To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73 /ecto-5′-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (−14%; P <0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl ₃ ) was significantly reduced by 20% in cd73 ^−/− mice ( P <0.05). Bleeding time after tail tip resection tended to be shorter in cd73 ^−/− mice (−35%). In vivo platelet cAMP levels were 0.96±0.46 in WT versus 0.68±0.27 pmol/10 ⁶ cells in cd73 ^−/− mice ( P <0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 μmol/L) was undistinguishable between the two strains. In the cremaster model of ischemia–reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73 ^−/− compared with WT littermates (WT 98% versus cd73 ^−/− 245%; P <0.005). The constitutive adhesion of monocytes in ex vivo–perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73 ^−/− mice ( P <0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.

Tubuloglomerular feedback (TGF) describes a sequence of events linking salt concentrations in tubular fluid at the macula densa to the vascular tone of the afferent arteriole and thus to the glomerular filtration rate (GFR) of the same nephron. The signal transduction pathways of TGF remain incompletely understood, but both ATP release from macula densa cells and local formation of adenosine were suggested to be involved in the process. To test the role of extracellular formation of adenosine by ecto-5′-nucleotidase (cd73) in TGF, in regulation of GFR, and in tubular reabsorption, renal clearance and micropunture experiments were performed in cd73 wild-type (cd73^+/+) and knockout mice (cd73^−/−). The cd73^−/− mice presented normal mean arterial blood pressure, but modestly lower whole kidney and single nephron GFR (SNGFR). Fractional reabsorption of Na⁺ and K⁺ up to the late proximal tubule, distal tubule, as well as urine were not significantly different between cd73^−/− and cd73^+/+ mice. Lack of cd73 resulted in a diminished TGF response, as indicated by smaller changes of stop-flow pressure in response to increasing loop of Henle perfusion from 0 to 25 nl/min, smaller differences in SNGFR determined from paired proximal and distal tubular collections, and by smaller fractional changes of distal SNGFR in response to adding 6 nl/min of artificial tubular fluid to free-flowing proximal tubules. The TGF response in cd73^+/+ mice and the residual TGF response in cd73^−/− mice were completely inhibited by adenosine A₁-receptor blockade. The results suggest that extracellular formation of adenosine by ecto-5′-nucleotidase (cd73) is dispensable for normal fluid, Na⁺, or K⁺ reabsorption along the nephron, but contributes to the regulation of GFR. Adenosine generated by both ecto-5′-nucleotidase (cd73)-dependent and -independent mechanisms participates in the mediation of TGF in vivo.

CD73 (ecto-5'-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A₁, A_2A, and A₃ in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A₁AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A₃AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy.

The activity of a cell‐surface ecto‐adenosine deaminase (eADA) is markedly increased in the endothelial activation and vascular inflammation leading to decreased adenosine concentration and alterations in adenosine signalling. Depending on the specific pathway activated, extracellular purines mediate host cell response or regulate growth and cytotoxicity on tumour cells. The aim of this study was to test the effects of adenosine deaminase inhibition by 2′deoxycoformycin (dCF) on the breast cancer development. dCF treatment decreased a tumour growth and a final tumour mass in female BALB/c mice injected orthotopically with 4T1 cancer cells. dCF also counteracted cancer‐induced endothelial dysfunction in orthotopic and intravenous 4T1 mouse breast cancer models. In turn, this low dCF dose had a minor effect on immune stimulation exerted by 4T1 cell implantation. In vitro studies revealed that dCF suppressed migration and invasion of 4T1 cells via A2a and A3 adenosine receptor activation as well as 4T1 cell adhesion and transmigration through the endothelial cell layer via A2a receptor stimulation. Similar effects of dCF were observed in human breast cancer cells. Moreover, dCF improved a barrier function of endothelial cells decreasing its permeability. This study highlights beneficial effects of adenosine deaminase inhibition on breast cancer development. The inhibition of adenosine deaminase activity by dCF reduced tumour size that was closely related to the decreased aggressiveness of tumour cells by adenosine receptor‐dependent mechanisms and endothelial protection.