Michael Wolzt

Ocular blood flow and associated functional deviations in diabetic retinopathy

Diabetic retinopathy is among the leading causes of blindness in the industrialised nations. The clinical course of diabetic retinopathy has been described in detail [1] and a grading system for the disease has been elaborated [2, 3]. The mechanisms underlying this severe complication of Type I (insulin-dependent) and Type II (non-insulin-dependent) diabetes mellitus are, however, still not clear. Whereas some of the mechanisms which lead to proliferation in the late stages of retinopathy have been explained, the early processes, which lead to the onset of the disease are still obscure. Several hypotheses have been proposed including biochemical dysfunction linked to hyperglycaemia, oxidative stress, humoral or genetic factors, thickening of the capillary basement membrane and rheological and haemodynamic factors. The concept that altered retinal blood flow may have a role in the development of diabetic retinopathy was emphasised more than 20 years ago. Since then haemodynamics of the retina in patients with diabetes mellitus as well as in animal models of diabetes has attracted considerable research interest. Much less attention has been paid to the choroidal circulation in diabetes, which is surprising as much of the retinal oxygen is supplied from this vascular bed. In part this may arise from the difficulties in assessing choroidal blood flow in humans. A role for the choroidal circulation in the pathogenesis of diabetic retinopathy has been proposed previously, based on histological studies of the diabetic choroid [4±6]. Excessive basement membrane thickening in the choroid has been shown in human and experimental diabetes [4, 5]. The exact nature of ocular blood flow abnormalities in the different stages of diabetic retinopathy is still a matter of controversy. This may at least partially be caused by the variety of techniques used to assess haemodynamics of the eye. Hence we will briefly review the different techniques used to assess ocular haemodynamics in animals and humans and discuss results from clinical and animal studies in diabetes.

Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes.

Background Muscular insulin resistance is frequently characterized by blunted increases in glucose-6-phosphate (G-6-P) reflecting impaired glucose transport/phosphorylation. These abnormalities likely relate to excessive intramyocellular lipids and mitochondrial dysfunction. We hypothesized that alterations in insulin action and mitochondrial function should be present even in nonobese patients with well-controlled type 2 diabetes mellitus (T2DM). Methods and Findings We measured G-6-P, ATP synthetic flux (i.e., synthesis) and lipid contents of skeletal muscle with 31P/1H magnetic resonance spectroscopy in ten patients with T2DM and in two control groups: ten sex-, age-, and body mass-matched elderly people; and 11 younger healthy individuals. Although insulin sensitivity was lower in patients with T2DM, muscle lipid contents were comparable and hyperinsulinemia increased G-6-P by 50% (95% confidence interval [CI] 39%–99%) in all groups. Patients with diabetes had 27% lower fasting ATP synthetic flux compared to younger controls (p = 0.031). Insulin stimulation increased ATP synthetic flux only in controls (younger: 26%, 95% CI 13%–42%; older: 11%, 95% CI 2%–25%), but failed to increase even during hyperglycemic hyperinsulinemia in patients with T2DM. Fasting free fatty acids and waist-to-hip ratios explained 44% of basal ATP synthetic flux. Insulin sensitivity explained 30% of insulin-stimulated ATP synthetic flux. Conclusions Patients with well-controlled T2DM feature slightly lower flux through muscle ATP synthesis, which occurs independently of glucose transport /phosphorylation and lipid deposition but is determined by lipid availability and insulin sensitivity. Furthermore, the reduction in insulin-stimulated glucose disposal despite normal glucose transport/phosphorylation suggests further abnormalities mainly in glycogen synthesis in these patients.

A Proinflammatory State Is Detectable in Obese Children and Is Accompanied by Functional and Morphological Vascular Changes

Background—Obesity is generally accepted as a risk factor for premature atherosclerosis. Subclinical inflammation as quantified by blood levels of C-reactive protein (CRP) contributes to the development and progression of atherosclerosis. We hypothesized that inflammation in obese children is related to functional and early morphological vascular changes. Methods and Results—Blood levels of high sensitivity (hs) CRP, hsIL-6, the soluble intercellular adhesion molecule1 (ICAM-1), vascular cell adhesion molecule (VCAM)-1, and E-selectin were measured in 145 severely obese (body mass index [BMI], 32.2±5.8 kg/m2) and 54 lean (BMI, 18.9±3.2 kg/m2) children 12±4 years old. Flow-mediated dilation (FMD) of the brachial artery and carotid intima-media thickness (IMT) measured by high-resolution ultrasound as markers of early vascular changes were assessed in 92 (77 obese and 15 lean) and 59 (50 obese and 9 lean) children, respectively. Obese children had significantly higher levels of hsCRP, hsIL-6, and E-selectin than healthy controls (4.1±4.8 versus 0.9±1.5 mg/L, P<0.001 for hsCRP; 1.99±1.30 versus 1.42±1.01 pg/mL, P=0.05 for hsIL-6; and 78±38 versus 59±29 ng/mL, P=0.01 for E-selectin). There were no differences in the levels of ICAM-1 and VCAM-1 between groups. Obese children had lower peak FMD response (7.70±6.14 versus 11.06±3.07%, P=0.006) and increased IMT (0.37±0.04 versus 0.34±0.03 mm, P=0.03) compared with controls. Morbidly obese children (n=14, BMI 44.1±3.9 kg/m2) had highest levels of hsCRP (8.7±0.7 mg/L), hsIL-6 (3.32±1.1 pg/mL), and E-selectin (83±40 ng/mL). Conclusions—A proinflammatory state is detectable in obese children, which is accompanied by impaired vascular endothelial function and early structural changes of arteries, even in young subjects at risk. It remains to be determined whether high hsCRP in obese children predicts cardiovascular events.

Simvastatin Blunts Endotoxin-Induced Tissue Factor In Vivo

Background—Beyond lipid lowering, various antiinflammatory properties have been ascribed to statins. Moreover, in vitro studies have suggested the presence of anticoagulant effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, as lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) was suppressed. In this study, we examined the role of statins in experimental endotoxemia on inflammatory and procoagulant responses in vivo. Methods and Results—In this double-blind, placebo-controlled, parallel-group study, 20 healthy, male subjects were randomized to receive either simvastatin (80 mg/d) or placebo for 4 days before intravenous administration of LPS (20 IU/kg IV). Plasma high-sensitive C-reactive protein (hsCRP), monocyte chemoattractant protein (MCP-1), sCD40L, sCD40, and prothrombin fragment F1+2 (F1.2) were determined by ELISAs at baseline and at 4 and 8 hours after LPS administration. Monocyte TF expression and monocyte-platelet aggregates were measured by whole-blood flow cytometry over the same time course. The increases in hsCRP and MCP-1, both known inducers of TF, were significantly suppressed by statin treatment after LPS challenge. Statin premedication blunted the increase of monocyte TF expression in response to LPS. In parallel, endotoxin-induced formation of F1.2 was significantly reduced by simvastatin after 4 and 8 hours. LPS infusion affected neither the formation and activation of monocyte-platelet aggregates nor plasma levels of sCD40 and sCD40L. Conclusions—Simvastatin suppresses the inflammatory response to endotoxin and blunts monocyte TF expression but does not affect platelet activation.

Role of NO in the O2 and CO2 responsiveness of cerebral and ocular circulation in humans

It is well known that changes in PCO2 or PO2 strongly influence cerebral and ocular blood flow. However, the mediators of these changes have not yet been completely identified. There is evidence from animal studies that NO may play a role in hypercapnia-induced vasodilation and that NO synthase inhibition modulates the response to hyperoxia in the choroid. Hence we have studied the effect of NO synthase inhibition by NG-monomethyl-L-arginine (L-NMMA, 3 mg/kg over 5 min as a bolus followed by a continuous infusion of 30 micrograms.kg-1.min-1) on the changes of cerebral and ocular hemodynamic parameters elicited by hypercapnia and hyperoxia in healthy young subjects. Mean flow velocities in the middle cerebral artery and the ophthalmic artery were measured with Doppler ultrasound, and ocular fundus pulsation amplitude, which estimates pulsatile choroidal blood flow, was measured with laser interferometry Administration of L-NMMA reduced ocular fundus pulsation. (-19%, P < 0.005) but only slightly reduced mean flow velocities in the larger arteries. Hypercapnia (PCO2 = 48 mmHg) significantly increased mean flow velocities in the middle cerebral artery (+26%, P < 0.01) and fundus pulsation amplitude (+16%, P < 0.005) but did not change mean flow velocity in the ophthalmic artery. The response to hypercapnia in the middle cerebral artery (P < 0.05) and in the choroid (P < 0.05) was significantly blunted by L-NMMA. On the contrary, L-NMMA did not affect hyperoxia-induced (PO2 = 530 mmHg) hemodynamic changes. The hemodynamic effects of L-NMMA (at baseline and during hypercapnia) were reversed by coadministration of L-arginine. The present study supports the concept that NO has a role in hypercapnia induced vasodilation in humans.It is well known that changes in [Formula: see text] or[Formula: see text] strongly influence cerebral and ocular blood flow. However, the mediators of these changes have not yet been completely identified. There is evidence from animal studies that NO may play a role in hypercapnia-induced vasodilation and that NO synthase inhibition modulates the response to hyperoxia in the choroid. Hence we have studied the effect of NO synthase inhibition by N G-monomethyl-l-arginine (l-NMMA, 3 mg/kg over 5 min as a bolus followed by a continuous infusion of 30 μg ⋅ kg-1 ⋅ min-1) on the changes of cerebral and ocular hemodynamic parameters elicited by hypercapnia and hyperoxia in healthy young subjects. Mean flow velocities in the middle cerebral artery and the ophthalmic artery were measured with Doppler ultrasound, and ocular fundus pulsation amplitude, which estimates pulsatile choroidal blood flow, was measured with laser interferometry. Administration ofl-NMMA reduced ocular fundus pulsations (-19%, P < 0.005) but only slightly reduced mean flow velocities in the larger arteries. Hypercapnia ([Formula: see text] = 48 mmHg) significantly increased mean flow velocities in the middle cerebral artery (+26%, P < 0.01) and fundus pulsation amplitude (+16%, P < 0.005) but did not change mean flow velocity in the ophthalmic artery. The response to hypercapnia in the middle cerebral artery ( P < 0.05) and in the choroid ( P < 0.05) was significantly blunted by l-NMMA. On the contrary,l-NMMA did not affect hyperoxia-induced ([Formula: see text] = 530 mmHg) hemodynamic changes. The hemodynamic effects ofl-NMMA (at baseline and during hypercapnia) were reversed by coadministration ofl-arginine. The present study supports the concept that NO has a role in hypercapnia-induced vasodilation in humans.

Nitric Oxide and Ocular Blood Flow in Patients With IDDM

Endothelial dysfunction has been implicated in the pathogenesis of diabetic vascular disorders such as diabetic retinopathy. We hypothesized that either local endogenous nitric oxide (NO) synthesis or local reactivity to endogenous NO might be impaired in patients with IDDM and that this may contribute to the development of diabetic retinopathy. Ten otherwise healthy patients with long-standing IDDM and ten healthy control subjects were studied according to an open randomized two-way cross-over design. Subjects received intravenous infusions of either NG-monomethyl-L-arginine, an inhibitor of NO-synthase, or L-arginine, the precursor of NO synthesis, on two separate study days. Ocular hemodynamics were assessed by laser interferometric measurement of fundus pulsations and Doppler sonographic measurement of blood flow velocity in the ophthalmic artery. NG-monomethyl-L-arginine decreased fundus pulsations and blood flow velocity in the ophthalmic artery and increased blood pressure in healthy subjects. The responses to NO-synthase inhibition were significantly less in diabetic subjects. In contrast, L-arginine caused a comparable increase in fundus pulsations and decrease in blood pressure in both cohorts. These results indicate that systemic and ocular hemodynamic reactivity to NO-synthase inhibition is reduced in patients with long-standing IDDM, compared with healthy control subjects. Thus, this study indicates that either NO-synthase activity is increased or NO sensitivity is decreased in patients with IDDM and supports the concept of an involvement of the Larginine–NO system in the pathophysiology of diabetic retinopathy.

Simvastatin Prevents Vascular Hyporeactivity During Inflammation

Background—There is growing evidence that statins exert anti-inflammatory and antioxidative vascular actions that are independent of lipid lowering. We tested whether hyporeactivity to the endothelium-dependent vasodilator acetylcholine (ACh) and the vasoconstrictor norepinephrine (NE) during acute experimental inflammation could be prevented by simvastatin. Methods and Results—In a randomized, placebo-controlled, parallel group study, forearm blood flow (FBF) responses to NE, ACh, and the endothelium-independent vasodilator nitroglycerin (NTG) were assessed at baseline, after 4 days of simvastatin 80 mg PO or placebo treatment, and during Escherichia coli endotoxin (lipopolysaccharide [LPS])–induced inflammation in 20 healthy volunteers. Additionally, markers of inflammation and neutrophil oxidative burst were assessed. Simvastatin and placebo had no effect on FBF or oxidative/inflammatory markers. LPS administration decreased the responses of FBF to NE by 43% (P<0.05) and decreased responses to ACh by 48% (P<0.05) but did not decrease FBF responses to NTG. Simvastatin completely preserved responses to NE and to ACh. The LPS-induced increases in neutrophil oxidative burst and plasma tumor necrosis factor-α concentrations were mitigated by simvastatin (P<0.05 versus placebo). Conclusions—This study demonstrates potent vasoprotective properties of high-dose simvastatin during endotoxemia that may be useful for patients with acute systemic inflammation and associated vascular hyporeactivity.

The adipokine visfatin is markedly elevated in obese children.

Objective: The insulin-mimetic adipocytokine visfatin has been linked to adiposity and the metabolic syndrome. Design Cross-sectional study. Subjects: Eighty-three nondiabetic obese children and 40 healthy controls. Measurements: We analyzed plasma visfatin concentrations to assess whether this adipokine is associated with adiposity. Results: Plasma visfatin concentrations were nearly 2-fold higher in obese children (mean, 1.1 ng/mL; 95% CI, 0.2-6.6) than in controls (0.6 ng/mL, 95% CI, 0.6 to 0.6; P < 0.001). No relationship was detectable between visfatin and other subject characteristics, hsCRP or the lipid profile. Conclusions: Visfatin may be involved in the development of metabolic derangements in obese children.

Effect of recombinant factor VIIa on melagatran-induced inhibition of thrombin generation and platelet activation in healthy volunteers

The objectives were to investigate whether activation of the extrinsic coagulation cascade by recombinant factor VIIa (rFVIIa) reverses the inhibition of thrombin generation and platelet activation by melagatran, the active form of the oral direct thrombin inhibitor ximelagatran. In a single-blind, randomized, parallel-group study, volunteers (20 per group) received a 5-hour intravenous (i.v.) infusion to achieve steady-state melagatran plasma concentrations of approximately 0.5 micromol/L, with a single i.v. bolus of rFVIIa (90 microg/kg) or placebo at 60 minutes. Prothrombin fragment 1+2, thrombin-anti-thrombin complex, fibrinopeptide A, beta-thromboglobulin, and thrombin-activatable fibrinolysis inhibitor were quantified for venous and shed blood. Activated partial thromboplastin time (APTT), prothrombin time (PT), endogenous thrombin potential, thrombus precursor protein (TpP), and plasmin-alpha(2)-antiplasmin complex concentrations were determined in venous blood. Shed blood volume was measured. Melagatran reduced markers of thrombin generation and platelet activation in shed blood and prolonged APTT. rFVIIa increased FVIIa activity, PT, and TpP in venous blood. All other parameters were unaffected. In conclusion, rFVIIa did not reverse the anticoagulant effects of high constant concentrations of melagatran. However, the potential value of higher, continuous or repeated doses of rFVIIa or its use with lower melagatran concentrations has not been excluded.

Asymmetric Dimethylarginine Enhances Cardiovascular Risk Prediction in Patients With Chronic Heart Failure

Objective—The purpose of this study was to investigate whether elevated asymmetrical dimethylorginine (ADMA) concentrations are associated with increased cardiovascular risk in chronic heart failure (HF) patients. Methods and Results—253 patients with symptomatic chronic HF and impaired left ventricular function (median age 70 years, 202 males) were followed for a median of 14.2 months (interquartile range 6.8 to 21.2). ADMA and N-terminal pro-brain natriuretic peptide (NT-proBNP) were assessed by high performance liquid chromatography and by an enzyme-linked immunosorbent assay, respectively. Subjects with ADMA concentrations in the highest tertile had a significantly higher adjusted hazard ratio (HR; 2.00; 95% confidence interval [CI] 1.01 to 3.97) for occurrence of an end point (cardiac decompensation, major adverse cardiovascular events or all-cause mortality) compared with patients in the lowest tertile (P=0.046) during the first 6 months of follow-up. NT-proBNP also identified subjects at risk before adjustment for confounders at 6 and 12 months of follow-up. HR for patients with ADMA and NT-proBNP in the highest tertile was significantly increased (3.68, CI 1.67 to 8.14; at 6 months follow-up) compared with patients without ADMA and NT-proBNP in the highest tertile (P<0.001). Conclusions—Elevated ADMA plasma concentrations are associated with adverse cardiovascular outcome in patients with chronic HF. Quantification of ADMA with NT-proBNP improves risk stratification in this cohort.