Frank Pistrosch

Endothelial dysfunction in patients with rheumatoid arthritis is associated with a reduced number and impaired function of endothelial progenitor cells

Background: Rheumatoid arthritis (RA) is associated with increased morbidity and mortality attributable to accelerated atherosclerosis and cardiovascular events. Objective: To determine the role played by endothelial progenitor cells (EPC) in the defence system against arteriosclerosis. Methods: The number and function of EPC in 13 young patients with RA with low disease activity (DAS28 3.5 (0.3)) and 13 healthy control subjects was studied. Endothelial function was investigated by agonist-induced, endothelium dependent vasodilatation measured by the forearm blood flow technique. Migratory activity and adhesion of EPC to tumour necrosis factor α (TNFα) activated mature endothelial cells and components of the extracellular matrix were tested in vitro. Putative precursor populations (CD34+, CD34+/CD133+, and CD34+/KDR+ haematopoietic stem cells) were measured by flow cytometric analysis. Results: Acetylcholine-induced, endothelium dependent vasodilatation was reduced by about 50% in patients with RA, indicating endothelial dysfunction, whereas endothelium-independent vasodilatation in response to glyceryl trinitrate was at control level. Significantly reduced numbers of EPC were found in the patients compared with controls. Migratory activity of EPC was decreased in patients with RA. Adhesion to mature endothelial cells after activation with TNFα was enhanced only in controls. The adhesion to matrix proteins and the number of putative precursor cell lineages was comparable in both groups. Conclusion: Endothelial dysfunction in patients with RA with low grade inflammation is associated with a reduced number and partial dysfunction of EPC. Further studies are needed to explore whether interventions that potentially ameliorate the number and function of EPC also improve endothelial function in these patients.

Endothelial dysfunction in young patients with long-term rheumatoid arthritis and low disease activity

OBJECTIVE Cardiovascular mortality is excessive in patients with rheumatoid arthritis (RA). It has been proposed that the chronic inflammatory state of RA contributes to accelerated atherosclerosis. The aim of this study was to determine whether endothelial dysfunction, an early sign of arteriosclerosis, is present in young, long-term RA patients receiving standard methotrexate (MTX) therapy. Furthermore, we tested whether etanercept (ENC), a TNF-alpha receptor blocker, resulted in improved endothelial function compared to MTX in the same patients. METHODS We studied eight RA patients twice: (1) on MTX and (2) after MTX washout and receiving ENC. Eight healthy volunteers matching for age, gender, height, weight and conventional cardiovascular risk factors served as control (C). All participants received intrabrachial infusions of increasing doses of acetylcholine (ACh, endothelium-dependent vasodilator) and glyceryl-trinitrate (GTN, endothelium-independent vasodilator). Forearm blood flow (FBF) was measured by bilateral venous occlusion plethysmography. RESULTS Disease activity of RA was comparably low during both MTX and ENC (DAS 28 3.9+/-0.3 and 3.5+/-0.3). FBF in response to ACh was reduced in RA compared to C (P<0.01). Switching from MTX to ENC failed to improve vascular responsiveness to ACh. GTN comparably increased FBF in all groups. CONCLUSIONS Our study for the first time demonstrates that long-term RA is associated with manifested endothelial dysfunction. Switching from MTX to ENC in stable RA patients has no beneficial effect on endothelial function.

Relationship Between Hypoglycemic Episodes and Ventricular Arrhythmias in Patients With Type 2 Diabetes and Cardiovascular Diseases: Silent Hypoglycemias and Silent Arrhythmias

OBJECTIVE In patients with type 2 diabetes and cardiovascular diseases (CVDs), intensive treatment with insulin and/or sulfonylurea (SU) may be associated with excessive increased risk of hypoglycemic episodes. To evaluate the risk of critical arrhythmias related to glycemic variability, we carried out an observational study in type 2 diabetes patients with CVD. RESEARCH DESIGN AND METHODS Thirty patients with type 2 diabetes and documented CVD who had been treated with insulin and/or SU underwent 5 days of monitoring with a continuous glucose measurement system along with parallel electrocardiogram recording for monitoring of ventricular arrhythmias. Twelve age-matched patients with documented CVD who received treatment with metformin and/or dipeptidyl peptidase-4 inhibitor served as the control group. Patients were receiving stable treatment, and were instructed to notice symptoms of arrhythmias and hypoglycemia, respectively. RESULTS We observed a high incidence of asymptomatic severe episodes of hypoglycemia (<3.1 mmol/L) in patients receiving treatment with insulin and/or SU, whereas severe hypoglycemia did not develop in any of the control subjects. Patients with severe hypoglycemia (n = 12) had a higher number of severe ventricular arrhythmias (patients with versus without severe hypoglycemia, respectively: ventricular couplets 41.7 ± 81.8 vs. 5.5 ± 16.7; ventricular tachycardia 1.0 ± 1.9 vs. 0.1 ± 0.3). No direct correlation could be found among different variables of glucose profile, corrected QT interval, and ventricular arrhythmias. CONCLUSIONS Our results suggest that severe episodes of hypoglycemia are associated with an increased risk of severe ventricular arrhythmias.

Platelet Sarcoplasmic Endoplasmic Reticulum Ca2+-ATPase and μ-Calpain Activity Are Altered in Type 2 Diabetes Mellitus and Restored by Rosiglitazone

Background— Platelets from patients with type 2 diabetes mellitus display hyperaggregability and increased thrombogenic potential. Methods and Results— In platelets from patients with type 2 diabetes mellitus, we found enhanced tyrosine nitration and inactivation of the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA-2), elevated platelet [Ca2+]i, and activation of &mgr;-calpain. The tyrosine nitration of SERCA-2 and the activation of &mgr;-calpain in vitro in platelets from healthy volunteers could be evoked in vitro by peroxynitrite. Platelet endothelial cell adhesion molecule-1 was identified as a &mgr;-calpain substrate; its in vitro degradation was stimulated by peroxynitrite and prevented by calpain inhibitors. Calpain activation also was linked to hyperresponsiveness to thrombin and the loss of platelet sensitivity to nitric oxide synthase inhibitors. Platelets from patients with type 2 diabetes mellitus (hemoglobin A1c >6.6%) contained little or no intact platelet endothelial cell adhesion molecule-1, whereas degradation products were detectable. The peroxisome proliferator–activated receptor-&ggr; agonist rosiglitazone increased SERCA-2 expression in megakaryocytes, and treating patients with type 2 diabetes mellitus with rosiglitazone for 12 weeks increased platelet SERCA-2 expression and Ca2+-ATPase activity, decreased SERCA-2 tyrosine nitration, and normalized platelet [Ca2+]i. Rosiglitazone also reduced &mgr;-calpain activity, normalized platelet endothelial cell adhesion molecule-1 levels, and partially restored platelet sensitivity to nitric oxide synthase inhibition. Conclusion— These data identify megakaryocytes/platelets as additional cellular targets for peroxisome proliferator–activated receptor-&ggr; agonists and highlight potential benefits of rosiglitazone therapy in cardiovascular diseases.

Reduced Agonist-Induced Endothelium-Dependent Vasodilation in Uremia Is Attributable to an Impairment of Vascular Nitric Oxide

Current concepts for the explanation of endothelial dysfunction and accelerated atherosclerosis in uremia propose a reduced vascular bioavailability of nitric oxide (NO). The aim of the present study was to test the contributions of NO and NO/prostacyclin (PGI(2))-independent mechanisms to both baseline vascular tone and agonist-induced endothelium-dependent vasodilation in patients on hemodialysis (HD). In 10 HD patients and eight matched healthy control subjects, forearm blood flow (FBF) was measured at rest and during intrabrachial infusions of norepinephrine (NE; endothelium-independent vasoconstrictor, 60, 120, and 240 pmol/min) and N-monomethyl-L-arginine (blocker of NO synthases, 16 micromol/min). After inhibition of cyclo-oxygenase by ibuprofen (1200 mg orally), endothelium-dependent and -independent vasodilation was assessed by infusion of acetylcholine (ACh; 1, 5, 10, 50, 100, and 300 nmol/min) and sodium-nitroprusside (2.5, 5, and 10 microg/min). NO/PGI(2)-independent vasodilation was tested by equal infusions of ACh during NO clamp. N-monomethyl-L-arginine reduced resting FBF to a comparable degree in both groups. Vascular responses to ACh were reduced in HD (P = 0.003 versus control by ANOVA), whereas those to sodium nitroprusside were mainly at control level. Infusion of ACh during NO clamp caused a similar increment of FBF in both groups. NO-mediated vasodilation as calculated by the difference between ACh-induced responses without and with NO clamp was substantially impaired in HD (P < 0.001) compared with control. In HD patients, baseline NO-mediated arteriolar tone is at control level. This study provides first evidence that endothelial dysfunction of uremic patients as shown by reduced agonist-induced endothelium-dependent vasodilation is attributable to reduced stimulation of NO, whereas the NO/PGI(2)-resistant portion of ACh-mediated vasodilation is unaffected.

Baseline blood flow and bradykinin-induced vasodilator responses in the human forearm are insensitive to the cytochrome P450 2C9 (CYP2C9) inhibitor sulphaphenazole.

A substantial portion of the vasodilator response elicited by bradykinin in the human forearm is unaffected by the combined inhibition of nitric oxide (NO) synthases and cyclo-oxygenases. The cytochrome P450 (CYP) 2C9 inhibitor sulphaphenazole was recently identified as a potent inhibitor of NO- and prostacyclin (PGI2)-independent relaxation in porcine coronary arteries. The aim of the present study was to determine the effect of sulphaphenazole on basal and bradykinin-induced NO/PGI2-independent changes in the forearm blood flow (FBF) of healthy subjects. Eleven healthy male volunteers participated in this placebo-controlled study. Test agents were infused into the brachial artery and FBF was measured by bilateral venous occlusion plethysmography. Sulphaphenazole (0.02-2 mg/min) alone did not affect basal blood flow. Inhibition of the NO synthases by NG-monomethyl-L-arginine (L-NMMA; 4 micromol/min) and cyclo-oxygenases by ibuprofen (1200 mg, orally) reduced FBF to 48 +/- 7% in the absence and 50 +/- 8% in the presence of sulphaphenazole (2 mg/min; P=not significant). After pretreatment with L-NMMA (16 micromol/min) and ibuprofen (1200 mg, orally), sulphaphenazole (6 mg/min) did not substantially inhibit bradykinin-induced vasodilation. We conclude that CYP2C9-derived metabolites (i) are not involved in the regulation of baseline blood flow, and (ii) do not mediate bradykinin-induced NO/PGI2-independent vasorelaxation in the human forearm. However, determining the contribution of this enzyme to regulation of blood flow in pathological conditions associated with endothelial dysfunction requires further studies.

Endothelial Progenitor Cells in Chronic Renal Insufficiency

There is growing evidence for a role of endothelial progenitor cells (EPCs) in the repair of damaged endothelium. It remains unclear which cell populations are most useful for clinical trials. Administration of drugs increasing EPC numbers and/or improving functional properties seems attractive. Further basic research is necessary to understand the mechanisms of mobilization, differentiation and homing of EPC in general and in particular under uremic conditions. Nephrologists should search for strategies to ameliorate EPC dysfunction of uremia. In this way it might be possible to test whether improved EPC biology is associated with decreased cardiovascular mortality in uremic humans. In any such studies the difficulties are going to be related to the complex procedures for EPC isolation, the testing of their identity and differentiation and their propagation before use.

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes

Platelets from patients with diabetes are hyperreactive and demonstrate increased adhesiveness, aggregation, degranulation, and thrombus formation, processes that contribute to the accelerated development of vascular disease. Part of the problem seems to be dysregulated platelet Ca(2+) signaling and the activation of calpains, which are Ca(2+)-activated proteases that result in the limited proteolysis of substrate proteins and subsequent alterations in signaling. In the present study, we report that the activation of μ- and m-calpain in patients with type 2 diabetes has profound effects on the platelet proteome and have identified septin-5 and the integrin-linked kinase (ILK) as novel calpain substrates. The calpain-dependent cleavage of septin-5 disturbed its association with syntaxin-4 and promoted the secretion of α-granule contents, including TGF-β and CCL5. Calpain was also released by platelets and cleaved CCL5 to generate a variant with enhanced activity. Calpain activation also disrupted the ILK-PINCH-Parvin complex and altered platelet adhesion and spreading. In diabetic mice, calpain inhibition reversed the effects of diabetes on platelet protein cleavage, decreased circulating CCL5 levels, reduced platelet-leukocyte aggregate formation, and improved platelet function. The results of the present study indicate that diabetes-induced platelet dysfunction is mediated largely by calpain activation and suggest that calpain inhibition may be an effective way of preserving platelet function and eventually decelerating atherothrombosis development.

The Metabolic Syndrome and Cancer

Epidemiological studies have demonstrated an association of many types of cancer with traits of the Metabolic syndrome. Data from case–control and cohort studies determined hazard ratios between 1.1 and 2.3 for cancer incidence in obesity and type 2 diabetes. About 10 % of the excess mortality in patients with these two conditions may be attributed to death from cancer. However, a coincidence of type 2 diabetes and obesity or additional traits of the metabolic syndrome may further increase the risk. Low-grade inflammation of adipose tissue—the common soil for the development of different traits of the metabolic syndrome—appears to be the most promising candidate for a causal relationship between cancer and the metabolic syndrome, too. Adipose tissue can produce different cytokines and hormones which influence cancer promotion and progression. On the other hand, there are concerns about an increase of cancer risk by commonly prescribed drugs against different traits of metabolic syndrome such as hyperlipidaemia or hyperglycaemia. The lack of prospective studies in this field of research is a major concern and forestalls a convincing evaluation.

Is Hyperglycemia a Cardiovascular Risk Factor

Patients with diabetes show an increased vascular morbidity and mortality that reduces their life expectancy by ~5–15 years (depending on the age at diagnosis) (1). There is convincing evidence from epidemiological and pathophysiological studies that hyperglycemia per se is largely responsible for the harmful effects of the disease. As recently shown by clinical trials, treatment of this condition may reduce cardiovascular events and mortality, and several therapies should be considered: initiating early and individualized treatment and avoiding hypoglycemia. As shown in the Multiple Risk Factor Intervention Trial, at any given level of major cardiovascular risk factors, diabetes is associated with an odds ratio of 2–4 for cardiovascular mortality compared with nondiabetic subjects (2). These results were confirmed by the European Prospective Investigation of Cancer and Nutrition (EPIC Norfolk) study (3) and a recent analysis of the Atherosclerosis Risk in Communities (ARIC) study (4). Furthermore, a recently published 18-year follow-up study from Finland demonstrated a similar impact of type 1 and type 2 diabetes on cardiovascular mortality. The adjusted hazard ratios compared with age-matched subjects without diabetes were 5.2 and 4.9 for type 1 and type 2 diabetes, respectively (5). Thus, today evidence exists on long-term follow-up population-based studies in patients with type 1 and type 2 diabetes. This evidence clearly suggests that hyperglycemia is a key risk factor not only for diabetes-related disease, but also for cardiovascular and all-cause mortality. On the basis of these long-term observations, one can assume an increment of cardiovascular disease per increase of 1 unit (%) A1C of ~18% (6). As shown in numerous prospective studies, the deleterious effects of dysglycemia (fasting and postprandial hyperglycemia) develop before diabetes is diagnosed. In the Glucose Tolerance in Acute Myocardial Infarction study of patients with acute coronary syndrome, abnormal glucose tolerance was the strongest independent …