Publications by authors named "Branislav Zagrapan"

An abdominal aortic aneurysm (AAA) is a multifactorial disease with a variety of genetic and environmental risk factors, but the exact mechanism of AAA formation and progression is still not well understood. The present study investigated the frequency of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and papillomavirus types 6 and 11 (HPV6 and HPV11), their impact on clinical manifestations of cardiovascular diseases, and their possible association with inflammation in patients with AAA and healthy volunteers. Genotyping of CMV UL75, EBV LMP-1, and HPV6 and HPV11 E6 was performed by polymerase chain reaction (PCR), while the viral DNA loads were measured by quantitative real-time PCR (qRT-PCR). Cytokine levels were determined by enzyme-linked immunosorbent assays (ELISAs). The CMV UL75 was detected more frequently in the blood of patients with AAA than in the blood of healthy volunteers (32.7% vs. 6.3%, p < 0.0001). Neither EBV LMP-1 nor HPV6 E6 was found in blood and aortic wall biopsies, while the HPV11 E6 was detected in 36.4% of AAA walls. The CMV infection in patients with AAA was associated with an increased risk of hypertension and coronary artery disease (CAD) (OR 9.057; 95% CI: 1.141 - 71.862, p = 0.037; OR 2.575; 95% CI: 1.002 - 6.615, p = 0.049, respectively). Additionally, CMV-infected patients with AAA had higher TNF-α levels compared with non-infected subjects (p = 0.017). Our findings suggest that CMV infection can stimulate local inflammation in the aorta but is not a direct cause of most abdominal aortic aneurysms. This article is protected by copyright. All rights reserved.

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of abdominal aortic aneurysms (AAAs). This study has addressed the notion that NET components might serve as AAA biomarkers or novel targets of AAA therapy. Thus, parameters of neutrophil activation and NET formation were measured in plasma. Their diagnostic marker value was explored in 41 AAA patients and 38 healthy controls. The NET parameter citrullinated histone H3 (citH3) was then validated in 63 AAA patients and 63 controls matched for cardiovascular disease. The prognostic marker potential was investigated in 54 observation periods of AAA growth over 6 months. NETs were further assessed in conditioned medium and sections of aortic tissue. CitH3 was found to be increased in blood (median 362 vs 304 ng/mL, P = 0.004) and aortic tissue (50 vs 1.5 ng/mg, P < 0.001) of AAA patients compared to healthy controls and accumulated in the intraluminal thrombus (629 ng/mg). The diagnostic potential of citH3 ranged at 0.705 area under the ROC curve (AUROC) and was validated with the independent sample set. Furthermore, plasma citH3 predicted AAA growth over the next 6 months (AUROC: 0.707, P = 0.015) and dropped significantly after surgical aneurysm repair. In an angiotensin II - based mouse model of experimental AAA, an inhibitor of histone citrullination was applied to block NET formation and AAA progression. Of note, further growth of an established aneurysm was prevented in mice treated with the NET inhibitor (P = 0.040). In conclusion, histone citrullination represents a promising AAA biomarker and potential therapeutic target to control disease progression.

Background: An abdominal aortic aneurysm (AAA) is a complex disease of the aging population that is associated with inflammation and the cellular immune response. To investigate the influence of interleukin (IL)-6 and tumor necrosis factor (TNF)-α single nucleotide polymorphisms (SNPs) on the risk of AAA formation and progression, the frequency of AAA and its associated risk factors were determined.

Method: Four SNPs in the IL-6 (-174G/C, rs1800795; -572G/C, rs1800796) and TNF-α (-238G/A, rs361525; -308G/A, rs1800629) genes were studied by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in patients with AAA and healthy volunteers. The mRNA expression and plasma IL-6 and TNF-α levels were also determined.

Results: A mutation detected in at least one allele of the IL-6 -174G/C SNP was associated with a 2-fold increased risk of AAA occurrence (OR: 2.08; 95% CI: 1.15-3.76; p = 0.014, in the dominant model). An increased risk of AAA incidence among heterozygous carriers of the TNF-α - 308G/A genotype was observed (OR: 2.06; 95% CI: 1.17-3.62; p = 0.011, in the overdominant model). The wild-type genotypes of the IL-6 -174G/C and the TNF-α -308G/A SNPs coexisted more frequently in healthy subjects than in AAA patients and was associated with decreased risk of AAA (p < 0.001). Moreover, elevated levels of IL-6 and TNF-α were associated with an increased risk of hypertension (p < 0.001 and p = 0.022, respectively).

Conclusions: The IL-6 -174G/C and the TNF-α -238G/A gene polymorphisms are associated with an increased risk of abdominal aortic aneurysm development.

In this observational case-control study, circulating levels of complement factors C3a and C5a and leukotriene B4 (LTB4) were analysed in abdominal aortic aneurysm (AAA) patients regarding their association with diagnosis and prognosis. Serum C5a was significantly raised in AAA patients compared to healthy controls-median 84.5 ng/ml (IQR = 37.5 ng/ml) vs. 67.7 ng/ml (IQR = 26.2 ng/ml), p = 0.007-but was not elevated in patients with athero-occlusive disease. Serum C5a levels correlated significantly with the increase in maximum AAA diameter over the following 6 months (r = 0.319, p = 0.021). The median growth in the lowest quartile of C5a (< 70 ng/ml) was 50% less compared to the highest C5a quartile (> 101 ng/ml): 1.0 mm/6 months (IQR = 0.8 mm) vs. 2.0 mm/6 months (IQR = 1.5 mm), p = 0.014. A log-linear mixed model predicted AAA expansion based on current diameter and C5a level. To our knowledge, this is the first study linking complement activation, in particular C5a serum level, with AAA progression.

TLRs are a family of signaling sensors that play a crucial role in the host immune response and are involved in the modulation of inflammatory processes. To study their contribution to abdominal aortic aneurysm (AAA) formation and development, we determined the frequency of , , , and single-nucleotide polymorphisms (SNPs) and investigated the association between polymorphisms and the risk of AAA incidence. A total of 104 patients with AAAs and 112 healthy, unrelated volunteers were screened for the presence of (2029C/T and 2258G/A), (1377C/T, 1234C/T, and -7C/A), (896A/G, 1196C/T, and 3266G/A), and (-1237T/C, -1486T/C, 1174G/A, and 2848C/T) SNPs by using PCR-RFLP analysis. The heterozygous genotype of the 2029C/T SNP was more common in patients with AAA than in healthy subjects ( < 0.0001) and was associated with at least an 8-fold increased risk of AAA incidence ( < 0.001). The wild-type genotype of the -7C/A SNP was associated with a 3-fold increased risk of hypertension ( = 0.026). The heterozygous genotype 1377C/T and -7C/A SNPs were less common in patients with AAA than in healthy subjects ( < 0.0001 and = 0.0004, respectively) and were associated with a decreased risk of AAA occurrence ( < 0.001 and = 0.0012, respectively). No relation to AAA risk was found for SNPs. Heterozygous genotypes of the 2029C/T and 1377C/T and -7C/A SNPs may serve as genetic biomarkers of AAA incidence.

The pathogenesis of abdominal aortic aneurysm (AAA) involves a central component of chronic inflammation which is predominantly mediated by myeloid cells. We hypothesized that the local inflammatory activity may be reflected in systemic alterations of neutrophil and monocyte populations as well as in soluble factors of myeloid cell activation and recruitment. To establish their marker potential, neutrophil and monocyte sub-sets were measured by flow cytometry in peripheral blood samples of 41 AAA patients and 38 healthy controls matched for age, sex, body mass index and smoking habit. Comparably, circulating factors reflecting neutrophil and monocyte activation and recruitment were assayed in plasma. Significantly elevated levels of CD16+ monocytes, activated neutrophils and newly released neutrophils were recorded for AAA patients compared with controls. In line, the monocyte chemoattractant C-C chemokine ligand 2 and myeloperoxidase were significantly increased in patients' plasma. The diagnostic value was highest for myeloperoxidase, a mediator which is released by activated neutrophils as well as CD16+ monocytes. Multivariable regression models using myeloid activation markers and routine laboratory parameters identified myeloperoxidase and D-dimer as strong independent correlates of AAA. These two biomarkers were combined to yield a diagnostic score which was subsequently challenged for confounders and confirmed in a validation cohort matched for cardiovascular disease. Importantly, the score was also found suited to predict rapid disease progression. In conclusion, D-dimer and myeloperoxidase represent two sensitive biomarkers of AAA which reflect distinct hallmarks (thrombus formation and inflammation) of the pathomechanism and, when combined, may serve as diagnostic and prognostic AAA score warranting further evaluation.

Thrombospondin-1 (TSP-1) is primarily expressed by platelets and endothelial cells (ECs) and rapidly released upon their activation. It functions in haemostasis as a bridging molecule in platelet aggregation, by promoting platelet adhesion to collagen and by protecting von Willebrand factor strings from degradation. In blood of patients undergoing surgery and in co-cultures of neutrophils with platelets or ECs, we observed proteolysis of the 185 kDa full-length TSP-1 to a 160-kDa isoform. We hypothesized that TSP-1 processing may alter its haemostatic properties. Selective enzyme inhibitors in co-cultures revealed that neutrophil proteases elastase and cathepsin G mediate TSP-1 processing. The cut site of cathepsin G was mapped to TSP-1 amino acids R237/T238 by Edman sequencing. Formation of neutrophil extracellular traps protected TSP-1 from complete degradation and promoted controlled processing to the 160-kDa isoform. Haemostatic properties were tested by platelet aggregation, adhesion, coagulation and string formation under flow. Platelets from TSP-1 deficient mice did not differ from wild-type in platelet aggregation but showed severe impairment of platelet adhesion to collagen and string formation under flow. Reconstitution experiments revealed that the 160-kDa TSP-1 isoform was markedly more potent than the 185-kDa full-length molecule in restoring function. Thus, TSP-1 processing by neutrophil proteases yields a 160-kDa isoform which shows enhanced potency to promote platelet adhesion and string formation. This finding reveals a novel mechanism of neutrophil-mediated thrombus formation and provides first evidence for the impact of TSP-1 proteolysis on its haemostatic properties.

Heme oxygenase-1 (HO-1), encoded by gene and regulated by Nrf2 transcription factor, is a cytoprotective enzyme. Its deficiency may exacerbate abdominal aortic aneurysm (AAA) development, which is also often associated with hyperlipidemia. Beneficial effects of statins, the broadly used antilipidemic drugs, were attributed to modulation of Nrf2/HO-1 axis. However, the effect of statins on Nrf2/HO-1 pathway in patients with AAA has not been studied yet. We analyzed AAA tissue from patients treated with simvastatin ( = 28) or without statins ( = 14). Simvastatin treatment increased HO-1 protein level in AAA, both in endothelial cells (ECs) and in smooth muscle cells (SMCs), but increased Nrf2 localization was restricted only to vasa vasorum. Nrf2 target genes , , and expression remained unchanged in AAA. studies showed that simvastatin raises HO-1 protein level slightly in ECs and to much higher extent in SMCs, which is not related to Nrf2/ARE activation, although expression is upregulated by simvastatin in both cell types. In conclusion, simvastatin-induced modulation of HO-1 level in ECs and SMCs is not related to Nrf2/ARE activity. Likewise, divergent HO-1 and Nrf2 localization together with stable expression of Nrf2 target genes, including , in AAA tissue denotes Nrf2 independency.

The oncogenic potential of the transcriptional repressor Bcl-6 (B-cell lymphoma 6) was originally discovered in non-Hodgkin patients and the soluble Bcl-6 inhibitor 79-6 was developed to treat diffuse large B-cell lymphomas with aberrant Bcl-6 expression. Since we found Bcl-6 and its co-repressor BCoR (Bcl-6 interacting co-repressor) to be regulated in human microvascular endothelium by colorectal cancer cells, we investigated their function in sprouting angiogenesis which is central to tumor growth. Based on Bcl-6/BCoR gene silencing we found that the transcriptional repressor complex in fact constitutes an endogenous inhibitor of vascular sprouting by supporting the stalk cell phenotype: control of Notch target genes (HES1, HEY1, DLL4) and cell cycle regulators (cyclin A and B1). Thus, when endothelial cells were transiently transfected with Bcl-6 and/or BCoR siRNA, vascular sprouting was prominently induced. Comparably, when the soluble Bcl-6 inhibitor 79-6 was applied in the mouse retina model of physiological angiogenesis, endothelial sprouting and branching were significantly enhanced. To address the question whether clinical treatment with 79-6 might therefore have detrimental therapeutic effects by promoting tumor angiogenesis, mouse xenograft models of colorectal cancer and diffuse large B-cell lymphoma were tested. Despite a tendency to increased tumor vessel density, 79-6 therapy did not enhance tumor expansion. In contrast, growth of colorectal carcinomas was significantly reduced which is likely due to a combined 79-6 effect on cancer cells and tumor stroma. These findings may provide valuable information regarding the future clinical development of Bcl-6 inhibitors.