Endothelial Dysfunction Disease Research Articles

A High-Efficiency AAV for Endothelial Cell Transduction Throughout the Central Nervous System.

Endothelial cells have a crucial role in nervous system function, and mounting evidence points to endothelial impairment as a major contributor to a wide range of neurological diseases. However, tools to genetically interrogate these cells in vivo remain limited. Here, we describe AAV-BI30, a capsid that specifically and efficiently transduces endothelial cells throughout the central nervous system. At relatively low systemic doses, this vector transduces the majority of arterial, capillary, and venous endothelial cells in the brain, retina, and spinal cord vasculature of adult C57BL/6 mice. Furthermore, we show that AAV-BI30 robustly transduces endothelial cells in multiple mouse strains and rats in vivo and human brain microvascular endothelial cells in vitro. Finally, we demonstrate AAV-BI30’s capacity to achieve efficient and endothelial-specific Cre-mediated gene manipulation in the central nervous system. This combination of attributes makes AAV-BI30 uniquely well-suited to address outstanding research questions in neurovascular biology and aid the development of therapeutics to remediate endothelial dysfunction in disease.

Regulation of vascular function and blood pressure by circadian variation in redox signalling.

There is accumulating evidence that makes the link between the circadian variation in blood pressure and circadian variations in vascular contraction. The importance of vascular endothelium-derived redox-active and redox-derived species in the signalling pathways involved in controlling vascular smooth muscle contraction are well known, and when linked to the circadian variations in the processes involved in generating these species, suggests a cellular mechanism for the circadian variations in blood pressure that links directly to the peripheral circadian clock. Relaxation of vascular smooth muscle cells involves endothelial-derived relaxing factor (EDRF) which is nitric oxide (NO) produced by endothelial NO synthase (eNOS), and endothelial-derived hyperpolarising factor (EDHF) which includes hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox). Both of these enzymes appear to be under the direct control of the circadian clock mechanism in the endothelial cells, and disruption to the clock results in endothelial and vascular dysfunction. In this review, we focus on EDRF and EDHF and summarise the recent findings on the influence of the peripheral circadian clock mechanism on processes involved in generating the redox species involved and how this influences vascular contractility, which may account for some of the circadian variations in blood pressure and peripheral resistance. Moreover, the direct link between the peripheral circadian clock and redox-signalling pathways in the vasculature, has a bearing on vascular endothelial dysfunction in disease and aging, which are both known to lead to dysfunction of the circadian clock.

Role of Circulating Endothelial Progenitor Cells in Micro VascularDamage in Sickle Cell Patients

The vascular wall participates in the pathogenesis of sickle cell disease (SCD). Circulating Endothelial progenitor cells (cEPCs) also play a key role in the vascular pathology of SCD, including the painful crisis. In previous investigations, reduced levels of cEPCs were found in conditions in which vascular injury is implicated such as myocardial infarction. The aim of this research is to study the role of cEPCs in the vascular pathology of SCD. cEPCs were enumerated using flow cytometry with the Flow Activated Cell Sorting machine (FACS). To differentiate the various genotypes-SS, SC, AS, AC and AA, Hb Electrophoresis was employed. Results showed that the median % cEPCs (CD34+/VEGF-2+) was lower in patients with SCD [0.555(0.4, 0.765)] than in healthy controls [(1.08(0.87, 1.39) (p=0.001)]. Patients in crisis had a higher cEPCs (0.65+0.39) than those in steady state (0.59+0.28) (p=0.522). SS group recorded the highest mean GGT (73.66+73.35). Only total cholesterol demonstrated a positive correlation (r=0.378, p=0.00814) with cEPCs in subjects; a trend unseen in healthy controls. Patients’ WBC, Hb and Liver enzymes- ALT, GGT, ALP showed no correlation with cEPCs. In healthy controls though, WBCs showed an inverse correlation with cEPCs (r=-0.6293, p=0.0003). SCD is as much a disease of endothelial dysfunction as it is a hemoglobinopathy that triggers erythrocyte polymerization: cEPC is a surrogate bio-marker for vascular function in SCD patients. The results suggest that SCD patients have depleted cEPCs compared with healthy controls. Oxidative stress, Nitric oxide activity, loss of CD133 during homing could influence progenitor cell populations. Total cholesterol positively correlates with cEPCs. High total cholesterol could spell the onset of painful crisis. Liver enzymes are not related to cEPCs correlatively even though the liver is involved in endothelial injury in SCD patients. SS individuals tend to have a high GGT. Overall, we have shown the direct correlation between total cholesterol and cEPCs in sickle cell patients.

Role of the endothelial glycocalyx in kidney disease and vascular dysfunction

BackgroundAlbuminuria, an early marker of kidney disease, is strongly and independently associated with adverse cardiovascular outcomes, including heart attacks and strokes. Nonetheless, the mechanisms linking albuminuria and adverse cardiovascular events remain unclear. The endothelial glycocalyx, a friable but protective lining of all blood vessels, is damaged in both atherosclerosis and kidney disease. We examined whether factors in the plasma of patients with kidney disease could damage the endothelial glycocalyx, and sought underlying mechanisms. MethodsMetabolic labelling of glycosaminoglycans (GAGs) was achieved by culturing human glomerular endothelial cells in media containing 20 μCi/mL D-[3H3] glucosamine for 24 h. Monolayers were washed and subsequently exposed either to conditioned serum free media containing 10% plasma from patients with either heavy albuminuria (from an intrinsic renal disease) or from healthy controls for 24h, or to 100 μM hydrogen peroxide or vehicle for 4 h. The supernatant was then collected and applied to a di-ethyl-amino-ethyl fast flow column connected to a fast protein liquid chromatography system. Samples were applied to the column at 1 mL/min and subsequently merged with column matrix for 15 min. Bound proteoglycans were eluted with 7M urea (pH 6) and collected in 1 mL fractions. All fractions were quantified by liquid scintillation counting. FindingsExposure of glomerular endothelial cells to plasma from three patients with albuminuria resulted in reduced levels of GAGs into the media (control plasma 112 514 disintegrations per min (DPM) [SE 6153], albuminuric plasma 74 743 DPM [9607]; p<0·05 unpaired t test), indicating altered endothelial cell GAG metabolism that might represent decreased turnover. Because oxidative stress is regarded as a key mediator of endothelial dysfunction in kidney disease, we generated reactive oxygen species by exposing cells to hydrogen peroxide. This exposure resulted in a different pattern of GAG turnover: shedding of radiolabelled GAGs into the media was increased (vehicle 1302 [684] DPM D-[3H3] glucosamine-labelled heparan sulphate GAG, hydrogen peroxide 25 053 [6635] DPM D-[3H3] glucosamine-labelled heparan sulphate GAG; p<0·005 unpaired t test). InterpretationDamage to the endothelial glycocalyx in response to factors in albuminuric plasma provides a novel concept in understanding endothelial dysfunction in disease states. This work provides evidence that the albuminuric state itself might lead to systemic alterations in endothelial cell function, although our preliminary studies indicate that reactive oxygen species are unlikely to be involved in these changes. We are now seeking alternative pathways underlying the effects of albuminuric plasma on the endothelial glycocalyx, assessing whether the endothelial glycocalyx is damaged in blood vessels in situ in patients with kidney disease, and examining whether the endothelial glycocalyx can be restored (and endothelial cell function improved) by manipulating these pathways. Our long-term goal is to manipulate endothelial glycocalyx regulation to develop an entirely new class of treatments for vasculoprotection in kidney disease and other conditions. FundingNational Institute for Health Research.

Sickle cell disease: looking back but towards the future

Sickle cell disease (SCD) is a multisystem disease associated with episodes of acute illness and progressive organ damage, and one of the most common monogenic disorders worldwide, affecting an estimated 30 million people. SCD represents a major public health problem because of its associated morbidity and mortality(1,2). Last year marked the 100th year since SCD was first described by James B. Herrick. The myriad clinical features of this hemolytic anemia result from a deceptively simple amino acid substitution of valine for glutamic acid in the sixth position of the s-subunits of hemoglobin (Hb). The result is intracellular polymerization under conditions of hypoxia and dehydration, which triggers the characteristic sickle-shaped erythrocytes(3). Modern advances in molecular and cellular biology have resulted in an accumulation of data on the sickle cell pathophysiology related to erythrocyte and extra-erythrocyte events(1). Since the seminal discovery by Dr. Herrick, we have learnt that SCD is as much a disease of endothelial dysfunction as it is a hemoglobinopathy. Oxidative stress, chronic endothelial damage and hemolysis initiate a cascade of events that result in episodic vaso-occlusion, subsequent ischemia-reperfusion injury, inflammation and organ dysfunction(3).

Chronic activation of AMP‐activated protein kinase prevents 20‐hydroxyeicosatetraenoic acid‐induced endothelial dysfunction

1. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor involved in vascular dysfunction and blood pressure regulation. Studies have revealed strong associations between 20-HETE and endothelial dysfunction; however, the signalling mechanisms are largely unknown. Therefore, the aim of the present study was to investigate the effect of 20-HETE on the association between endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (Hsp90). 2. In mouse aortic rings, 20-HETE significantly enhanced the constriction to phenylephrine and inhibited the relaxation to acetylcholine (P=0.05 vs control rings). In mice with chronic AMP-activated protein kinase (AMPK) activation, this protected against the negative effects of 20-HETE (P<0.05). Immunoprecipitation of eNOS in human umbilical vein endothelial cells treated with 20-HETE revealed a decrease in basal and vascular endothelial growth factor-stimulated Hsp90 association with eNOS (P<0.05). Pretreatment of cells with 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR; a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. Treatment with 20-HETE for 24 h induced an increase in eNOS phosphorylation that was not seen following acute treatment (30 min). The increased eNOS phosphorylation was accompanied by transient changes in Akt phosphorylation. 3. In conclusion, 20-HETE impairs eNOS-Hsp90 association, which can be reversed by chronic activation of AMPK. This provides a mechanism for reduced nitric oxide bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.

Over‐expression of mitogen‐activated protein kinase phosphatase‐2 enhances adhesion molecule expression and protects against apoptosis in human endothelial cells

We assessed the effects of over-expressing the dual-specific phosphatase, mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2), in human umbilical vein endothelial cells (HUVECs) on inflammatory protein expression and apoptosis, two key features of endothelial dysfunction in disease. We infected HUVECs for 40 h with an adenoviral version of MKP-2 (Adv.MKP-2). Tumour necrosis factor (TNF)-α-stimulated phosphorylation of MAP kinase and protein expression was measured by Western blotting. Cellular apoptosis was assayed by FACS. Infection with Adv.MKP-2 selectively abolished TNF-α-mediated c-Jun-N-terminal kinase (JNK) activation and had little effect upon extracellular signal-regulated kinase or p38 MAP kinase. Adv.MKP-2 abolished COX-2 expression, while induction of the endothelial cell adhesion molecules, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), two NFκB-dependent proteins, was not affected. However, when ICAM and VCAM expression was partly reduced by blockade of the NFκB pathway, Adv.MKP-2 was able to reverse this inhibition. This correlated with enhanced TNF-α-induced loss of the inhibitor of κB (IκB)α loss, a marker of NFκB activation. TNF-α in combination with NFκB blockade also increased HUVEC apoptosis; this was significantly reversed by Adv.MKP-2. Protein markers of cellular damage and apoptosis, H2AX phosphorylation and caspase-3 cleavage, were also reversed by MKP-2 over-expression. Over-expression of MKP-2 had different effects upon the expression of inflammatory proteins due to a reciprocal effect upon JNK and NFκB signalling, and also prevented TNF-α-mediated endothelial cell death. These properties may make Adv.MKP-2 a potentially useful future therapy in cardiovascular diseases where endothelial dysfunction is a feature.

Abstract 224: 20-hydroxyeicosatetraenoic Acid Mediated Amp Activated Protein Kinase Suppression Induces Endothelial Nitric Oxide Synthase Uncoupling

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 product of arachidonic acid metabolism, is a potent vasoconstrictor thought to be involved in vascular dysfunction and the regulation of blood pressure. 20-HETE has been demonstrated to play an inhibitory role in eNOS activity and both animal and human studies have revealed strong associations between 20-HETE and endothelial dysfunction. However, the signalling mechanisms are largely unknown. Previous studies have shown that AMPK activity is involved in the Hsp90-eNOS association. Therefore we sought to investigate the effect of 20-HETE on eNOS and Hsp90 association, and whether this was mediated by AMPK. Treatment of human umbilical vein endothelial cells (HUVECs) with 10μM 20-HETE for 24h induces an increase in eNOS phosphorylation, which is not observed following acute treatment (30mins). This was accompanied by transient changes in Akt phosphorylation. Conversely, upstream AMPK activity increased rapidly following 20-HETE treatment (&lt;1min) before decreasing by 30mins. Treatment with 20-HETE also resulted in an acute increase in the production of reactive oxygen species (ROS), which returned to control levels at &gt;2hr post-20-HETE treatment. Immunoprecipitation of eNOS in cells treated with 20-HETE revealed a decrease in basal, VEGF and A23187 stimulated Hsp90 association with eNOS, indicating an uncoupling mechanism. Pre-treatment of the cells with AICAR (a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. To further investigate the functional effect of Hsp90 dissociation, we utilised an endothelial-smooth muscle cell system to measure cGMP production. Chronic 20-HETE treatment appeared to reduce both basal and VEGF stimulated cGMP levels. Pre-treatment of the cells with AICAR protected against the 20-HETE induced decrease in cGMP levels, both basally and following VEGF stimulation. In conclusion, 20-HETE suppression of AMPK activity impairs eNOS-Hsp90 association, providing a mechanism for reduced NO bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.

Characterization of the brachial artery shear stress following walking exercise

Habitual exercise provides repeated episodes of elevated vascular shear stress (SS), which may be a mechanism for repair of endothelial dysfunction in disease. Our aim was to determine the brachial artery SS during the 3-hour period following single bouts of low, moderate, and high-intensity walking exercise. In a randomized crossover design, 14 men walked for 45 minutes on a treadmill at 25%, 50% and 75% of VO2peak separated by 2-7 days. Using Doppler ultrasonography, brachial artery SS was assessed immediately after exercise and then hourly for 3 hours. High-intensity walking elicited greater (p<0.05) post-exercise SS compared with low and moderate intensity. In addition, a 3x4 (intensity x time) ANOVA indicated an absence of interaction (p=0.369) and a decline in post-exercise SS over time (p<0.0001) which was abolished after 2 hours. Thus, we found that brachial artery SS is greatest following high-intensity walking and that the rate of decline in SS is similar across all walking intensities.

Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis

Plasminogen activator (PA) inhibitor-1 (PAI-1) has been recognized as a surrogate marker of endothelial dysfunction in diseases associated with impaired angiogenesis, including atherosclerosis, diabetic vasculopathy, and nephropathy. To establish the necessary and sufficient components of the PA system [PAI-1, urokinase-type PA (uPA), or tissue-type PA (tPA), and plasminogen (Plg)] for angiogenesis, we examined angiogenic competence of vascular explant cultures obtained from mice deficient in PAI-1, tPA, uPA, and Plg. To gain insight into the requirement for different matrix-degrading systems during endothelial cell migration across plasmin-degradable basement membranes compared with profibrotic areas containing plasmin-nondegradable collagen, we contrasted vascular sprouting in collagen with Matrigel lattices. PAI-1(-/-) vessels showed an increased capillary sprouting in both collagen and Matrigel. Deficiency of uPA significantly reduced the rate of sprouting, whereas tPA(-/-) vessels showed a profound inhibition of capillary sprouting. The Plg(-/-) vessels failed to sprout, a defect that was restored not only by exogenous Plg, but also by the addition of PAs; a nonproteolytic effect of tPA was observed in Matrigel. Zymography revealed no differences in the activity of metalloproteinase (MMP)-2 and -9 in wild-type and PAI-1(-/-) vessels, but demonstrated reduced MMP-9 activity in all angiogenesis-deficient vessels. In summary, 1) PAI-1 by itself is a modest inhibitor of endothelial sprouting, 2) tPA and Plg are indispensable for angiogenesis in this model, 3) Plg is not the only substrate for PAs, and 4) the activity of MMP-9 is undetectable in explant cultures from tPA and Plg knockout mice.

Endothelial transport processes and tissue metabolism: evidence for microvascular endothelial dysfunction in insulin-resistant diseases?

Clinical manifestations of the insulin resistance syndrome are accompanied by endothelial dysfunction at the level of conductance and resistance vessels. Other conditions, such as smoking, also associated with endothelial dysfunction, exhibit insulin resistance too. Hypercholesterolaemia, in contrast, despite being the classical disease of endothelial dysfunction, is not associated with insulin resistance. Furthermore, only in insulin-resistant diseases, markers of microvascular alteration, such as microalbuminuria or increased plasma concentrations of von Willebrand factor, are found. At capillary sites, in contrast, transendothelial transport of both insulin and glucose takes place. These transport processes are thought to contribute to overall insulin sensitivity. In this article, I not only hypothesize but also summarize existing experimental and clinical evidence that a distinct capillary endothelial dysfunction is closely involved in the insulin resistance syndrome.

Endothelium-derived vasoactive factors and regulation of vascular tone in human blood vessels

The endothelium releases a variety of factors which can affect vascular tone. Endothelium-derived relaxing factor or nitric oxide is a very potent vasodilator and inhibitor of platelet function. Its release has been demonstrated in a variety of human blood vessels. In most human vascular preparations, prostacyclin does not significantly contribute to the endothelium-dependent relaxations. Prostacyclin is, however, an endothelium-derived product which can evoke vasodilation and inhibition of platelet aggregation. In addition, the endothelium of human veins can release endothelium-derived contracting factors produced by the cyclooxygenase pathway. Endothelin is an endothelium-derived vasoactive peptide which has profound vasoconstrictor properties in human arteries and particularly in veins. Its action can only be partially inhibited by calcium antagonists, while endothelium-derived nitric oxide and exogenous nitrovasodilators are effective antagonists of the peptide. The mechanisms and amounts of endothelin released in human blood vessels remains to be defined. Under physiological conditions, endothelium-derived relaxing factors appear to dominate. The release of endothelium-derived nitric oxide is reduced in atherosclerotic human arteries. This indicates that in cardiovascular disease endothelial dysfunction occurs; this may contribute in the pathogenesis of coronary artery disease, pulmonary hypertension and stroke.