Edge restenosis: impact of low dose irradiation on cell proliferation and ICAM-1 expression

BACKGROUND: Low dose irradiation (LDI) of uninjured segments is the consequence of the suggestion of many authors to extend the irradiation area in vascular brachytherapy to minimize the edge effect. Atherosclerosis is a general disease and the uninjured segment close to the intervention area is often atherosclerotic as well, consisting of neointimal smooth muscle cells (SMC) and quiescent monocytes (MC). The current study imitates this complex situation in vitro and investigates the effect of LDI on proliferation of SMC and expression of intercellular adhesion molecule-1 (ICAM-1) in MC. METHODS: Plaque tissue from advanced primary stenosing lesions of human coronary arteries (9 patients, age: 61 ± 7 years) was extracted by local or extensive thrombendarterectomy. SMC were isolated and identified by positive reaction with smooth muscle α-actin. MC were isolated from buffy coat leukocytes using the MACS cell isolation kit. For identification of MC flow-cytometry analysis of FITC-conjugated CD68 and CD14 (FACScan) was applied. SMC and MC were irradiated using megavoltage photon irradiation (CLINAC2300 C/D, VARIAN, USA) of 6 mV at a focus-surface distance of 100 cm and a dose rate of 6 Gy min(-1 )with single doses of 1 Gy, 4 Gy, and 10 Gy. The effect on proliferation of SMC was analysed at day 10, 15, and 20. Secondly, total RNA of MC was isolated 1 h, 2 h, 3 h, and 4 h after irradiation and 5 μg of RNA was used in standard Northern blot analysis with ICAM-1 cDNA-probes. RESULTS: Both inhibitory and stimulatory effects were detected after irradiation of SMC with a dose of 1 Gy. At day 10 and 15 a significant antiproliferative effect was found; at day 20 after irradiation cell proliferation was significantly stimulated. Irradiation with 4 Gy and 10 Gy caused dose dependent inhibitory effects at day 10, 15, and 20. Expression of ICAM-1 in human MC was neihter inhibited nor stimulated by LDI. CONCLUSION: Thus, the stimulatory effect of LDI on SMC proliferation at day 20 days after irradiation may be the in vitro equivalent of a beginning edge effect. Extending the irradiation area in vascular brachytherapy in vivo may therefore merely postpone and not inhibit the edge effect. The data do not indicate that expression of ICAM-1 in quiescent MC is involved in the process

Pathogenic variants in cardiomyopathy disorder genes underlie pediatric myocarditis - Further impact of heterozygous immune disorder gene variants?

Myocarditis is an inflammatory disease of the heart. Pediatric myocarditis with the dilated cardiomyopathy (DCM) phenotype may be caused by likely pathogenic or pathogenic genetic variants [(L)P] in cardiomyopathy (CMP) genes. Systematic analysis of immune disorder gene defects has not been performed so far. We analyzed 12 patients with biopsy-proven myocarditis and the DCM phenotype together with their parents using whole-exome sequencing (WES). The WES data were filtered for rare pathogenic variants in CMP (n = 89) and immune disorder genes (n = 631). Twelve children with a median age of 2.9 (1.0–6.8) years had a mean left ventricular ejection fraction of 28% (22–32%) and myocarditis was confirmed by endomyocardial biopsy. Patients with primary immunodeficiency were excluded from the study. Four patients underwent implantation of a ventricular assist device and subsequent heart transplantation. Genetic analysis of the 12 families revealed an (L)P variant in the CMP gene in 8/12 index patients explaining DCM. Screening of recessive immune disorder genes identified a heterozygous (L)P variant in 3/12 index patients. This study supports the genetic impact of CMP genes for pediatric myocarditis with the DCM phenotype. Piloting the idea that additional immune-related genetic defects promote myocarditis suggests that the presence of heterozygous variants in these genes needs further investigation. Altered cilium function might play an additional role in inducing inflammation in the context of CMP

Colocalization of connexin 36 and corticotropin-releasing hormone in the mouse brain

<p>Abstract</p> <p>Background</p> <p>Gap junction proteins, connexins, are expressed in most endocrine and exocrine glands in the body and are at least in some glands crucial for the hormonal secretion. To what extent connexins are expressed in neurons releasing hormones or neuropeptides from or within the central nervous system is, however, unknown. Previous studies provide indirect evidence for gap junction coupling between subsets of neuropeptide-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Here we employ double labeling and retrograde tracing methods to investigate to what extent neuroendocrine and neuropeptide-containing neurons of the hypothalamus and brainstem express the neuronal gap junction protein connexin 36.</p> <p>Results</p> <p>Western blot analysis showed that connexin 36 is expressed in the PVN. In bacterial artificial chromosome transgenic mice, which specifically express the reporter gene Enhanced Green Fluorescent Protein (EGFP) under the control of the connexin 36 gene promoter, EGFP expression was detected in magnocellular (neuroendocrine) and in parvocellular neurons of the PVN. Although no EGFP/connexin36 expression was seen in neurons containing oxytocin or vasopressin, EGFP/connexin36 was found in subsets of PVN neurons containing corticotropin-releasing hormone (CRH), and in somatostatin neurons located along the third ventricle. Moreover, CRH neurons in brainstem areas, including the lateral parabrachial nucleus, also expressed EGFP/connexin 36.</p> <p>Conclusion</p> <p>Our data indicate that connexin 36 is expressed in subsets of neuroendocrine and CRH neurons in specific nuclei of the hypothalamus and brainstem.</p

Time course study of oxidative and nitrosative stress and antioxidant enzymes in K(2)Cr(2)O(7)-induced nephrotoxicity

BACKGROUND: Potassium dichromate (K(2)Cr(2)O(7))-induced nephrotoxicity is associated with oxidative and nitrosative stress. In this study we investigated the relation between the time course of the oxidative and nitrosative stress with kidney damage and alterations in the following antioxidant enzymes: Cu, Zn superoxide dismutase (Cu, Zn-SOD), Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT). METHODS: Nephrotoxicity was induced in rats by a single injection of K(2)Cr(2)O(7). Groups of animals were sacrificed on days 1,2,3,4,6,8,10, and 12. Nephrotoxicity was evaluated by histological studies and by measuring creatinine clearance, serum creatinine, blood urea nitrogen (BUN), and urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and total protein. Oxidative and nitrosative stress were measured by immunohistochemical localization of protein carbonyls and 3-nitrotyrosine, respectively. Cu, Zn-SOD, Mn-SOD, and CAT were studied by immunohistochemical localization. The activity of total SOD, CAT, GPx, and GR was also measured as well as serum and kidney content of chromium and urinary excretion of NO(2 )(-)/NO(3)(-). Data were compared by two-way analysis of variance followed by a post hoc test. RESULTS: Serum and kidney chromium content increased reaching the highest value on day 1. Nephrotoxicity was made evident by the decrease in creatinine clearance (days 1–4) and by the increase in serum creatinine (days 1–4), BUN (days 1–6), urinary excretion of NAG (days 1–4), and total protein (day 1–6) and by the structural damage to the proximal tubules (days 1–6). Oxidative and nitrosative stress were clearly evident on days 1–8. Urinary excretion of NO(2)(-)/NO(3)(- )decreased on days 2–6. Mn-SOD and Cu, Zn-SOD, estimated by immunohistochemistry, and total SOD activity remained unchanged. Activity of GPx decreased on days 3–12 and those of GR and CAT on days 2–10. Similar findings were observed by immunohistochemistry of CAT. CONCLUSION: These data show the association between oxidative and nitrosative stress with functional and structural renal damage induced by K(2)Cr(2)O(7). Renal antioxidant enzymes were regulated differentially and were not closely associated with oxidative or nitrosative stress or with kidney damage. In addition, the decrease in the urinary excretion of NO(2)(-)/NO(3)(- )was associated with the renal nitrosative stress suggesting that nitric oxide was derived to the formation of reactive nitrogen species involved in protein nitration

Quantification of the Temporal Evolution of Collagen Orientation in Mechanically Conditioned Engineered Cardiovascular Tissues

Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network

Low Drift Thrust Balance with High Resolution

In this paper we present a small thrust balance for thrusters up to 2 kg developed by AST Advanced Space Technologies GmbH in cooperation with Deutsches Zentrum für Luft- und Raumfahrt e.V. (DLR). The measurement range is optimized for 0.1mN to 250mN while the full range is up to 1 N. In high dynamic mode the balance is able to resolve a pulsed force of 100mN with a duration of 100ms at a repetition rate of 1 Hz. By design the thrust balance is very insensitive to external disturbances. This leads to a very low long term drift which could be demonstrated to be smaller than 250 µN over 27 hours within a non-vacuum laboratory environment with only rudimentary decoupling from external influences