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Chronic kidney disease (CKD) promotes development of cardiac abnormalities and is highly prevalent in patients with heart failure, particularly in those with preserved ejection fraction. CKD is associated with endothelial dysfunction, however, whether CKD can induce impairment of endothelium-to-cardiomyocyte crosstalk leading to impairment of cardiomyocyte function is not known. The sodium-glucose co-transporter 2 inhibitor, empagliflozin, reduced cardiovascular events in diabetic patients with or without CKD, suggesting its potential as a new treatment for heart failure with preserved ejection fraction. We hypothesized that uremic serum from patients with CKD would impair endothelial control of cardiomyocyte relaxation and contraction, and that empagliflozin would protect against this effect. Using a co-culture system of human cardiac microvascular endothelial cells with adult rat ventricular cardiomyocytes to measure cardiomyocyte relaxation and contraction, we showed that serum from patients with CKD impaired endothelial enhancement of cardiomyocyte function which was rescued by empagliflozin. Exposure to uremic serum reduced human cardiac microvascular endothelial cell nitric oxide bioavailability, and increased mitochondrial reactive oxygen species and 3-nitrotyrosine levels, indicating nitric oxide scavenging by reactive oxygen species. Empagliflozin attenuated uremic serum-induced generation of endothelial mitochondrial reactive oxygen species, leading to restoration of nitric oxide production and endothelium-mediated enhancement of nitric oxide levels in cardiomyocytes, an effect largely independent of sodium-hydrogen exchanger-1. Thus, empagliflozin restores the beneficial effect of cardiac microvascular endothelial cells on cardiomyocyte function by reducing mitochondrial oxidative damage, leading to reduced reactive oxygen species accumulation and increased endothelial nitric oxide bioavailability.

Insulin-mediated microvascular recruitment (IMVR) regulates delivery of insulin and glucose to insulin-sensitive tissues. We have previously proposed that perivascular adipose tissue (PVAT) controls vascular function through outside-to-inside communication and through vessel-to-vessel, or "vasocrine" signaling. However, direct experimental evidence supporting a role of local PVAT in regulating IMVR and insulin sensitivity in vivo is lacking. Here, we studied muscles with and without PVAT in mice using combined contrast-enhanced ultrasonography and intravital microscopy to measure IMVR and gracilis artery (GA) diameter at baseline and during the hyperinsulinemic-euglycemic clamp. We show, using microsurgical removal of PVAT from the muscle microcirculation, that local PVAT depots regulate insulin-stimulated muscle perfusion and glucose uptake in vivo We discovered direct microvascular connections between PVAT and the distal muscle microcirculation, or adipomuscular arterioles, removal of which abolished IMVR. Local removal of intramuscular PVAT altered protein clusters in the connected muscle, including upregulation of a cluster featuring heat shock proteins 90ab1 and 70 and downregulation of a cluster of mitochondrial protein components of complexes III, IV and V. These data highlight the importance of PVAT in vascular and metabolic physiology, and are likely relevant for obesity and diabetes.

Visual Abstract, Highlights • CMECs exert a direct positive effect on cardiomyocyte contraction and relaxation, which is mainly mediated by endothelial-derived NO. • Pro-inflammatory stimulation of CMECs by pre-incubation with TNF-α or interleukin-1β abrogates the positive regulatory function of these cells on cardiomyocyte contractile property. • Mechanistically, pro-inflammatory activation of CMECs leads to mitochondrial and cytoplasmic ROS accumulation that results in the scavenging of NO. • Empagliflozin directly restores the beneficial effect of CMECs on cardiomyocyte contraction and relaxation by reducing TNF-α-induced mitochondrial and cytoplasmic ROS accumulation, which leads to reinstatement of CMEC-derived NO delivery., Summary The positive findings of the EMPA-REG OUTCOME trial (Randomized, Placebo-Controlled Cardiovascular Outcome Trial of Empagliflozin) on heart failure (HF) outcome in patients with type 2 diabetes mellitus suggest a direct effect of empagliflozin on the heart. These patients frequently have HF with preserved ejection fraction (HFpEF), in which a metabolic risk-related pro-inflammatory state induces cardiac microvascular endothelial cell (CMEC) dysfunction with subsequent cardiomyocyte (CM) contractility impairment. This study showed that CMECs confer a direct positive effect on contraction and relaxation of CMs, an effect that requires nitric oxide, is diminished after CMEC stimulation with tumor necrosis factor-α, and is restored by empagliflozin. Our findings on the effect of empagliflozin on CMEC-mediated preservation of CM function suggests that empagliflozin can be used to treat the cardiac mechanical implications of microvascular dysfunction in HFpEF.

Reduced vasodilator properties of insulin in obesity are caused by changes in perivascular adipose tissue and contribute to microvascular dysfunction in skeletal muscle. The causes of this dysfunction are unknown. The effects of a short-term Western diet on JNK2-expressing cells in perivascular adipose tissue (PVAT) on insulin-induced vasodilation and perfusion of skeletal muscle were assessed. In vivo, 2 wk of Western diet (WD) reduced whole body insulin sensitivity and insulin-stimulated muscle perfusion, determined using contrast ultrasonography during the hyperinsulinemic clamp. Ex vivo, WD triggered accumulation of PVAT in skeletal muscle and blunted its ability to facilitate insulin-induced vasodilation. Labeling of myeloid cells with green fluorescent protein identified bone marrow as a source of PVAT in muscle. To study whether JNK2-expressing inflammatory cells from bone marrow were involved, we transplanted JNK2−/− bone marrow to WT mice. Deletion of JNK2 in bone marrow rescued the vasodilator phenotype of PVAT during WD exposure. JNK2 deletion in myeloid cells prevented the WD-induced increase in F4/80 expression. Even though WD and JNK2 deletion resulted in specific changes in gene expression of PVAT; epididymal and subcutaneous adipose tissue; expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, or protein inhibitor of STAT1 was not affected. In conclusion, short-term Western diet triggers infiltration of JNK2-positive myeloid cells into PVAT, resulting in PVAT dysfunction, nonclassical inflammation, and loss of insulin-induced vasodilatation in vivo and ex vivo. NEW & NOTEWORTHY We demonstrate that in the earliest phase of weight gain, changes in perivascular adipose tissue in muscle impair insulin-stimulated muscle perfusion. The hallmark of these changes is infiltration by inflammatory cells. Deletion of JNK2 from the bone marrow restores the function of perivascular adipose tissue to enhance insulin’s vasodilator effects in muscle, showing that the bone marrow contributes to regulation of muscle perfusion.

Background: It has been proposed that diabetes and chronic kidney disease cause heart failure with preserved ejection fraction (HFpEF) through low-grade inflammation and microvascular endothelial dysfunction. We have previously shown that endothelial enhancement of cardiomyocyte contraction and relaxation is mediated by NO, impaired by cytokines, and restored upon treatment with the SGLT2 inhibitor Empagliflozin. Chronic kidney disease (CKD) is linked to diabetes and HFpEF and causes systemic endothelial dysfunction. Objective: To test the hypothesis that uremic serum from patients with CKD impairs endothelial control of cardiomyocyte contraction and relaxation, and that Empagliflozin protects against this effect. Methods and Results: Co-culture of rat cardiomyocytes (CM) with human cardiac microvascular endothelial cells (CMECs) enhanced their contraction and relaxation. Pre-treatment with serum from patients with CKD abrogated these effects of CMEC, and treatment of CMECs with Empagliflozin restored the effects of CMECs on CM function. Exposure of CMECs to the uremic serum reduced endothelial NO bioavailability and increased intracellular reactive oxygen species ROS by increasing ROS generation by mitochondria. Uremic serum also increased 3-nitrotyrosine (3-NT) level, indicating scavenging of NO by ROS. Empagliflozin attenuated uremic serum-induced mitochondrial and intracellular ROS, leading to restoration of endothelial NO bioavailability. Conclusions: Exposure of CMECs to serum from CKD patients impairs CMEC-mediated enhancement of CM contraction and relaxation. Empagliflozin restores the beneficial effect of CMECs on CM function by reducing mitochondrial oxidative damage, leading to a decrease in intracellular ROS accumulation and increased endothelial NO bioavailability. Disclosure R. Juni: None. R. Al-Shama: None. D. Kuster: None. J. van der Velden: None. M. Vervloet: None. W. Paulus: None. E.C. Eringa: None. P. Koolwijk: None. V.W. van Hinsbergh: None. Funding Netherlands Heart Foundation

Early atherosclerosis features functional and structural changes in the endothelial barrier function that affect the traffic of molecules and solutes between the vessel lumen and the vascular wall. Such changes are mechanistically related to the development of atherosclerosis. Proatherogenic stimuli and cardiovascular risk factors, such as dyslipidaemias, diabetes, obesity, and smoking, all increase endothelial permeability sharing a common signalling denominator: an imbalance in the production/disposal of reactive oxygen species (ROS), broadly termed oxidative stress. Mostly as a consequence of the activation of enzymatic systems leading to ROS overproduction, proatherogenic factors lead to a pro-inflammatory status that translates in changes in gene expression and functional rearrangements, including changes in the transendothelial transport of molecules, leading to the deposition of low-density lipoproteins (LDL) and the subsequent infiltration of circulating leucocytes in the intima. In this review, we focus on such early changes in atherogenesis and on the concept that proatherogenic stimuli and risk factors for cardiovascular disease, by altering the endothelial barrier properties, co-ordinately trigger the accumulation of LDL in the intima and ultimately plaque formation.

In the original article, due to an XML tagging error the equal contribution statement “Joana Amado-Azevedo, Anne-Marieke D. van Stalborch, Erik T. Valent have contributed equally to this work (shared first author), Jurjan Aman and Coert Margadant have contributed equally to this work (shared last author).” was not correctly reflected in the PubMed. The original article has been corrected.

Obesity is a global epidemic, accompanied by increased risk of type 2 diabetes and cardiovascular disease. Adipose tissue hypertrophy is associated with adipose tissue inflammation, which alters secretion of adipose tissue-derived bioactive products, known as adipokines. Adipokines determine vessel wall properties such as smooth muscle tone and vessel wall inflammation. Exercise is a mainstay of prevention of chronic, non-communicable diseases, type 2 diabetes and cardiovascular disease in particular. Aside from reducing adipose tissue mass, exercise has been shown to reduce inflammatory activity in this tissue. Mechanistically, contracting muscles release bioactive molecules known as myokines, which have been shown to alter adipose tissue metabolic phenotype. In adipose tissue, myokines induce browning, enhance fatty acid oxidation and improve insulin sensitivity. In the past years, so-called perivascular adipose tissue surrounding the vasculature (PVAT) has been shown to control vascular tone and inflammation through local release of adipokines. In obesity, an increase in mass and inflammation of PVAT culminate in dysregulation of adipokine secretion, which contributes to vascular dysfunction. This review describes our current understanding of the mechanisms by which active muscles interact with adipose tissue and improve vascular function. Aside from the exercise-dependent regulation of canonical adipose tissue function, we will focus on interaction between skeletal muscle and PVAT and the role of novel myokines, such as interleukin-15, fibroblast growth factor 21 and irisin, in this cross talk.

Objectives Mast cells (MCs) may play an important role in plaque destabilization and atherosclerotic coronary complications. Here, we have studied the presence of MCs in the intima and media of unstable and stable coronary lesions at different time points after myocardial infarction (MI). Methods Coronary arteries were obtained at autopsy from patients with acute MI (up to 5 days old; n = 27) and with chronic MI (5–14 days old; n = 18), as well as sections from controls without cardiac disease ( n = 10). Herein, tryptase-positive MCs were quantified in the intima and media of both unstable and stable atherosclerotic plaques in infarct-related and non-infarct-related coronary arteries. Results In the media of both acute and chronic MI patients, the number of MCs was significantly higher than in controls. This was also found when evaluating unstable and stable plaques separately. In patients with chronic MI, the number of MCs in unstable lesions was significantly higher than in stable lesions. This coincided with a significant increase in the relative number of unstable plaques in patients with chronic MI compared with control and acute MI. No differences in MC density were found between infarct-related and non-infarct-related coronary arteries in patients with MI. Conclusion The presence of MCs in the media of both stable and unstable atherosclerotic coronary lesions after MI suggests that MCs may be involved in the onset of MI and, on the other hand, that MI triggers intra-plaque infiltration of MCs especially in unstable plaques, possibly increasing the risk of re-infarction.

Decreased tissue perfusion increases the risk of developing insulin resistance and cardiovascular disease in obesity, and decreased levels of globular adiponectin (gAdn) have been proposed to contribute to this risk. We hypothesized that gAdn controls insulin's vasoactive effects through AMP-activated protein kinase (AMPK), specifically its α2 subunit, and studied the mechanisms involved. In healthy volunteers, we found that decreased plasma gAdn levels in obese subjects associate with insulin resistance and reduced capillary perfusion during hyperinsulinemia. In cultured human microvascular endothelial cells (HMEC), gAdn increased AMPK activity. In isolated muscle resistance arteries gAdn uncovered insulin-induced vasodilation by selectively inhibiting insulin-induced activation of ERK1/2, and the AMPK inhibitor compound C as well as genetic deletion of AMPKα2 blunted insulin-induced vasodilation. In HMEC deletion of AMPKα2 abolished insulin-induced Ser(1177) phosphorylation of eNOS. In mice we confirmed that AMPKα2 deficiency decreases insulin sensitivity, and this was accompanied by decreased muscle microvascular blood volume during hyperinsulinemia in vivo. This impairment was accompanied by a decrease in arterial Ser(1177) phosphorylation of eNOS, which closely related to AMPK activity. In conclusion, globular adiponectin controls muscle perfusion during hyperinsulinemia through AMPKα2, which determines the balance between NO and ET-1 activity in muscle resistance arteries. Our findings provide a novel mechanism linking reduced gAdn-AMPK signaling to insulin resistance and impaired organ perfusion.

Insulin-mediated microvascular recruitment (IMVR) regulates delivery of insulin and glucose to insulin-sensitive tissues. We have previously proposed that perivascular adipose tissue (PVAT) controls glucose metabolism and vascular function through outside-to-inside communication and through vessel-to-vessel, or “vasocrine” signaling. Here, we studied this hypothesis in mice by examining effects of removal of local intramuscular PVAT on muscle blood flow and glucose metabolism. Using the hyperinsulinemic, euglycemic clamp (HEC) in combination with positron emission tomography, we found that local PVAT removal transiently reduces muscle glucose uptake by ±50 percent. Contrast-enhanced ultrasonography and intravital microscopy of the gracilis artery (GA) during the HEC showed that PVAT removal abolishes insulin-induced increases in GA diameter and abrogated insulin-stimulated muscle blood volume (microvascular recruitment or IMVR). The effect of PVAT on IMVR was mediated by distinct microvessels or anastomoses, which we showed using lightsheet microscopy of mice expressing mCherry in endothelial cells. Proteomics analysis revealed that PVAT removal significantly alters expression of 109 of 1719 detected proteins in muscle. Observed changes in protein expression included reduction of a mitochondrial protein cluster and of vesicle-associated membrane protein 5 (Vamp5), involved in Glut4 trafficking. In conclusion, we have found that PVAT within muscle regulates muscle perfusion, glucose uptake and muscle protein expression, communicating with the distal microcirculation via microvascular anastomoses. These data highlight the importance of PVAT in vascular and metabolic physiology, and are relevant for type 2 diabetes and associated muscle dysfunction. Disclosure A.H. Turaihi: None. E.H. Serne: None. C. Molthoff: None. M.T. Goumans: None. C.J. Jimenez: None. J.S. Yudkin: None. Y.M. Smulders: None. V.W. van Hinsbergh: None. E.C. Eringa: None.

Perivascular adipose tissue (PVAT) surrounds most arteries and enhances insulin-induced vasodilatation through local release of adipokines. Obesity impairs the vasoregulatory function of PVAT on insulin-induced vasodilation, thus contributing to cardiovascular risk. Exercise enhances insulin-induced vasodilatation, and we evaluated whether exercise preconditions the muscle microcirculation against HFD-induced impairment of insulin-induced vasodilatation. C57BL/6 mice were exercised for 4 weeks (EX), or fed a high fat diet (HFD) for 2 weeks. Exercise preconditioning was performed by exercising mice for 4 weeks prior to HFD treatment (EX+HFD) or before and during HFD (EX+HFD/EX). HFD increased femoral PVAT and exercise attenuated this effect [(Control (C) 9±1mg vs. HFD 64±7mg - p In conclusion, exercise reduces the HFD-induced increase in PVAT and preserves insulin-induced vasodilatation by increasing mitochondrial uncoupling and improving adipokine expression in PVAT. Disclosure B.C.S. Boa: None. F.M. Ferr o: None. I.M. Gonzaga: None. L.R. Pinto: None. V.W.M. van Hinsbergh: None. E.C. Eringa: None.

Introduction: Insulin signaling in adipose tissue has been shown to regulate insulin's effects in muscle. In muscle, perivascular adipose tissue (PVAT) and vascular insulin signaling regulate muscle perfusion. Insulin receptor substrate (IRS) 2 has been shown to control adipose tissue function and glucose metabolism, and here we tested the hypothesis that IRS2 mediates insulin's actions on the vessel wall as well as the vasoactive properties of PVAT. Methods: We studied PVAT and muscle resistance arteries (RA) from littermate IRS2+/+ and IRS2-/- mice and vasoreactivity by pressure myography, vascular insulin signaling, adipokine expression, and release and PVAT morphology. As insulin induced constriction of IRS2+/+ RA in our mouse model, we also exposed RA's of C57/Bl6 mice to PVAT from IRS2+/+ and IRS2-/- littermates to evaluate vasodilator properties of PVAT. Results: IRS2-/- RA exhibited normal vasomotor function, yet a decreased maximal diameter compared to IRS2+/+ RA. IRS2+/+ vessels unexpectedly constricted endothelin-dependently in response to insulin, and this effect was absent in IRS2-/- RA due to reduced ERK1/2activation. For evaluation of PVAT function, we also used C57/Bl6 vessels with a neutral basal effect of insulin. In these experiments insulin (10.0 nM) increased diameter in the presence of IRS2+/+ PVAT (17 ± 4.8, p = 0.014), yet induced a 10 ± 7.6% decrease in diameter in the presence of IRS2-/- PVAT. Adipocytes in IRS2-/- PVAT (1314 ± 161 μm2) were larger (p = 0.0013) than of IRS2+/+ PVAT (915 ± 63 μm2). Adiponectin, IL-6, PAI-1 secretion were similar between IRS2+/+ and IRS2-/- PVAT, as were expression of pro-inflammatory genes (TNF-α, CCL2) and adipokines (adiponectin, leptin, endothelin-1). Insulin-induced AKT phosphorylation in RA was similar in the presence of IRS2-/- and IRS2+/+ PVAT. Conclusion: In muscle, IRS2 regulates both insulin's vasoconstrictor effects, mediating ERK1/2-ET-1 activation, and its vasodilator effects, by mediating the vasodilator effect of PVAT. The regulatory role of IRS2 in PVAT is independent from adiponectin secretion.

This editorial refers to ‘C1q/tumour necrosis factor-related protein 1: an adipokine marking and promoting atherosclerosis’, by L. Lu et al ., doi:10.1093/eurheartj/ehv649. Approximately 30% of the global population are overweight or obese, posing a major threat to healthcare in developed and developing countries. Obesity decreases life expectancy, mainly by increasing the risk of type 2 diabetes and cardiovascular disease. A mechanistic explanation for these intertwined metabolic and cardiovascular risks has been offered by adipokines, a large and diverse group of molecules secreted by adipose tissue that affect both glucose metabolism and cardiovascular function. Importantly, the perivascular depot of adipose tissue is situated around the majority of the arterial tree, and a large number of studies have shown that local adipokine expression controls vascular function and dysfunction.1 Well-known adipokines include cytokines such as interleukin-1β and tumour necrosis factor α (TNFα) (resulting in the term adipose tissue-derived cytokines), leptin, and fatty acids. Among the most intriguing adipokines are the 16-member family of C1q/TNF-related proteins (CTRPs) that includes the intensively studied adiponectin,2 also synthesized in arterial perivascular adipose tissue (PVAT).3 The CTRPs share the globular adiponectin domain in their molecular structure, which binds and activates the adiponectin receptors AdipoR1 and AdipoR2, widely expressed in the cardiovascular system.4 CTRPs circulate as monomers but also as heterotrimers, enabling diverse biological effects.2 Figure 1 The adipokine CTRP1 (C1q/tumour necrosis factor-related protein 1) is produced in adipose tissue (yellow area) as well by oxidized LDL (OxLDL)- or interleukin-1β (IL-1β)-stimulated macrophages and endothelial cells in the atherosclerotic plaque (purple area). In the plaque, CTRP1 activates endothelial cells and macrophages themselves to produce leucocyte adhesion molecules and tumour necrosis factor α (TNFα), …

BACKGROUND: Complement is an important mediator in arterial blood pressure-induced vein graft failure. Previously, we noted activation of cell protective mechanisms in human saphenous veins too. Here we have analyzed whether C4b-binding protein (C4bp), an endogenous complement inhibitor, is present in the vein wall.METHODS: Human saphenous vein segments obtained from patients undergoing coronary artery bypass grafting (n = 55) were perfused in vitro at arterial blood pressure with either autologous blood for 1, 2, 4, or 6 hr or with autologous blood supplemented with reactive oxygen species scavenger N-acetylcysteine. The segments were subsequently analyzed quantitatively for presence of C4bp and complement activation product C3d using immunohistochemistry.RESULTS: Perfusion induced deposition of C3d and C4bp within the media of the vessel wall, which increased reproducibly and significantly over a period of 4 hr up to 3.8% for C3d and 81% for C4bp of the total vessel area. Remarkably after 6 hr of perfusion, the C3d-positive area decreased significantly to 1.3% and the C4bp-positive area to 19% of the total area of the vein. The areas positive for both C4bp and C3d were increased in the presence of N-acetylcysteine.CONCLUSIONS: Exposure to arterial blood pressure leads to a transient presence of C4bp in the vein wall. This may be part of a cell-protective mechanism to counteract arterial blood pressure-induced cellular stress and inflammation in grafted veins.

A 53-year-old obese, but otherwise healthy, woman, participated in a study on impaired muscle perfusion in obesity. A muscle biopsy taken from this patient shows the fi rst identifi cation of perivascular adipose tissue (PVAT) in human muscle. In the past decade, evidence has accumulated that PVAT controls various vascular functions, including infl ammation, smooth-muscle tone, and endothelial function. Perivascular fat has been proposed to control insulin sensitivity through regulation of insulin-stimulated muscle perfusion. In 2015, PVAT was shown to determine insulin’s vasoactive eff ect on muscle arterioles; this regulation is impaired in obesity. The discovery of PVAT in human muscle and its vascular functions opens new avenues for the treatment of vascular disease and insulin resistance in obesity.

This editorial refers to ‘Thromboxane A2 induces blood flow recovery via platelet adhesion to ischaemic regions’ by H. Amano et al. , doi:10.1093/cvr/cvv139. The reconstitution of oxygen and nutrient supply in cases of disturbed blood flow caused by tissue damage or vascular occlusion is driven by sprouting angiogenesis, arteriogenesis as an adaptation of pre-collateral vessels into collateral conduit vessels, and vasculogenesis characterized by invasion of circulating monocytic and endothelial progenitor cells.1 To study these mechanisms, a mouse model is often used, in which ligation of the femoral artery causes interruption of blood flow in the hind leg and hypoxia in the distal tissue.2 To save the hind leg tissue, the body responds immediately by the stimulation of a collateral circulation that starts proximal to the ligation. In a healthy C57/BL6 mouse, the transformation of pre-existing narrow collaterals into arterial conduit vessels with a many-fold wider diameter restores the blood supply and tissue perfusion to the hind leg within 7–10 days. In the occluded hind leg model, arteriogenesis is usually accompanied by angiogenesis in the ischaemic muscle, which results in an increase in local microvascular density. This further facilitates blood distribution and oxygen diffusion to meet the increased oxygen demand of the injured tissue. Figure 1 Involvement of platelets and TPs in arteriogenesis in mice.6,7 After femoral occlusion, altered shear forces and oxygen availability modulate endothelial functioning, after which platelets start adhering the endothelium of pre-existing collaterals and vessels that became ischaemic distal of the occlusion. These …

Endothelial dysfunction associated with diabetes and cardiovascular disease is characterized by changes in vasoregulation, enhanced generation of reactive oxygen intermediates, inflammatory activation, and altered barrier function. These endothelial alterations contribute to excess cardiovascular disease in diabetes, but may also play a role in the pathogenesis of diabetes, especially type 2. The mechanisms underlying endothelial dysfunction in diabetes differ between type 1 (T1D) and type 2 diabetes (T2D): hyperglycemia contributes to endothelial dysfunction in all individuals with diabetes, whereas the causative mechanisms in T2D also include impaired insulin signaling in endothelial cells, dyslipidemia and altered secretion of bioactive substances (adipokines) by adipose tissue. The close association of so-called perivascular adipose tissue with arteries and arterioles facilitates the exposure of vascular endothelium to adipokines, particularly if inflammation activates the adipose tissue. Glucose and adipokines activate specific intracellular signaling pathways in endothelium, which in concert result in endothelial dysfunction in diabetes. Here, we review the characteristics of endothelial dysfunction in diabetes, the causative mechanisms involved and the role of endothelial dysfunction(s) in the pathogenesis of T2D. Finally, we will discuss the therapeutic potential of endothelial dysfunction in T2D.

Background Arterial blood pressure–induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A 2 (sPLA 2 -IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA 2 -IIA herein. Methods The effects of PX-18, an inhibitor of both sPLA 2 -IIA and apoptosis, on residual endothelium and the presence of sPLA 2 -IIA were studied in human saphenous vein segments ( n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs. Results The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA 2 -IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA 2 -IIA. Conclusions In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure–induced death, possibly also independent of sPLA 2 -IIA inhibition.

Objective: Exogenous insulin use in patients with type 2 diabetes (DM2) has been associated with an increased risk of cardiovascular events. Through which mechanisms insulin may increase atherosclerotic plaque vulnerability is currently unclear. Because insulin has been suggested to promote angiogenesis in diabetic retinopathy and tumors, we hypothesized that insulin enhances intra-plaque angiogenesis. Methods: An in vitro model of pathological angiogenesis was used to assess the potential of insulin to enhance capillary-like tube formation of human microvascular endothelial cells (hMVEC) into a three dimensional fibrin matrix. In addition, insulin receptor expression within atherosclerotic plaques was visualized in carotid endarterectomy specimens of 20 patients with carotid artery stenosis, using immunohistochemical techniques. Furthermore, microvessel density within atherosclerotic plaques was compared between 68 DM2 patients who received insulin therapy and 97 DM2 patients who had been treated with oral glucose lowering agents only. Results: Insulin, at a concentration of 10(-8) M, increased capillary-like tube formation of hMVEC 1.7-fold (p < 0.01). Within human atherosclerotic plaques, we observed a specific distribution pattern for the insulin receptor: insulin receptor expression was consistently higher on the endothelial lining of small nascent microvessels compared to more mature microvessels. There was a trend towards an increased microvessel density by 20% in atherosclerotic plaques derived from patients using insulin compared to plaques derived from patients using oral glucose lowering agents only (p = 0.05). Conclusion: Exogenous insulin use in DM2 patients may contribute to increased plaque vulnerability by stimulating local angiogenesis within atherosclerotic plaques. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Obesity is associated with insulin resistance, hypertension, and cardiovascular disease, but the mechanisms underlying these associations are incompletely understood. Microvascular dysfunction may play an important role in the pathogenesis of both insulin resistance and hypertension in obesity. Adipose tissue-derived substances (adipokines) and especially inflammatory products of adipose tissue control insulin sensitivity and vascular function. In the past years, adipose tissue associated with the vasculature, or perivascular adipose tissue (PAT), has been shown to produce a variety of adipokines that contribute to regulation of vascular tone and local inflammation. This review describes our current understanding of the mechanisms linking perivascular adipose tissue to vascular function, inflammation, and insulin resistance. Furthermore, we will discuss mechanisms controlling the quantity and adipokines secretion by PAT.

Hypertension is a common side effect in cancer patients treated with inhibitors of vascular endothelial growth factor/vascular endothelial growth factor receptor-2 signaling and may represent a marker of clinical benefit. Functional rarefaction (a decrease in perfused microvessels) or structural rarefaction (a reduction in anatomic capillary density) may play an important role in the development of hypertension. We investigated whether sunitinib caused impairment of microvascular function and/or reduction of capillary density in patients with metastatic renal cell cancer (mRCC). Sixteen mRCC patients were treated with sunitinib (50 mg/day). Assessments of 24-h ambulatory blood pressure, microvascular endothelial function by laser Doppler fluxmetry, and capillary density by capillary microscopy were performed at baseline and days 14 and 28. Median blood pressure had increased on day 14 (systolic 10 mmHg, P

Disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications. This study focused on the role of angiogenic factors in the first trimester in the pathogenesis of preeclampsia (PE) and/or fetal growth restriction (FGR). First-trimester decidua was obtained during chorionic villous sampling.The expression of the angiogenic factors was determined by reverse transcriptase polymerase chain reaction and related to the pregnancy outcome. First-trimester decidua expressed all angiogenic factors.The differential expression of angiogenic factors appeared to be more prominent in FGR than in PE. These first-trimester samples provided a unique opportunity to obtain information regarding the onset of PE and FGR. First-trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. This, in turn, may unintentionally set the stage for increased angiogenesis and altered decidual/placental vascular adaptation, which may be part of the pathogenesis of PE and/or FGR. © 2008 by the Society for Gynecologic Investigation. Chemicals / CAS: angiopoietin, 250740-90-0; vasculotropin receptor, 301253-48-5; vasculotropin, 127464-60-2

Proteolysis is essential for decidual development during embryonic implantation, but little is known regarding the expression and functions of membrane-type matrix metalloproteinases (MT-MMPs) and urokinase-type plasminogen activator (uPA) and its receptor uPAR in decidua. Therefore, their protein and mRNA levels were analysed in three first trimester decidual tissues, decidual secretory endometrium (DSE), decidua parietalis (DP) and basalis (DB). Decidua was obtained during first trimester pregnancy termination. uPA, uPAR, and MT1/2/3/5-MMP expression were studied by RT-PCR and immunohistochemistry, and CD56-positive uNK cells and CD68-positive macrophages were quantified in serial sections. The mRNAs and antigens of all proteases and uPAR were detectable in the decidual tissues and extravillous trophoblasts (EVT). mRNA levels of all proteases and uPAR, except MT5-MMP, were elevated in both DB and DP compared to DSE, being significant for MT1-MMP and uPAR in DP. MT2- and MT3-MMP mRNAs in DB were 24- and 10-fold higher than in DSE, and 19- and 7-fold increased compared to DP. At the protein level uPA and uPAR were particularly elevated in DB, while pro-angiogenic MT1- and MT3-MMPs were elevated in both DB and DP compared to DSE. MT2-MMP was prominently present in all conditions. The number of uNK cells was increased in DB and DP versus DSE, while a comparable increase in macrophages did not reach statistical significance. These data are consistent with a differential regulation of pericellular proteases in decidua by pregnancy-induced hormones, immune cells and EVT. © The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Aims/hypothesis Obesity increases the risk of cardiovascular disease and type 2 diabetes, partly through reduced insulin-induced microvascular vasodilation, which causes impairment of glucose delivery and uptake. We studied whether perivascular adipose tissue (PVAT) controls insulin-induced vasodilation in human muscle, and whether altered properties of PVAT relate to reduced insulin-induced vasodilation in obesity. Methods Insulin-induced microvascular recruitment was measured using contrast enhanced ultrasound (CEU), before and during a hyperinsulinaemic–euglycaemic clamp in 15 lean and 18 obese healthy women (18–55 years). Surgical skeletal muscle biopsies were taken on a separate day to study perivascular adipocyte size in histological slices, as well as to study ex vivo insulin-induced vasoreactivity in microvessels in the absence and presence of PVAT in the pressure myograph. Statistical mediation of the relation between BMI and microvascular recruitment by PVAT was studied in a mediation model. Results Obese women showed impaired insulin-induced microvascular recruitment and lower metabolic insulin sensitivity compared with lean women. Microvascular recruitment was a mediator in the association between obesity and insulin sensitivity. Perivascular adipocyte size, determined in skeletal muscle biopsies, was larger in obese than in lean women, and statistically explained the difference in microvascular recruitment between obese and lean women. PVAT from lean women enhanced insulin-induced vasodilation in isolated skeletal muscle resistance arteries, while PVAT from obese women revealed insulin-induced vasoconstriction. Conclusions/interpretation PVAT from lean women enhances insulin-induced vasodilation and microvascular recruitment whereas PVAT from obese women does not. PVAT adipocyte size partly explains the difference in insulin-induced microvascular recruitment between lean and obese women. Electronic supplementary material The online version of this article (doi:10.1007/s00125-015-3606-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Cholesterol-lowering statins can ameliorate severe forms of vascular hyperper-meability in experimental studies, and may thereby amellorate acute lung injury and sepsis. It is unknown whether this also applies to humans. This study aimed to define whether or not prior statin therapy reduces mild post-operative increases in pulmonary capillary protein permeability associated with acute lung injury after cardiac or major vascular surgery. A prospective observational study was performed in an intensive care unit of a university hospital on 64 patients, 37 after elective cardiac and 27 after major vascular surgery, of whom 68 and 44%, respectively, had received prior statin therapy. A mobile probe system was used to measure the pulmonary leak index (PLI), i.e. the transvascular transport rate of gallium-67-radiolabelled transferrin. For all of the patients together, the mean PLI did not differ between the statin and control groups (22.9 versus 24.4 × 10-3 min-1). The prevalence of an elevated PLI was 57% in the statin and 59% in the control group. Subgroup analysis did not reveal significant differences caused by statins in the PLI of these patients. Prior statin therapy neither has an adverse effect on mildly increased pulmonary capillary permeability in patients after cardiac or major vascular surgery nor does it ameliorate this increased capillary permeability.

SummaryIn diabetes mellitus an increased risk exists for vascular complications. A role for advanced glycation endproducts (AGEs) in the acceleration of vascular disease has been suggested. Nε(carboxymethyl)lysine (CML)- and methylglyoxal (MGO)-modified proteins have been identified as majorAGEs. The interaction of these AGEs with the human endothelial cells and macrophages was studied. Changes in adhesion molecule expression, i. e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin were determined by cell-bound Elisa on human endothelial cells after incubation with CML-modified albumin and MGO-modified albumin. The presence of the full-length receptor of AGEs (RAGE) and splice variants of RAGE was determined by specific RT-PCR. In addition, binding studies were performed with CML- and MGOmodified albumin to endothelial cells and P388D1 macrophages. We demonstrated that CML-albumin or MGO-albumin did not induce activation of endothelial cells as measured by the expression of adhesion molecules, while, under the same conditions, TNF-α did. No specific binding of CML-albumin and MGO-albumin on these cells was found. In contrast to endothelial cells, a specific binding of MGO-albumin to P388D1 macrophages was demonstrated, which could be competed by ligands of scavenger receptors. In human umbilical vein and microvascular endothelial cells we found the N-truncated and C-truncated splice variants of RAGE.In conclusion, under our experimental conditions no CML- or MGO-albumin-induced increase in adhesion molecule expression was found on endothelial cells. In agreement with this, no binding of these AGEs was found to endothelial cells. The existence of splice variants of RAGE in endothelial cells might explain the lack of interaction of extracellular AGEs with these cells.

In a combination of in vivo and in vitro studies, we investigated mechanisms via which alpha-tocopherol, a lipid soluble form of vitamin E, can directly affect endothelial activation as induced by H(2)O(2) and TNFalpha.We measured effects of alpha-tocopherol on H(2)O(2)-induced lipid peroxidation as determined with a fluorescent C-11 BODIPY(581/591) probe and on adhesion molecule expression in cultured endothelial cells. In 20 healthy volunteers treated with increasing doses of alpha-tocopherol up to 800 IU/ml for 12 weeks, plasma levels of soluble cell adhesion molecules (sCAMs) and C-reactive protein were measured.We showed that alpha-tocopherol protects cultured endothelial cells against H(2)O(2)-induced lipid peroxidation, while TNFalpha did not induce lipid peroxidation. Moreover, alpha-tocopherol attenuated H(2)O(2)-, but not TNFalpha-induced increases in adhesion molecule expression. In healthy persons, alpha-tocopherol decreased plasma levels of sE-selectin from 65+/-6 to 60+/-6 ng/ml (P=0.002), sVCAM from 893+/-31 to 853+/-23 ng/ml (P=0.022), and sICAM from 483+/-21 to 463+/-16 ng/ml (P=0.048). C-Reactive protein, as a sensitive marker of low grade inflammation, was not significantly affected.alpha-Tocopherol specifically inhibits lipid peroxidation-induced endothelial activation in vitro. The observed vitamin E-induced decrease in sCAMs in control subjects suggests that lipid peroxidation can take place in healthy individuals. Although vitamin E supplementation may be especially effective in specific groups of patients exposed to increased oxidative stress, our study suggests that vitamin E supplementation can be of benefit in healthy individuals as well.

SummaryThe transmigration of leukocytes across the endothelium is a prerequisite for the inflammatory process. Leukocyte-endothelium interaction is regulated by several endothelial adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Their expression is enhanced by inflammatory mediators, such as TNFα. In vivo a small part of these adhesion molecules is shed by proteases, and can be detected in the circulation. In this study, the time course of the TNFα-induced accumulation in the blood of three circulating soluble adhesion molecules, sE-selectin, sICAM-1 and sVCAM-1, was studied in plasma samples of tumor patients enrolled in a phase I trial receiving TNFα. Two different cohorts were studied. Eleven patients received a continuous 24-hour TNFα infusion while 10 other patients received a 5-day continuous TNFα infusion. After 24 hours of TNFα infusion sE-selectin levels increased by 1985 ± 312 %, sVCAM-1 by 301± 19 % and sICAM-1 by 445 ± 82 %. Differences in accumulation patterns were observed after 5 days of continuous TNFα infusion. sVCAM-1 and sICAM-1 levels showed an increase during the infusion with a maximum at 3 to 5 days and stayed elevated after discontinuation of the TNFα infusion. In contrast, sE-selectin reached its peak at day 1 and declined thereafter. In conclusion, sVCAM-1 and sICAM-1 show a different accumulation pattern upon TNFα infusion as compared to sE-selectin in man.

Background Several studies have reported differences in the mortality risk between diabetic subjects detected by screening and known diabetic patients. We studied mortality in relation to diabetes duration, and the contribution of other cardiovascular risk factors to the elevated risk. Materials and methods Participants were type 2 diabetic subjects ( n = 174) of a population-based cohort study. Of these, 95 were diagnosed by screening. Known diabetic subjects were grouped into two categories of diabetes duration, with a median duration of 2·4 and 11·2 years, respectively. We assessed the contribution of classical cardiovascular risk factors (dyslipidaemia, hypertension, and prior myocardial infarction), and of new cardiovascular risk factors (microalbuminuria, von Willebrand factor, sVCAM-1 and Creactive protein) to the mortality risk during nearly 10 years of follow up. Cox’s proportional hazards model was used to study the association of diabetes duration and mortality. Results The age- and sex-adjusted relative risks of mortality were 2·06 (95% C.I. 1·04‐ 4·10) and 3·19 (1·64‐6·20) for the patients with short- and long-term diabetes compared with the screening-detected diabetic subjects, respectively. Adjustment for cardiovascular risk factors resulted in a reduction of mortality risk in both groups: 1·13 (0·51‐2·50) and 2·39 (1·18‐4·83), respectively. Mortality risk significantly increased with increasing diabetes duration, even after multiple adjustment ( P -value for trend ranged from < 0·001‐0·018). Conclusions Mortality risk increased with increasing diabetes duration. In subjects with short diabetes duration the mortality risk could largely be attributed to other risk factors. In subjects with a longer diabetes duration, however, the elevated mortality risk was independent of these cardiovascular risk factors.

Background Amadori-albumin, a major glycated protein, is involved in experimental hyperglycaemia-induced microvascular complications, and is associated with advanced nephropathy in Type I diabetic patients in humans. Our aim was to assess the association of Amadori-albumin with early nephropathy and with retinopathy in Type I diabetic patients and the involvement of chronic low-degree inflammation therein. Design and Methods Amadori-albumin, the Amadori product of haemoglobin (HbA1c), C-reactive protein, and fibrinogen levels were measured in the EUCLID study, a 2-year randomised, double-blind, placebo-controlled trial of lisinopril in 447 Type I diabetic patients. Retinal photographs were taken in 341 patients at baseline and 294 at follow up. Results Amadori-albumin was positively associated with albumin the excretion rate and retinopathy status (P = 0·0001 and P = 0·02 for trend, respectively) and with the progression from normoalbuminuria to (micro)albuminuria (38·6 U mL−1 in nonprogressors, 44·3 U mL−1 in progressors; P = 0·02), but not with the development or progression of retinopathy during a 2-year follow up. Amadori-albumin levels at baseline were associated with C-reactive protein and fibrinogen (P = 0·0007 and P = 0·0001, respectively). C-reactive protein and fibrinogen were also associated with albumin excretion rates (P = 0·03 and P = 0·01, respectively) and retinopathy status (P = 0·02 and P = 0·0006, respectively). Adjustment for these inflammatory markers did not markedly attenuate the association between Amadori-albumin and the albumin excretion rate, while adjustment for fibrinogen, but not C-reactive protein, abolished the association between Amadori-albumin and retinopathy. Lisinopril had no impact on the association between the levels of Amadori-albumin and albumin excretion rates or retinopathy. Conclusions Amadori-albumin was associated with early nephropathy and with retinopathy in Type I diabetic patients and preceded an increase in albumin excretion rate, but not retinopathy. A chronic low-degree inflammation does not appear to be involved in Amadori-albumin-associated microvascular complications in Type I diabetes.

Increased plasma levels of soluble adhesion molecules are associated with an increased risk of atherothrombosis. The pathophysiological mechanisms responsible for these associations are not known. The aim of the present study was to investigate the association of soluble intercellular adhesion molecule-1 (sICAM-1) concentration and risk of cardiovascular and all-cause mortality among individuals with and without type 2 diabetes. In addition, we assessed potential pathophysiological mechanisms by which sICAM-1 may promote mortality. Six hundred and thirty-one subjects taken from a general population of the middle-aged and elderly participated in this prospective cohort study. Baseline data collection was performed from 1989 to 1992; subjects were followed until 1 January 2000. Subjects who died had higher levels of sICAM-1 than those who survived (506(164) vs. 477(162) ng mL(-1), respectively). After adjustment for age, gender and glucose tolerance status, subjects with sICAM-1 levels in the upper quartile (> or =550 ng mL(-1)) had a relative risk of cardiovascular mortality of 2.05 (95% confidence interval, 1.10-3.81) compared to subjects with sICAM-1 levels in the other quartiles. Further adjustment for classical cardiovascular risk factors or indicators of (sub)clinical atherosclerosis, endothelial dysfunction, inflammation and renal function did not materially alter this relative risk. A high sICAM-1 level was more frequent in subjects with type 2 diabetes than in subjects with a normal glucose tolerance (33.3 vs. 17.8%). Individuals with a plasma concentration of sICAM-1 higher than 550 ng mL(-1) had a cardiovascular mortality risk that was twice that of individuals with a lower concentration. Classical cardiovascular risk factors (sub)clinical atherosclerosis, endothelial dysfunction and inflammation do not explain this excess risk.

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-α (TNF-α) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-α was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the α(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against α(v)β3 and α(v)β5-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (C) 2000 by The American Society of Hematology. Chemicals/CAS: fibrin, 9001-31-4; phorbol 13 acetate 12 myristate, 16561-29-8; plasmin, 9001-90-5, 9004-09-5; plasminogen activator, 9039-53-6; tissue plasminogen activator, 105913-11-9; urokinase, 139639-24-0; vasculotropin, 127464-60-2; Antigens, CD; Culture Media; Culture Media, Conditioned; DNA, Complementary; Endothelial Growth Factors; Fibrin, 9001-31-4; Fibroblast Growth Factor 2, 103107-01-3; Integrin alphaV; integrin alphaVbeta5; Integrins; Lymphokines; Oxygen, 7782-44-7; Plasmin, EC 3.4.21.7; plasminogen activator, urokinase receptors; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases, EC 2.7.1.112; Receptors, Cell Surface; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor, EC 2.7.1.112; Receptors, Vitronectin; RNA, Messenger; Tumor Necrosis Factor; Urinary Plasminogen Activator, EC 3.4.21.73; Vascular Endothelial Growth Factor Receptor-1, EC 2.7.1.112; vascular endothelial growth factor

Background: Hyperhomocysteinaemia may constitute an independent risk factor for cardiovascular disease, but it is still unclear by which pathophysiological mechanisms homocysteine (tHcy) may promote atherothrombosis. The aim of this study was firstly to examine whether tHcy is associated with endothelial dysfunction, increased adherence of leukocytes, and/or chronic low-grade inflammation, as estimated from plasma levels of von Willebrand factor (vWf), soluble vascular cell adhesion molecule 1 (sVCAM-1) and C-reactive protein (CRP), respectively. Secondly we investigated whether the presence of type 2 diabetes modifies these associations. Materials and Methods: Six hundred and ten subjects of a general population of middle-aged and elderly subjects, 170 of whom had type 2 diabetes, participated in this cross-sectional study. Linear regression analyses were used to study whether tHcy was associated with vWf, sVCAM-1 and CRP, and whether the presence of diabetes modified these associations. Results: After adjustment for confounders, tHcy was significantly but weakly associated with vWf (β=0·15, P=0·05) and sVCAM-1 (β=0·082, P=0·04). tHcy was not significantly associated with CRP (β=0·02, P=0·91). The presence of diabetes did not significantly modify these associations. Conclusions: This study provides evidence that tHcy is, at most, weakly associated with endothelial dysfunction as estimated from plasma vWf, and with leukocyte adhesion as estimated from plasma sVCAM-1. tHcy was not significantly associated with chronic low-grade inflammation as estimated from plasma CRP. Our data thus suggest that the link between tHcy and atherothrombosis cannot be explained by associations of tHcy with vWf, sVCAM-1 or CRP. Chemicals/CAS: C-Reactive Protein, 9007-41-4; Cell Adhesion Molecules; Homocysteine, 454-28-4; von Willebrand Factor

The hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children. The role of a verocytotoxin (VT)-producing Escherichia coli has been strongly implicated in the epidemic form of HUS. Although direct toxicity of VT on glomerular endothelial cells has been demonstrated, it remained still unclear how the VT is transported from the intestine to the target organs. In this study we demonstrate that VT, when incubated in whole blood, binds rapidly and completely to human polymorphonuclear leukocytes (PMNs) and not to other components of blood. Binding studies with 125I-VT-1 showed a single class of binding sites on freshly isolated, non-stimulated human PMNs. The K(d) of VT-binding to PMNs was 10-8 mol/L, 100-fold less than that of the VT-receptor globotriaosylceramide. On incubation of VT-preloaded PMNs with human glomerular microvascular endothelial cells (GMVECs), transfer of VT-1 to the endothelial cells occurred. Incubation of non-stimulated GMVECs with VT- preloaded PMNs, but not with PMNs or VT-1 alone, caused inhibition of protein synthesis and cell death. Our data are in concert with a role of PMNs in the transfer of VT from the intestine to the kidney endothelium. This transfer occurs by selective binding to a specific receptor on PMNs and subsequent passing of the ligand VT to the VT-receptor on GMVECs, which causes cell damage. This new mechanism further underpins the important role of PMNs in HUS. (C) 2000 by The American Society of Hematology.

The retinal pigment epithelium (RPE) maintains the choriocapillaris (CC) in the normal eye and is involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Vascular endothelial growth factor-A (VEGF) is produced by differentiated human RPE cells in vitro and in vivo and may be involved in paracrine signaling between the RPE and the CC. We investigated whether there is a polarized secretion of VEGF by RPE cells in vitro. Also, the localization of VEGF receptors in the human retina was investigated. We observed that highly differentiated human RPE cells, cultured on transwell filters in normoxic conditions, produced two- to sevenfold more VEGF toward their basolateral side as compared to the apical side. In hypoxic conditions, VEGF-A secretion increased to the basal side only, resulting in a three- to 10-fold higher basolateral secretion. By immunohistochemistry in 30 human eyes and in two cynomolgus monkey eyes, KDR (VEGFR-2) and flt-4 (VEGFR-3) were preferentially localized at the side of the CC endothelium facing the RPE cell layer, whereas flt-1 (VEGFR-1) was found on the inner CC and on other choroidal vessels. Our results indicate that RPE secretes VEGF toward its basal side where its receptor KDR is located on the adjacent CC endothelium, suggesting a role of VEGF in a paracrine relation, possibly in cooperation with flt-4 and its ligand. This can explain the known trophic function of the RPE in the maintenance of the CC and its fenestrated permeable phenotype and points to a role for VEGF in normal eye functioning. Up-regulated basolateral VEGF secretion by RPE in hypoxia or loss of polarity of VEGF production may play a role in the pathogenesis of choroidal neovascularization.

Angiogenesis, the formation of new capillary blood vessels, is a feature of a variety of pathological processes. To study the effects of a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor-α. This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and tumor stroma. Our results show that testosterone and dexamethasone are strong inhibitors and all-trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) are potent stimulators of the formation of capillary-like tubular structures. These effects are mediated by their respective nuclear hormone receptors as demonstrated by the use of specific synthetic receptor agonists and antagonists. 17β-estradiol, progesterone, and 1,25-dihydroxyvitamin D3 did not affect or only weakly affected in vitro angiogenesis, which may be related to the lack of significant nuclear receptor expression. Although hMVEC express both thyroid hormone receptors α and β, no effect of thyroid hormone on tube formation was found. The effects of testosterone, dexamethasone,at-RA, and 9-cis RA on tube formation were accompanied by parallel changes in urokinase-type plasminogen activator (u-PA) expression, at both mRNA and antigen levels. Exogenous suppletion of the medium with single chain u-PA enhances tube formation in our in vitro model, whereas quenching of u-PA activity (but not of tissue-type plasminogen activator activity) or of u-PA binding to u-PA receptor by specific antibodies suppressed basal and retinoid-stimulated tube formation. Moreover, addition of scu-PA to testosterone- or dexamethasone-treated hMVEC restored the suppressed angiogenic activity for a substantial part. Aprotinin, an inhibitor of plasmin activity, completely inhibited tube formation, indicating that the proteolytic properties of the u-PA/u-PA receptor complex are crucial in this process. Our results show that steroid hormones (testosterone and dexamethasone) and retinoids have strong, but opposite effects on tube formation in a human in vitro model reflecting pathological angiogenesis in the presence of fibrin and inflammatory mediators. These effects can be explained by hormone-receptor–mediated changes in u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.© 1998 by The American Society of Hematology.

SummaryDysfunction of the vascular endothelium is considered an early step in the development of diabetic angiopathy. Hyperglycaemia results in endothelial dysfunction, both through direct effects of glucose and through formation of advanced glycosylation end-products (AGEs). We hypothesized that the effects of glucose and AGEs on endothelial function in insulin-dependent diabetes mellitus (IDDM) are distinct and are reflected by distinct plasma markers of endothelial function. We therefore measured plasma levels of von Willebrand factor (vWF), soluble (s) E-selectin and vascular cell adhesion molecule-1 (sVCAM-1), and evaluated the relationship with HbA1c and urinary excretion of pentosidine, an AGE product, in 56 patients with IDDM. Urinary pentosidine excretion was higher in the diabetic than in a control group (n = 60) of similar age (P

Objectives. To compare percutaneous transluminal coronary angioplasty (PTCA) and stent implantation with respect to the long-term changes they induce in the newly formed endothelium in porcine coronary arteries by studying both morphological and functional parameters of the endothelium at 2 weeks and 3 months after intervention.Background. Problems affecting PTCA or stent implantation have been overcome to a large extent by means of better techniques and the availability of new drugs. Late problems, however, still exist in that restenosis affects a large number of patients. With an increasing number of patients being treated with stents, the problem of in-stent restenosis is of even greater concern, as this seems difficult to treat. A functional endothelial lining is thought to be important in controlling the growth of the underlying vascular tissue. We hypothesized that the enhanced neointimal hyperplasia observed after stenting is associated with a more pronounced and prolonged endothelial dysfunction.Methods. Arteries were analyzed using a dye-exclusion test and planimetry of permeable areas. Thereafter, the arteries were processed for light and scanning electron microscopy for assessment of morphology and proliferative response.Results. Leakage of the endothelium for molecules such as Evans blue-albumin as well as prolonged endothelial proliferation is observed as late as 3 months after the intervention, and is more pronounced after stenting. Permeability is associated with distinct morphologic characteristics: endothelial retraction, the expression of surface folds, and the adhesion of leukocytes.Conclusions. Stenting especially decreases long-term vascular integrity with respect to permeability and endothelial proliferation, and is associated with distinct morphologic characteristics.

Acute renal failure is one of the hallmarks of the hemolytic uremic syndrome (HUS). Infection with a verocytotoxin (VT)- or Shiga-like toxin (SLT)-producing Escherichia coli has been strongly implicated in the etiology of the epidemic form of HUS. The functional receptor for these closely related toxins appears to be a glycosphingolipid, globotriaosylceramide (Gb3). Endothelial damage in the glomeruli and arterioles of the kidney induced by VT is believed to play a crucial role in the pathogenesis of HUS. However, little information is available regarding the effects of VT on mesangial cells, which also play an important role in glomerular function. In this study, the effects of VT on human mesangial cells in vitro were investigated. Mesangial cells were enriched by collecting hillock-shaped outgrowths derived from adult human glomeruli and subsequently purified by elimination of contaminating epithelial cells by immunoseparation with ulex europaeus lectin-I (UEA-I)-coated dynabeads. The obtained and subcultured mesangial cell populations were98% pure. Their mesangial nature was established by the presence of a-smooth muscle cell actin in highly confluent cultures and the absence of cytokeratin or platelet/endothelial cell adhesion molecule-1. Mesangial cells bound VT to bands of Gb3 and a closely related glycolipid, which is similar to a glycolipid involved in the VT-dependent cytokine production in monocytes. VT did not induce the release of cytokines or chemokines in mesangial cells. In VT-susceptible cells, binding of VT to Gb3 causes cell death by the inhibition of protein synthesis. Although protein synthesis was inhibited in mesangial cells, all cells remained viable, both under basal and tumor necrosis factor-alpha-stimulated conditions. However, the marked reduction in protein synthesis may impair a proper response of the cells in conditions of increased demand of newly synthesized proteins. Furthermore, VT markedly inhibited DNA synthesis and proliferation of mesangial cells. The inhibition of mitogenesis was also found with the B-subunit of VT-1 alone, albeit to a lesser extent, without a significant effect on protein synthesis. Because the inhibition of protein synthesis involves the A-subunit, this suggests that two distinct mechanisms contribute to the effects of VT on protein synthesis and mitogenesis. Intracellular routing of VT (A- and B-subunits) may vary between cell types and result in differential effects on human mesangial cells when compared with other cell types.

Objective and Methods: To review, from the clinical perspective, the contribution of dysfunction of the vascular endothelium to the pathogenesis of diabetic micro- and macroangiopathy. Results: Available data indicate that endothelial dysfunction in diabetes complicated by micro- or macroalbuminuria (renal microangiopathy) is generalised. The close linkage between microalbuminuria and endothelial dysfunction is an attractive explanation for the fact that microalbuminuria is a risk marker for atherosclerotic cardiovascular disease in diabetes. Endothelial dysfunction precedes the occurrence of even early diabetic microangiopathy. However, it is not clear whether endothelial dysfunction is a feature of the diabetic state per se or whether additional factors are required to induce endothelial dysfunction given the presence of diabetes. Convincing data from animal and in vitro models exist to indicate that endothelial dysfunction in diabetes may be related to hyperglycaemic pseudohypoxia, activation of protein kinase C, increased expression of transforming growth factor-β and vascular endothelial growth factor, non-enzymatic glycation, oxidative stress, activation of the coagulation cascade, increased expression of tumour necrosis factor-α, and high levels of insulin and insulin precursor molecules. However, the importance of these proposed mechanisms have not yet been extensively assessed in diabetes in man. Conclusions: Endothelial dysfunction plays a key role in the pathogenesis of diabetic angiopathy in man. The biochemical basis of endothelial dysfunction in diabetic man, however, has yet to be fully elucidated.

IL-6 −/− mice showed impaired leukocyte accumulation in subcutaneous air pouches. Defective leukocyte accumulation was not due to a reduced migratory capacity of IL-6 −/− leukocytes and was associated with a reduced in situ production of chemokines. These observations led to a reexamination of the interaction of IL-6 with endothelial cells (EC). EC express only the gp130 signal transducing chain and not the subunit-specific IL-6R and are therefore unresponsive to IL-6. However, EC are responsive to a combination of IL-6 and soluble IL-6R as measured by the activation of STAT3, chemokine expression, and augmentation of ICAM-1. Activation by IL-6–IL-6R complexes was inhibited by an IL-6 receptor antagonist and potentiated by a superagonist. Hence, in vivo and in vitro evidence supports the concept that the IL-6 system plays an unexpected positive role in local inflammatory reactions by amplifying leukocyte recruitment.

Effects of TNFα and verocytotoxin cytotoxicity in human glomerular microvascular endothelial cells. In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelial damage of glomeruli and arterioles of the kidney appears to play a central role. Previous studies have shown that verocytotoxin-1 (VT-1) cytotoxicity on human vein endothelial cells require additional stimuli, in particular the inflammatory mediator tumor necrosis factor alpha (TNFα). In this study the effects of VT on human glomerular microvascular endothelial cells (GMVEC) were examined. A reproducible method was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their endothelial origin was demonstrated by the expression of the endothelial antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNFα the cells expressed the endothelial-specific adhesion molecule E-selectin. A limited number of fenestral structures was observed by scanning electron microscopy (SEM), suggesting glomerular origin of the endothelial cells. Cytotoxicity of VT-1 to GMVEC was evaluated by determination of the number of viable adherent cells and by assay of overall protein synthesis after exposure to varying concentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 was inversely related to the degree and duration of confluence, subconfluent cells being the most sensitive. In highly confluent GMVEC, VT cytotoxicity required pre-exposure of the cells to the inflammatory mediator TNFα, which induced an increase in the number of VT receptors on GMVEC. Thin layer chromatography of extracted glycolipids from the GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known to be the functional receptor for VT. There were no major differences in protein synthesis inhibition with equal concentrations VT-1 and VT-2. In conclusion, in this study we provide a reproducible method to isolate, purify and culture well characterized human GMVEC on a routine basis. In vitro studies with these GMVEC demonstrate that VT cytotoxicity depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNFα. These observations provide further insight into the complex events that may occur in glomeruli in the pathogenesis of HUS.

Objective We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S -Adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent. Methods and results Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l -Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l -Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l -Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47 phox and nitrotyrosine, and H 2 O 2 . Both Hcy and ADA significantly increased PS exposure ( n = 5), caspase-3 activity ( n = 6) and cytochrome c release ( n = 3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47 phox expression and local reactive oxygen species (ROS) ( n = 3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction ( n = 3) and ROS production ( n = 3). Conclusion SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.

Vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors are effective agents in the treatment of metastatic renal cell cancer (mRCC). We here investigated whether inhibition of VEGFR signalin by sunitinib causes changes in plasma proteins associated with tumor endothelium. Forty-three patients with mRCC received sunitinib 50 mg/day in a 4-weeks on 2-weeks off schedule. Sequential plasma samples were obtained before treatment (C1D1), on C1D14, on C1D28, and on C2D1 before start of cycle 2. Plasma levels were assessed for VEGF, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular cell adhesion molecule-1 (sICAM-1), von Willebrand factor (vWF), circulating angiopoietin-2 (Ang-2) and soluble Tie-2 (sTie-2). Total tumor burden was calculated at baseline and at first evaluation. Progression-free survival (PFS) and overall survival (OS) were determined. Tumor burden was positively associated with baseline circulating Ang-2 [Spearman's rho (ρ) = 0.378, p = 0.028] and vWF (ρ = 0.417, p = 0.008). During sunitinib treatment, circulating Ang-2 and sTie-2 significantly decreased (p < 0.001 for both), plasma levels of sVCAM-1 and VEGF significantly increased (p = 0.022 and p < 0.001), whereas those of sICAM-1 and vWF remained stable. These protein changes had recovered on C2D1. The reduction in circulating Ang-2 levels on C1D28 was positively correlated with the percentage decrease in tumor burden (ρ = 0.605; p = 0.002). Baseline protein levels and subsequent changes were not associated with PFS or OS. In conclusion, sunitinib-induced changes in Ang-2, sTie-2, sVCAM-1 and VEGF are related to the administration schedule, while reduction in Ang-2 is also associated with decrease in tumor burden.

Objectives: Arterial pressure induced vein graft injury can result in endothelial loss, accelerated atherosclerosis and vein graft failure. Inflammation, including complement activation, is assumed to play a pivotal role herein. Here, we analyzed the effects of C1-esterase inhibitor (C1inh) on early vein graft remodeling. Methods: Human saphenous vein graft segments (n = 8) were perfused in vitro with autologous blood either supplemented or not with purified human C1inh at arterial pressure for 6 h. The vein segments and perfusion blood were analyzed for cell damage and complement activation. In addition, the effect of purified C1inh on vein graft remodeling was analyzed in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery. Results: Application of C1inh in the in vitro perfusion model resulted in significantly higher blood levels and significantly more depositions of C1inh in the vein wall. This coincided with a significant reduction in endothelial loss and deposition of C3d and C4d in the vein wall, especially in the circular layer, compared to vein segments perfused without supplemented C1inh. Administration of purified C1inh significantly inhibited vein graft intimal thickening in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery. Conclusion: C1inh significantly protects against early vein graft remodeling, including loss of endothelium and intimal thickening. These data suggest that it may be worth considering its use in patients undergoing coronary artery bypass grafting. (C) 2011 Elsevier Ireland Ltd. All rights reserved

A small amount of adipose tissue associated with small arteries and arterioles is encountered both in mice and man. This perivascular adipose tissue (PVAT) has a paracrine effect on the vascular tone regulation. PVAT is expanded in obesity and in diabetes. This expansion not only involves enlargement of fat cells, but also the accumulation of inflammatory cells and a shift in the production of adipokines and cytokines. This effect is illustrated in this review by the effect of PVAT-derived factors of insulin-mediated vasoregulation in mouse resistance arteries. Insulin sensitivity of endothelial cells is also involved in the insulin-mediated regulation of muscle glucose uptake. Insulin affects vasoregulation by acting on different signaling pathways regulating NO and endothelin-1 release. This process is influenced by various adipokines and inflammatory mediators released from PVAT, and is affected by the degree of expansion and content of inflammatory cells. It is modulated by adiponectin (via 5' adenosine monophosphate-activated protein kinase, AMPK), TNFα (via c-jun N-terminal kinase) and free fatty acids (via protein kinase C-θ). PVAT thus provides an important site of control of vascular (dys)function in obesity and type 2 diabetes. An altered profile of adipokine and cytokine production by PVAT of resistance arteries may also contribute to or modulate hypertension, but a causal role in hypertension has still to be established.

Fibrinolytic parameters and von Willebrand factor (vWF) antigen were measured in the plasma of 10 patients with hemolytic uremic syndrome (HUS). Samples were taken at presentation and again 2 wk later, before and after infusion of 1-desamino-8-arginine vasopressin. Compared with the plasma values of healthy control children, levels of tissue-plasminogen activator (t-PA) antigen, plasminogen activator inhibitor type I (PAI-1) activity, and vWF as well as fibrin(ogen) degradation products were significantly elevated in the plasma of HUS patients on admission. No response of the fibrinolytic parameters and vWF were seen when 1-desamino-8-arginine vasopressin infusion was given on admission. After 2 wk, t-PA antigen and vWF had partially returned to basal values, and t-PA antigen increased rapidly again after 1- desamino-8-arginine vasopressin infusion. To investigate whether verocytotoxin contributes to the alteration of the fibrinolytic system found in HUS patients, purified verocytotoxin-1 (VT-1) was added to the media of cultured human endothelial cells. Addition of VT-1 alone did not change the production of t-PA, plasminogen activator inhibitor type I, and vWF antigen in these cells. However, when the endothelial cells were preincubated with tumor necrosis factor-α to increase the number of VT-1 receptors, VT-1 induced a marked decrease of the synthesis of t-PA, plasminogen activator inhibitor type I, and vWF. This was caused by a decrease in overall protein synthesis in the tumor necrosis factor-α- and VT-1-treated endothelial cells. We conclude from this study that the systemic fibrinolytic parameters measured in the plasma of HUS patients are probably not a direct effect of VT-1 on the endothelium but are sequelae of the disease in which the intestine and the kidney are predominantly affected. Chemicals/CAS: argipressin[1 deamino], 113-81-5; plasminogen activator inhibitor 1, 140208-23-7; tissue plasminogen activator, 105913-11-9; verotoxin, 108066-86-0; von Willebrand factor, 109319-16-6; Bacterial Toxins; Desmopressin, 16679-58-6; Fibrin Fibrinogen Degradation Products; Plasminogen Activator Inhibitor 1; Shiga-Like Toxin I; Tissue Plasminogen Activator, EC 3.4.21.68; Tumor Necrosis Factor; von Willebrand Factor

To evaluate the diagnostic value of plasma protein levels for pulmonary vascular permeability and acute respiratory distress syndrome. During acute lung injury and acute respiratory distress syndrome, increased vascular permeability induces protein-rich fluid extravasation. We hypothesized that plasma protein levels predict increased vascular permeability and acute respiratory distress syndrome.A prospective, observational study.Eighty-three consecutive, mechanically ventilated patients with or at risk for acute lung injury/acute respiratory distress syndrome, of whom 18 had sepsis. Patients with increased pulmonary capillary wedge pressures or central venous pressures were excluded.Patients were subjected to pulmonary capillary wedge pressure/central venous pressure-guided fluid loading with saline or colloid fluids.We measured plasma albumin and transferrin levels and determined the Gallium-transferrin pulmonary leak index, the American European Consensus Conference criteria, and the lung injury score. Measurements were performed before and after fluid loading to evaluate effects of fluid loading. Plasma albumin and transferrin levels were approximately 30% lower in acute respiratory distress syndrome than patients with acute lung injury (p.01) and patients without lung injury (p.05). Protein levels inversely related to the pulmonary leak index (standardized regression coefficient -0.28, p.001 for albumin; standardized regression coefficient -0.30, p = .003 for transferrin) and the lung injury score (standardized regression coefficient -0.19, p = .01 for albumin), independently of presence of sepsis, severity of disease, and fluid loading. Albumin and transferrin levels had a high sensitivity (77-93%) and negative predictive value (80-98%) for elevated pulmonary vascular permeability and acute respiratory distress syndrome (American European Consensus Conference criteria and lung injury score). The addition of hypoalbuminemia (17.5 g/L) and hypotransferrinemia (0.98 g/L) as criteria to the American European Consensus Conference criteria or the lung injury score increased their predictive values for elevated pulmonary vascular permeability.In critically ill patients, decreased plasma albumin and transferrin levels parallel increased pulmonary vascular permeability irrespective of underlying disease and fluid status. While normal levels help to exclude acute respiratory distress syndrome, hypoalbuminemia and hypotransferrinemia increase the diagnostic accuracy of the American European Consensus Conference criteria and lung injury score for elevated pulmonary vascular permeability.