Increased Corneal Epithelial Turnover Contributes to Abnormal Homeostasis in the Pax6+/? Mouse Model of Aniridia

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6^+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6^+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6^+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6^+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6^+/− heterozygotes but BrdU-LRCs were also present in Pax6^+/− corneas. It seems likely that Pax6^+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6^+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6^+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6^+/− than WT mice. This implies that epithelial cell loss is higher in Pax6^+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6^+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.     

Bone Marrow�Cderived Endothelial Progenitor Cells and Endothelial Cells May Contribute to Endothelial Repair in the Kidney Immediately After Ischemia�CReperfusion

In ischemic acute kidney injury, renal blood flow is decreased. We have previously shown that reperfused, transplanted kidneys exhibited ischemic injury to vascular endothelium and that preservation of peritubular capillary endothelial integrity may be critical to recovery from ischemic injury. We hypothesized that bone marrow–derived (BMD) endothelial progenitor cells (EPCs) might play an important role in renal functional recovery after ischemia. We tested this hypothesis in recipients of cadaveric renal allografts before and for 2 weeks after transplantation. We found that the numbers of circulating CD34-positive EPCs and CD146-positive endothelial cells (ECs) decreased immediately after ischemia–reperfusion. In renal allograft tissues obtained 1 hr after reperfusion, CD34-positive cells were more frequently observed along the endothelial lining of peritubular capillaries compared with non-ischemic controls. Moreover, 0–17.5% of peritubular capillary ECs were of recipient origin. In contrast, only 0.1–0.7% of tubule cells were of recipient origin. Repeat graft biopsy samples obtained 35 and 73 days after transplant did not contain capillary ECs of recipient origin, whereas 1.4% and 12.1% of tubule cells, respectively, were of recipient origin. These findings suggest that BMD EPCs and ECs may contribute to endothelial repair immediately after ischemia–reperfusion. (J Histochem Cytochem 58:687–694, 2010)     

Tetraspanin CD63 Promotes Vascular Endothelial Growth Factor Receptor 2-β1 Integrin Complex Formation,Thereby Regulating Activation and Downstream Signaling in Endothelial Cells in Vitro and in Vivo

CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of β1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs. Mechanistically, CD63 associated with both β1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between β1 integrin and VEGFR2 because CD63 silencing disrupted VEGFR2-β1 integrin complex formation identified using proximity ligation assays. Signaling downstream of β1 integrin and VEGFR2 was attenuated in CD63-silenced cells, although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.     

A Complex Interaction between Rickettsia conorii and Dickkopf-1 – Potential Role in Immune Evasion Mechanisms in Endothelial Cells

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heat-inactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelial-derived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)α in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection.     

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A₂ mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44^mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH₄) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O₂^•–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O₂^•– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH₄ level. Insulin and tempol blocked the high D-glucose–increased p42/44^mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O₂^•–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.     

Astrocyte-Derived Sonic Hedgehog Contributes to Angiogenesis in Brain Microvascular Endothelial Cells via RhoA/ROCK Pathway After Oxygen–Glucose Deprivation

The human adult brain possesses intriguing plasticity, including neurogenesis and angiogenesis, which may be mediated by the activated sonic hedgehog (Shh). By employing a coculture system, brain microvascular endothelial cells (BMECs) cocultured with astrocytes, which were incubated under oxygen–glucose deprivation (OGD) condition, we tested the hypothesis that Shh secreted by OGD-activated astrocytes promotes cerebral angiogenesis following ischemia. The results of this study demonstrated that Shh was mainly secreted by astrocytes and the secretion was significantly upregulated after OGD. The proliferation, migration, and tube formation of BMECs cocultured with astrocytes after OGD were significantly enhanced, but cyclopamine (a Shh antagonist) or 5E1 (an antibody of Shh) reversed the change. Furthermore, silencing Ras homolog gene family, member A (RhoA) of BMECs by RNAi and blocking Rho-dependent kinase (ROCK) by Y27632, a specific antagonist of ROCK, suppressed the upregulation of proliferation, migration, and tube formation of BMECs after OGD. These findings suggested that Shh derived from activated astrocytes stimulated RhoA/ROCK pathway in BMECs after OGD, which might be involved in angiogenesis in vitro.     

ALK5 and ALK1 Play Antagonistic Roles in Transforming Growth Factor β-Induced Podosome Formation in Aortic Endothelial Cells

Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-β-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.     

MicroRNA-3646 Contributes to Docetaxel Resistance in Human Breast Cancer Cells by GSK-3β/β-Catenin Signaling Pathway

Acquisition of resistance to docetaxel (Doc) is one of the most important problems in treatment of breast cancer patients, but the underlying mechanisms are still not fully understood. In present study, Doc-resistant MDA-MB-231 and MCF-7 breast cancer cell lines (MDA-MB-231/Doc and MCF-7/Doc) were successfully established in vitro by gradually increasing Doc concentration on the basis of parental MDA-MB-231 and MCF-7 cell lines (MDA-MB-231/S and MCF-7/S). The potential miRNAs relevant to the Doc resistance were screened by miRNA microarray. We selected 5 upregulated miRNAs (has-miR-3646, has-miR-3658, has-miR-4438, has-miR-1246, and has-miR-574-3p) from the results of microarray for RT-qPCR validation. The results showed that expression level of miR-3646 in MDA-MB-231/Doc cells was significantly higher than in MDA-MB-231/S cells. Compared to MCF-7/S cells, miR-3646 expression was up-regulated in MCF-7/Doc cells. Further studies revealed that transfection of miR-3646 mimics into MDA-MB-231/S or MCF-7/S cells remarkably increased their drug resistance, in contrast, transfection of miR-3646 inhibitors into MDA-MB-231/Doc or MCF-7/Doc cells resulted in significant reduction of the drug resistance. By the pathway enrichment analyses for miR-3646, we found that GSK-3β/β-catenin signaling pathway was a significant pathway, in which GSK-3β was an essential member. RT-qPCR and Western blot results demonstrated that miR-3646 could regulate GSK-3β mRNA and protein expressions. Furthermore, a marked increase of both nuclear and cytoplasmic β-catenin expressions (with phosphorylated-β-catenin decrease) was observed in MDA-MB-231/Doc cells compared with MDA-MB-231/S cells, and their expression were positively related to miR-3646 and negatively correlated with GSK-3β. Taken together, our results suggest that miR-3646-mediated Doc resistance of breast cancer cells maybe, at least in part, through suppressing expression of GSK-3β and resultantly activating GSK-3β/β-catenin signaling pathway.     

The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Sch?nlein Purpura

Objective

In addition to IgA, the deposition of complement (C)3 in dermal vessels is commonly found in Henoch-Schönlein purpura (HSP). The aim of this study is to elucidate the role of circulating complement proteins in the pathogenesis of childhood HSP.

Methods

Plasma levels of C3a, C4a, C5a, and Bb in 30 HSP patients and 30 healthy controls were detected by enzyme-linked immunosorbent assay (ELISA). The expression of C3a receptor (C3aR), C5a receptor (CD88), E-selectin, intercellular adhesion molecule 1 (ICAM-1), C3, C5, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, and RANTES by human dermal microvascular endothelial cells (HMVEC-d) was evaluated either by flow cytometry or by ELISA.

Results

At the acute stage, HSP patients had higher plasma levels of C3a (359.5 ± 115.3 vs. 183.3 ± 94.1 ng/ml, p < 0.0001), C5a (181.4 ± 86.1 vs. 33.7 ± 26.3 ng/ml, p < 0.0001), and Bb (3.7 ± 2.6 vs. 1.0 ± 0.6 μg/ml, p < 0.0001), but not C4a than healthy controls. Although HSP patient-derived acute phase plasma did not alter the presentation of C3aR and CD88 on HMVEC-d, it enhanced the production of endothelial C3 and C5. Moreover, C5a was shown in vitro to up-regulate the expression of IL-8, MCP-1, E-selectin, and ICAM-1 by HMVEC-d with a dose-dependent manner.

Conclusion

In HSP, the activation of the complement system in part through the alternative pathway may have resulted in increased plasma levels of C3a and C5a, which, especially C5a, may play a role in the disease pathogenesis by activating endothelium of cutaneous small vessels.     

Mouse Hepatic Oval Cells Require Met-Dependent PI3K to Impair TGF-β-Induced Oxidative Stress and Apoptosis

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met^−/− oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Met^flx/flx), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Met^flx/flx and Met^−/− oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met^−/− oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met^−/− oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Met^flx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met^−/− oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Met^flx/flx oval cells, whereas no effect was observed in Met^−/− oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis.     

The Interaction of Amyloid β and the Receptor for Advanced Glycation Endproducts Induces Matrix Metalloproteinase-2 Expression in Brain Endothelial Cells

The pathological hallmarks of Alzheimer’s disease (AD) include formation of extracellular amyloid-β peptide (Aβ) and inflammatory responses. Numerous studies have reported that cerebral microvascular Aβ deposition promotes neuroinflammation in AD. Matrix metalloproteinases (MMPs) are involved in the cleavage of extracellular matrix proteins and regulation of growth factors, receptors, and adhesion molecules. Relatively little is known about the involvement of MMPs as inflammatory mediators in the pathological processes of AD. In this study, we explored the signaling pathway of MMP-2 up-regulation by Aβ in brain endothelial cells (BECs) of mice. Using Western blots, we found that inhibitors of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) significantly decreased Aβ-induced MMP-2 expression in BECs. Furthermore, antibody neutralization of the receptor for advanced glycation endproducts effectively blocked Aβ-induced activation of ERK and JNK and their contribution to elevated MMP-2 expression in BECs. Our results suggest that increased MMP-2 expression induced by the interaction of Aβ with RAGE in BECs may contribute to enhanced vascular inflammatory stress in Aβ-related vascular disorders, such as cerebral amyloid angiopathy and AD. This study offers new insights into neuroinflammation in the progression of AD.     

Effects of Preconditioning with Sevoflurane on TNF-α-Induced Permeability and Activation of p38 MAPK in Rat Pulmonary Microvascular Endothelial Cells

Preconditioning with sevoflurane (SPC) diminishes effusion of rat alveolar membrane during inflammation. It is not clear whether this preconditioning directly inhibits permeability of pulmonary microvascular endothelial cell (PMVEC) monolayer. In this article, we evaluated effects of SPC on permeability of PMVEC monolayer and identified signaling pathways involved in these effects. PMVEC monolayer was exposed to different conditions (5-hydroxydecanoate (5-HD), TNF-α, SPC, SPC with subsequent exposure to TNF-α and 5-HD, and SPC with subsequent exposure to TNF-α alone), and the permeability of PMVEC monolayer was assessed using FITC-bovine serum albumin (ELISA). Expression of ICAM-1 (Western blot and RT-PCR) and activation of p38 MAPK (Western blot) were also assessed. Compared to the TNF-α group, permeability of PMVEC monolayer in the SPC + TNF-α group was significantly lower. Activation of p38 MAPK was also diminished in the TNF-α group. Pre-treatment with 5-HD reverted beneficial effects of SPC. Expression of ICAM-1 was not modulated by any of the tested experimental exposures. The results of this study demonstrate that SPC is capable of diminishing the TNF-α-induced increase of permeability of PMVEC monolayer, and that this beneficial effect is partly reversed by 5-HD. Further, SPC suppresses activation of p38 MAPK.     

EMILIN1/α9β1 Integrin Interaction Is Crucial in Lymphatic Valve Formation and Maintenance

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometric in vitro and in vivo functional assays, we conclude that this impaired phenotype depends on the lack of α9β1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a “guiding” molecule in migration of lymphatic endothelial cells.     

Neuropilin-1/GIPC1 Signaling Regulates α5β1 Integrin Traffic and Function in Endothelial Cells

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes α5β1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with α5β1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.     

4-Pyridone-3-Carboxamide-1β-D-Ribonucleoside Metabolism in Endothelial Cells and Its Impact on Cellular Energetic Balance

4-Pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) is a naturally occurring compound related to nicotinamide that could be metabolized to mono-, di-, and triphosphates of 4PYR (4PYMP, 4PYDP, 4PYTP) and nicotinamide adenine dinucleotide (NAD) analogue (4PYRAD) in all types of cells. Previous studies demonstrated that formation of 4PYMP and 4PYTP was dependent on adenosine kinase activity. Pathway of 4PYRAD production is not yet identified, but most likely this process involves production of 4PYMP. This study aimed to evaluate influence of 4PYR on metabolism of endothelial cells and to test effect of nucleoside transport inhibitors. 4PYR was obtained by chemical synthesis. Endothelial cell line (HMEC-1) was incubated for 24 or 48 hours with 100 μM 4PYR. After incubation, cells were separated from medium and analyzed for concentrations of ATP, NAD, and 4PYR metabolites by using reversed-phase high performance liquid chromatography. We demonstrated progressive accumulation of 4PYR metabolites in endothelium that reached 33.2 ± 0.8 nmol/mg protein for 4PYMP and 5.25 ± 0.17 nmol/mg protein for 4PYRAD after 48-hour incubation with 4PYR. Dipyridamole protected from accumulation of 4PYR metabolites in endothelial cells. We conclude that endothelium is capable to convert 4PYR into intracellular metabolites and this causes disruption of cell energy balance. Nucleoside transport inhibition with dipyridamole could protect endothelium from this effect. This finding could be of clinical relevance in conditions associated with accumulation of 4PYR such as chronic renal disease.