Markus Exner

Capillary Deposition of Complement Split Product C4d in Renal Allografts is Associated with Basement Membrane Injury in Peritubular and Glomerular Capillaries: A Contribution of Humoral Immunity to Chronic Allograft Rejection

Endothelial deposition of the complement split product C4d is an established marker of antibody-mediated acute renal allograft rejection. A contribution of alloantibody-dependent immune reactions to chronic rejection is under discussion. In this study, the association of immunohistochemically detected endothelial C4d deposition in peritubular capillaries (PTC) with morphologic features of chronic renal allograft injury was investigated in a large study cohort. C4d deposits in PTC were detected in 73 (34%) of 213 late allograft biopsies performed in 213 patients more than 12 mo after transplantation (median, 4.9 yr) because of chronic allograft dysfunction. Endothelial C4d deposition was found to be associated with chronic transplant glomerulopathy (CG) (P < 0.0001), with basement membrane multilayering in PTC (P = 0.01), and with an accumulation of mononuclear inflammatory cells in PTC (P < 0,001). Furthermore, C4d deposits in PTC (in biopsies with normal glomerular morphology) were associated with development of CG in follow-up biopsies. Other morphologic features of chronic allograft nephropathy (with exception of tubular atrophy) were not associated with C4d deposits in PTC. Analyses of previous and follow-up biopsies revealed that C4d deposits may occur de novo and may also disappear at any time after transplantation. In conclusion, the data suggest that complement activation in renal microvasculature, indicating humoral alloreactivity, contributes to chronic rejection characterized by chronic transplant glomerulopathy and basement membrane multilayering in PTC.

Mean platelet volume is an independent risk factor for myocardial infarction but not for coronary artery disease.

Summary.  After rupture of an arteriosclerotic plaque in a coronary artery, platelets play a crucial role in the subsequent thrombus formation, leading to myocardial infarction. An increased mean platelet volume (MPV), as an indicator of larger, more reactive platelets, may represent a risk factor for myocardial infarction. However, this hypothesis is still controversial and most studies addressing the role of MPV were performed comparing patients suffering from myocardial infarction with healthy controls. We intended to identify patients at high risk of suffering myocardial infarction in a group of patients with known coronary artery disease. One hundred and eighty‐five consecutive patients with stable coronary artery disease were compared with 188 individuals who had suffered myocardial infarction. Patients within the highest quintile of MPV (≥ 11·6 fl) had a significantly higher risk of experiencing a myocardial infarction compared with patients within the lowest quintile (OR = 2·6, 95% CI 1·3–5·1) in a multivariate analysis that included sex, age, body mass index, hyperlipidaemia, hypertension, smoking and diabetes mellitus. Our results indicate that patients with pre‐existing coronary artery disease and an increased MPV (≥ 11·6 fl) are at higher risk of myocardial infarction. These patients can be easily identified during routine haematological analysis and could possibly benefit from preventive treatment.

Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis

Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P41–49) with 100% homology to the bacterial adhesin FimH, with which they cross-react. Rats immunized with FimH develop pauci-immune FNGN and also develop antibodies to rat and human LAMP-2. Finally, we show that infections with fimbriated pathogens are common before the onset of FNGN. Thus, FimH-triggered autoimmunity to LAMP-2 provides a previously undescribed clinically relevant molecular mechanism for the development of pauci-immune FNGN.

Leptin Induces Endothelin-1 in Endothelial Cells In Vitro

Leptin, a protein encoded by the obese gene, is produced by adipocytes and released into the bloodstream. In obese humans, serum leptin levels are increased and correlate with the individual’s body mass index and blood pressure. Elevated serum concentrations of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, were also observed in obese subjects. The pathomechanisms underlying this ET-1 increase in obesity are poorly understood. In the present study, we investigated the influence of the ob gene product leptin on the expression of ET-1 in human umbilical vein endothelial cells (HUVECs). Binding studies using 125I-radiolabeled leptin revealed high- and low-affinity leptin binding sites on HUVECs (Kd1=13.1±3.1 nmol/L and Kd2=1390±198 nmol/L, respectively), mediating a time- and dose-dependent increase of ET-1 mRNA expression and protein secretion after incubation of HUVECs with leptin. This leptin-induced ET-1 expression was inhibited by preincubation of HUVECs with 0.75 &mgr;mol/L antisense phosphorothioate oligonucleotides directed against the leptin receptor Ob-Rb. Furthermore, after incubation with leptin, increased nuclear staining of c-fos and c-jun, the major components of the transcription factor AP-1, and increased AP-1 DNA binding were observed. Transient transfection studies with ET-1 promoter constructs showed that leptin-induced promoter activity was abolished in the absence of AP-1 binding sites or by cotransfection with a plasmid overexpressing a mutated jun, which is able to bind c-fos but not DNA. Thus, leptin upregulates ET-1 production in HUVECs via a mechanism potentially involving jun binding members of the bZIP family.

Medical conditions increasing the risk of chronic thromboembolic pulmonary hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by organized thromboemboli that obstruct the pulmonary vascular bed. Although CTEPH is a serious complication of acute symptomatic pulmonary embolism in 4% of cases, signs, symptoms and classical risk factors for venous thromboembolism are lacking. The aim of the present study was to identify medical conditions conferring an increased risk of CTEPH. We performed a case-control-study comparing 109 consecutive CTEPH patients to 187 patients with acute pulmonary embolism that was confirmed by a high probability lung scan. Splenectomy (odds ratio=13, 95% CI 2.7-127), ventriculo-atrial (VA-) shunt for the treatment of hydrocephalus (odds ratio=13, 95% CI 2.5-129) and chronic inflammatory disorders, such as osteomyelitis and inflammatory bowel disease (IBD, odds ratio=67, 95% CI 7.9-8832) were associated with an increased risk of CTEPH.

Inflammation and Carotid Artery—Risk for Atherosclerosis Study (ICARAS)

Background—Compelling evidence suggests that inflammation is fundamentally involved in the pathogenesis of atherosclerosis; however, temporal correlation between inflammation and morphological features of atherosclerosis progression has not been demonstrated unequivocally. Methods and Results—We prospectively studied 1268 consecutive patients who were initially asymptomatic with respect to carotid artery disease. Patients underwent serial carotid ultrasound investigations at baseline and after a follow-up interval of a median of 7.5 months (range 6 to 9 months), with measurement of carotid flow velocities and categorization of carotid arteries as 0% to 29%, 30% to 49%, 50% to 69%, 70% to 89%, or 90% to 99% stenosed or occluded. High-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA) were measured at baseline and follow-up. Progression of carotid atherosclerosis was found in 103 (8.1%) of 1268 patients. Hs-CRP and SAA, respectively, at baseline (P=0.004 and P=0.014) and follow-up (P<0.001 and P<0.001) and the change from baseline to follow-up (P<0.001 and P<0.001) were significantly associated with progressive atherosclerosis. Adjusted ORs (95% CI) for atherosclerosis progression with increasing quintiles of baseline hs-CRP were 1.65 (0.71 to 3.84), 1.87 (0.8 to 4.37), 3.32 (1.49 to 7.39), and 3.65 (1.65 to 8.08), and with increasing quintiles of baseline SAA, they were 0.86 (0.38 to 1.92), 0.99 (0.49 to 1.99), 1.72 (0.91 to 3.28), and 2.28 (1.24 to 4.20), respectively, compared with the lowest quintiles. Conclusions—These findings supply evidence for a close temporal correlation between inflammation and morphological features of rapidly progressive carotid atherosclerosis, which suggests that elevation or increase of the inflammatory biomarkers hs-CRP and SAA identifies the presence of active atherosclerotic disease.

Expression of Heme Oxygenase-1 in Human Vascular Cells Is Regulated by Peroxisome Proliferator-Activated Receptors

Objective—Activation of peroxisome proliferator-activated receptors (PPARs) by lipid-lowering fibrates and insulin-sensitizing thiazolidinediones inhibits vascular inflammation, atherosclerosis, and restenosis. Here we investigate if the vasculoprotective and anti-inflammatory enzyme heme oxygenase-1 (HO-1) is regulated by PPAR ligands in vascular cells. Methods and Results—We show that treatment of human vascular endothelial and smooth muscle cells with PPAR ligands leads to expression of HO-1. Analysis of the human HO-1 promoter in transient transfection experiments together with mutational analysis and gel shift assays revealed a direct transcriptional regulation of HO-1 by PPAR&agr; and PPAR&ggr; via 2 PPAR responsive elements. We demonstrate that a clinically relevant polymorphism within the HO-1 promoter critically influences its transcriptional activation by both PPAR isoforms. Moreover, inhibition of HO-1 enzymatic activity reversed PPAR ligand-mediated inhibition of cell proliferation and expression of cyclooxygenase-2 in vascular smooth muscle cells. Conclusion—We demonstrate that HO-1 expression is transcriptionally regulated by PPAR&agr; and PPAR&ggr;, indicating a mechanism of anti-inflammatory and antiproliferative action of PPAR ligands via upregulation of HO-1. Identification of HO-1 as a target gene for PPARs provides new strategies for therapy of cardiovascular diseases and a rationale for the use of PPAR ligands in the treatment of other chronic inflammatory diseases.

Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with restenosis after percutaneous transluminal angioplasty.

Purpose: To determine if an association exists between postdilation restenosis and heme oxygenase-1 (HO-1), which is induced by balloon injury and inhibits neointimal formation through the action of endogenous carbon monoxide. A dinucleotide repeat in the promoter region of the HO-1 gene shows a length polymorphism that modulates the level of gene transcription. Methods: This cohort study included 96 consecutive patients (64 men; median age 69 years, interquartile range 60–75) who underwent successful balloon dilation in the femoropopliteal segment. Six-month patency was evaluated using oscillography, ankle-brachial index, and duplex sonography. The association of patency and the length of (GT) repeats in the HO-1 gene promoter was assessed in univariate and multivariate analyses. Results: Restenosis was found in 23 (24%) patients within the first 6 months. Patients with short (<25 GT) dinucleotide repeats in the HO-1 gene promoter on either allele had restenosis significantly less often than patients with longer (≥25 GT) dinucleotide repeats (p = 0.01). Multivariate analysis revealed a significantly reduced risk for restenosis in these patients compared to patients without the short allele (odds ratio 0.2, 95% CI 0.06 to 0.70, p = 0.007). Conclusions: Genetic risk factors for restenosis after percutaneous transluminal angioplasty have not been investigated. In this patient population, short repeat alleles of the heme oxygenase-1 gene promoter polymorphism were associated with reduced postdilation restenosis at 6 months. Upregulation of HO-1 may be an important protective factor after balloon angioplasty by inhibition of vascular smooth muscle cell proliferation.

Joint Effects of C-Reactive Protein and Glycated Hemoglobin in Predicting Future Cardiovascular Events of Patients With Advanced Atherosclerosis

Background—C-reactive protein (CRP) and glycohemoglobin (HbA1c) are established risk factors for the development of cardiovascular disease. We investigated the joint effects of these parameters on cardiovascular outcome of patients with advanced atherosclerosis. Methods and Results—We studied 454 patients with advanced atherosclerosis (median age, 69 years; 264 male). Cardiovascular risk profile, high-sensitivity CRP (hs-CRP), and HbA1c were obtained at baseline, and patients were followed for a median of 21 months (interquartile range, 13 to 26) for the occurrence of major adverse cardiovascular events (MACE) (myocardial infarction, percutaneous coronary interventions, coronary artery bypass graft, carotid revascularization, stroke, and death). We observed 166 MACE in 128 patients (28%). Cumulative event-free survival rates at 6, 12, and 24 months were 91%, 85%, and 73%, respectively. Adjusted hazard ratios for the occurrence of MACE according to increasing quartiles of hs-CRP and HbA1c were 1.35 (P =0.31), 1.90 (P =0.026) and 2.13 (P =0.007), and 1.40 (P =0.26), 1.81 (P =0.059), and 2.36 (P =0.023), respectively, compared with the lowest quartiles. Considering both parameters jointly, we found that patients with hs-CRP >0.44 mg/dL and HbA1c >6.2% (upper quartiles) were at highest risk for MACE, with each parameter adding to the prognostic information of the other. Conclusions—Inflammation, indicated by hs-CRP, and hyperglycemia, indicated by HbA1c, jointly contribute to the cardiovascular risk of patients with advanced atherosclerosis. Patients with both hs-CRP and HbA1c in the upper quartiles (>0.44 mg/dL and >6.2%, respectively) are at particularly high risk for poor cardiovascular outcome.

Glomerular overproduction of oxygen radicals in Mpv17 gene-inactivated mice causes podocyte foot process flattening and proteinuria : A model of steroid-resistant nephrosis sensitive to radical scavenger therapy

Focal segmental glomerulosclerosis is a steroid-resistant glomerular disease characterized by foot process flattening and heavy proteinuria. A similar disease was found to occur spontaneously in mice in which the Mpv17 gene was inactivated by retroviral insertion (Mpv17-/- mice). Here evidence is provided that glomerular damage in this murine model is due to overproduction of oxygen radicals and accumulation of lipid peroxidation adducts that were found in isolated glomeruli of Mpv17-/- mice. The development of glomerular disease in Mpv17-/- mice was inhibited by scavengers of oxygen radicals (dithiomethylurea) and lipid peroxidation (probucol), but not by steroid treatment. Although the glomerular polyanion was greatly reduced in proteinuric Mpv17-/- mice, it was preserved by antioxidative therapy. These results indicate that the glomerular disease in Mpv17-/- mice qualifies as a model of steroid-resistant focal segmental glomerulosclerosis and that experimental therapies with scavengers of oxygen radicals and lipid peroxidation efficiently ameliorate glomerular damage.