mice deficient in endothelial ADK displayed increased angiogenesis as evidenced by the rapid development of the retinal and hindbrain vasculature, increased healing of skin wounds, and prompt recovery of arterial blood flow in the ischemic hindlimb. Mechanistically, hypomethylation of the promoters of a series of pro-angiogenic genes, especially for VEGFR2 in ADK KD cells, was demonstrated by the Infinium methylation assay. Methylation-specific PCR, bisulfite sequencing, and methylated DNA immunoprecipitation further confirmed hypomethylation in the promoter region of VEGFR2 in ADK-deficient endothelial cells. Accordingly, loss or inactivation of ADK increased VEGFR2 expression and signaling in endothelial cells. Based on these findings, we propose that ADK downregulation-induced elevation of intracellular adenosine levels in endothelial cells in the setting of hypoxia is one of the crucial intrinsic mechanisms that promote angiogenesis.

Background and Purpose: Excess nutrient‐induced endothelial cell inflammation is a hallmark of high fat diet (HFD)‐induced metabolic syndrome. Pharmacological activation of the protein kinase AMP‐activated α1 (PRKAA1) also known as AMPKα1, shows its beneficial effects in many studies of cardiometabolic disorders. However, AMPKα1, as a major cellular sensor of energy and nutrients in endothelial cells, has not been studied for its physiological role in excess nutrient‐induced endothelial cell (EC) inflammation. Experimental Approach Wild‐type and EC‐specific Prkaa1 knockout mice were fed with an HFD. Body weight, fat mass composition, glucose, and lipid levels were monitored regularly. Insulin sensitivity was analysed systemically and in major metabolic organs/tissues. Inflammation status in metabolic organs/tissues were examined with quantitative RT‐PCR and flow cytometry. Additionally, metabolic status, inflammation severity, and signalling in cultured ECs were assayed with multiple approaches at the molecular level. Key Results: EC Prkaa1 deficiency unexpectedly alleviated HFD‐induced metabolic syndromes including decreased body weight and fat mass, enhanced glucose clearance and insulin sensitivity, and relieved adipose inflammation and hepatic steatosis. Mechanistically, PRKAA1 knockdown in cultured ECs reduced endothelial glycolysis and fatty acid oxidation, decreased levels of acetyl‐CoA and suppressed transcription of inflammatory molecules mediated by ATP citrate lyase and histone acetyltransferase p300. Conclusions and Implications: This unexpected pro‐inflammatory effect of endothelial AMPKα1/PRKAA1 in a metabolic context provides additional insight in AMPKα1/PRKAA1 activities. An in‐depth study and thoughtful consideration should be applied when AMPKα1/PRKAA1 is used as a therapeutic target in the treatment of metabolic syndrome. [ABSTRACT FROM AUTHOR]