Maria Kokozidou

Chronic kidney disease aggravates arteriovenous fistula damage in rats.

Neointimal hyperplasia (NIH) and impaired dilatation are important contributors to arteriovenous fistula (AVF) failure. It is unclear whether chronic kidney disease (CKD) itself causes adverse remodeling in arterialized veins. Here we determined if CKD specifically triggers adverse effects on vascular remodeling and assessed whether these changes affect the function of AVFs. For this purpose, we used rats on a normal diet or on an adenine-rich diet to induce CKD and created a fistula between the right femoral artery and vein. Fistula maturation was followed noninvasively by high-resolution ultrasound (US), and groups of rats were killed on 42 and 84 days after surgery for histological and immunohistochemical analyses of the AVFs and contralateral femoral vessels. In vivo US and ex vivo morphometric analyses confirmed a significant increase in NIH in the AVFs of both groups with CKD compared to those receiving a normal diet. Furthermore, we found using histological evaluation of the fistula veins in the rats with CKD that the media shrank and their calcification increased significantly. Afferent artery dilatation was significantly impaired in CKD and the downstream fistula vein had delayed dilation after surgery. These changes were accompanied by significantly increased peak systolic velocity at the site of the anastomosis, implying stenosis. Thus, CKD triggers adverse effects on vascular remodeling in AVFs, all of which contribute to anatomical and/or functional stenosis.

ADAM12 is expressed by astrocytes during experimental demyelination

A disintegrin and metalloproteinase (ADAM) 12 represents a member of a large family of similarly structured multi-domain proteins. In the central nervous system (CNS), ADAM12 has been suggested to play a role in brain development, glioblastoma cell proliferation, and in experimental autoimmune encephalomyelitis. Furthermore, ADAM12 was reported to be almost exclusively expressed by oligodendrocytes and could, therefore, be considered as suitable marker for this cell type. In the present study, we investigated ADAM12 expression in the healthy and pathologically altered murine CNS. As pathological paradigm, we used the cuprizone demyelination model in which myelin loss during multiple sclerosis is imitated. Besides APC(+) oligodendrocytes, SMI311(+) neurons and GFAP(+) astrocytes express ADAM12 in the adult mouse brain. ADAM12 expression was further analyzed in vitro. After the induction of demyelination, we observed that activated astrocytes are the main source of ADAM12 in brain regions affected by oligodendrocyte loss. Exposure of astrocytes in vitro to either lipopolysaccharides (LPS), tumor necrosis factor alpha (TNFalpha), glutamate, or hydrogen peroxide revealed a highly stimulus-specific regulation of ADAM12 expression which was not seen in microglial BV2 cells. It appears that LPS- and TNFalpha-induced ADAM12 expression is mediated via the classic NFkappaB pathway. In summary, we demonstrated that ADAM12 is not a suitable marker for oligodendrocytes. Our results further suggest that ADAM12 might be implicated in the course of distinct CNS diseases such as demyelinating disorders.

C-Terminal truncations of syncytin-1 (ERVWE1 envelope) that increase its fusogenicity

Abstract Syncytin-1, the envelope protein of ERVWE1, an endogenous retrovirus of the HERV-W family, plays an important role in regulating fusion of the placental trophoblast. At least one of its receptors is expressed on a variety of human cell types. Its ability to fuse cells makes it an attractive candidate molecule in gene therapy against cancer. We studied the relevance of sequences in the cytoplasmic tail of syncytin-1 for inducing cell-cell fusion. We generated a series of C-terminally truncated syncytin-1 variants. Sequences immediately adjacent to the transmembrane region of syncytin-1 were necessary for inducing optimal fusion, whereas the extreme C-terminus of syncytin-1 partially inhibited its fusogenicity. Two variants of syncytin-1, truncated after residues 483 and 515, were significantly hyperfusogenic compared to wild-type syncytin-1. Cellular and cell-surface expression levels of these two variant proteins were similar to those of wild-type syncytin-1. In testing the latter we found that only a very minor portion of recombinantly expressed cellular syncytin-1 was fully mature and expressed on the cell surface. Our results contribute to the understanding of the structure-function relationship of syncytin-1, and might have implications for the use of this molecule in gene therapy.

Complex patterns of ADAM12 mRNA and protein splice variants in the human placenta

AIMS Trophoblast fusion in the placenta is prerequisite to successful pregnancy and the pathological conditions related to it. The presence of syncytin-1, is not sufficient to explain the complete event and ADAM12 is a major co-player candidate. Via differential splicing, the ADAM12 gene produces a short and a long form, being the ADAM12-S and the ADAM12-L respectively. METHODS AND RESULTS We investigated the localisation of both variants in the human placenta using whole mount in situ hybridisation, immunohistochemistry and Northern blotting in 1st (n=8) and 3rd (n=8) trimester placentae and in the case of NB in several cell lines. In Northern blotting, 1st and 3rd trimester placentae were positive for the ADAM12-S and Bewo, 293HEK, JAR, leucocytes, macrophages, 1st and 3rd trimester placentae were positive for ADAM12-L. In whole mount in situ hybridisation, the 1st and 3rd trimester placental syncytium was positive for both variants. In immunohistochemistry, ADAM12-L localised in the cytotrophoblast of both 1st and 3rd trimester placentae, while ADAM12-S localised in the complete syncytium, often including the cytotrophoblast. CONCLUSION The different localisation of ADAM12-S and ADAM12-L indicates a possible different role making ADAM12-L a candidate for the fusion event, while the syncytial localisation of the ADAM12-S makes it a candidate for cell-cell and cell-matrix interactions between the placental syncytium and the maternal interface.

Chemoselective Oxime Reactions in Proteins and Peptides by Using an Optimized Oxime Strategy: The Demise of Levulinic Acid

form a special imine known as an oxime (3). Advantages of this strategy include chemoselectivity, as a ketone is inert to most other reactions, and the mild conditions in which the reaction can be performed. Oxime formation has been thoroughly investigated and was found to proceed in a step-wise manner, depending on pH. Although the reaction proceeds faster in acidic conditions (pH 4–5), oximes will also form at neutral pH. Furthermore, oxime formation can be accelerated by addition of the catalyst aniline, and oxime linkages are stable at neutral pH. Taken together, these properties make the oxime linkage one of the preferred methods for the chemoselective modification of peptides and proteins. Levulinic acid (LA) is one of the most frequently used ketones for oxime formation, and is generally introduced by attachment to a lysine side chain e-NH2 or the N-terminal NH2 moiety. The reaction between the protein–LA complex and an aminooxy moiety proceeds well at millimolar concentrations; however, the relatively low quantity and high molecular weight of most proteins result in sub-millimolar protein concentrations and limitations of the oxime reaction. Under these conditions, we and others have found that oxime reaction yields are low because of the formation of a levulinoyl-derived side-product that competes with oxime bond formation. As oxime ligations are increasingly attractive and provide an orthogonal approach to label proteins, we explored alternative ketone moieties for bioconjugation. Our hypothesis was that the levulinoyl side-product (corresponding to a mass loss of 18 Da) is the result of intramolecular cyclization of LA, thus preventing the LA ketone group from reacting with the aminooxy moiety. The formation of this by-product is mainly seen at low concentrations, because under these conditions the concentration-independent cyclization side-reaction benefits from the slow oxime formation. To study the molecular mechanism underlying levulinoyl cyclization, the pentapeptide LYRAK was synthesized with LA coupled to the lysine e-amine (LYRAK(LA)). Lyophilized LYRAK(LA) was dissolved in water under acidic conditions (pH 4.5) and was left to cyclize spontaneously for 72 h at room temperature. The conversion to cyclized derivatives was monitored by ESI-MS and NMR (both in [D6]DMSO and D2O) and structurally characterized by 2D NMR experiments (Figures S1 and S2). The cyclization of LA was tested at three concentrations and was shown to be concentration independent (Figure S3). Based on these results, the following reaction mechanism for intramolecular cyclization of the levulinoyl moiety was proposed (Scheme 2). The amide nitrogen in the linear levulinoyl peptide 4 performs a nucleophilic attack on the ketone carbonyl group resulting in the cyclic intermediate 5. This reaction was previously reported to be favored by protonation of the carbonyl. Spontaneous dehydration of 5 leads to the iminium species 6, which is stabilized by TFA in the solution. Spontaneous isomerization of 6 leads to 7 and 8, as can determined by 2D NMR methods (Figure S1). Conversion from 5 to the exocyclic-double-bond-containing species 7 has been reported, but formation of the endocyclic double bond has not. Here, 8 was observed but it was formed as a minor product compared Scheme 1. Overall oxime reaction. A ketone or aldehyde (1) reacts with an aminooxy (2) to form an oxime bond (3).

Cardiovascular remodeling during arteriovenous fistula maturation in a rodent uremia model

Purpose The aim of this study was to evaluate cardiovascular remodeling after arteriovenous fistula (AVF) surgery and to characterize the effect of chronic kidney disease (CKD) in a rodent femoral AVF model. Methods Sixteen rats (8 healthy; 8 CKD) underwent femoral AVF surgery; 4 animals served as controls. AVF and cardiac morphology as well as function were assessed during the fistula maturation process (until day 84 after surgery) using magnetic resonance imaging and histopathological analyses. Results Histopathological analysis revealed that a glomerular and interstitial nephropathy caused CKD. In healthy and CKD animals, AVF surgery resulted in progressive downstream vein dilation and a subsequent cardiac adaptation. This vein dilation during maturation was less in CKD rats during the early postoperative course (day 21: p=0.0475) and similar thereafter until day 84. The dilation was accompanied by an aggravation of neointimal hyperplasia (NIH) and calcification in AVFs of CKD rats. The chronic volume overload resulted in both groups in a significantly increased end-diastolic volume (healthy rats: p=0.0087; CKD rats: p=0.0333). Simultaneously, cardiac output increased 195% in healthy and 244% in uremic rats, which was caused by both a significantly increased stroke volume and heart rate. The left ventricular mass rose in AVF animals and was increased at the end of the study period, indicating a distinct cardiac hypertrophy. Conclusion Our rat model showed typical cardiovascular features of the AVF maturation process, which strongly resemble clinical findings in patients. Uremia caused inferior dilation in the early phase after surgery and an exacerbation of NIH. This model should help to identify the cellular and molecular mechanisms that contribute to AVF failure.

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.(1-5) The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. (6-8) The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia. Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.(9-11) This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., (9) as well as others.(12-14) Nevertheless, these cited small animal protocols are complicated. To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8±3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Cardiovascular remodeling after AVF surgery in rats assessed by a clinical MRI scanner

PURPOSE To evaluate a cardiovascular magnetic resonance imaging (MRI) technique which allows the longitudinal analysis of cardiovascular remodeling in a rodent femoral arteriovenous fistula (AVF) model by means of a clinical scanner. MATERIALS AND METHODS Eight rats underwent femoral AVF surgery and four rats served as controls. Vascular and cardiac morphology as well as cardiac function was assessed from Week 3 to 12 using contrast-enhanced, time-resolved magnetic resonance angiography (MRA) and cardiac MRI (cine gradient-echo sequence) at 3 T in one imaging session. RESULTS Arteriovenous surgery resulted in progressive venous dilation and a subsequent cardiac adaptation. This procedure led to downstream vasodilation of the iliac vein and inferior vena cava of 179% and 188%, respectively (3 weeks). To accommodate the increased returning blood volume, cardiac output (CO) increased significantly (P=.014; 6 weeks). This was caused by increased end-diastolic volume (EDV), stroke volume (SV) and heart rate (HR) consistent with an increased volume load. A continuous increase in heart weight peaked at 12 weeks. This increase combined with a distinct end-diastolic left ventricular dilation implied eccentric hypertrophy. CONCLUSION Small rodent MRI is feasible and clearly depicts fistula maturation and cardiac alterations. This technique proved to be a valuable tool for longitudinal in vivo monitoring in this model, which strongly resembles clinical findings in hemodialysis patients.

Selective renal blood perfusion induces renal tubules injury in a porcine model

OBJECTIVE Extracorporeal circulation is routinely used in thoracoabdominal aortic aneurysm repair to preserve blood perfusion. Despite this protective measure, acute and chronic kidney disorders can develop. Therefore, the aim of this study was to establish a new large-animal model to assess the efficacy of selective renal perfusion (SRP) with extracorporeal circulation in a setting of thoracoabdominal aortic aneurysm repair. METHODS Eighteen pigs underwent a thoracolaparotomy, during with the aorta and renal arteries were exposed. The animals were divided into three cohorts of six pigs each: cohort I--control; cohort II--thoracic aortic clamping with distal aortic perfusion (DAP) using a roller pump; and cohort III--thoracic aortic clamping with DAP plus SRP. Kidney metabolism, kidney injury, and red blood cell damage were measured by oxygen extraction ratio (O2ER), neutrophil gelatinase-associated lipocalin, a marker for acute kidney damage, and serum free hemoglobin. RESULTS With normal mean arterial blood pressures, flow rates in the renal arteries during perfusion decreased to 75% (group II) with DAP and to 50% (group III) with SRP compared with the control animals (group I; P = .0279 for I vs II; P = .0002 for I vs III). Microcirculation, measured by microspheres, did not differ significantly among the groups. In contrast, O2ER (P = .0021 for I vs III) and neutrophil gelatinase-associated lipocalin (P = .0083 for I vs III) levels were significantly increased in group III, whereas free hemoglobin was increased in groups II and III (P = .0406 for I vs II; P = .0018 for I vs III). CONCLUSIONS SRP with a roller pump induces kidney tubule injury. Thus, distal aortic and SRP in our model does not provide adequate kidney protection. Furthermore, the perfusion system provokes red blood cell damage with increased free hemoglobin. Hence, the SRP perfusion technique should be revised and tested.