An integrative systems approach identifies novel candidates in Marfan syndrome-related pathophysiology.

Marfan syndrome (MFS) is an autosomal dominant genetic disorder caused by mutations in the FBN1 gene. Although many peripheral tissues are affected, aortic complications, such as dilation, dissection and rupture, are the leading causes of MFS-related mortality. Aberrant TGF-beta signalling plays a major role in the pathophysiology of MFS. However, the contributing mechanisms are still poorly understood. Here, we aimed at identifying novel aorta-specific pathways involved in the pathophysiology of MFS. For this purpose, we employed the Fbn1 under-expressing mgR/mgR mouse model of MFS. We performed RNA-sequencing of aortic tissues of 9-week-old mgR/mgR mice compared with wild-type (WT) mice. With a false discovery rat

Translational Medicine: Towards Gene Therapy of Marfan Syndrome

Marfan syndrome (MFS) is one of the most common inherited disorders of connective tissue caused by mutations of the fibrillin-1 gene (FBN1). Vascular abnormalities, such as the enlargement of the aorta with the risk of life-threatening rupture are frequently observed. However, current treatment is limited and therapeutic options focus solely on symptomatic therapy. Gene therapy focuses on genetically modifying cells to produce a therapeutic effect and may be a promising treatment option for MFS. Here, we first provide an overview of the historical background and characterization of MFS. Subsequently, we summarise current gene therapy options and possible translational concepts for this inherited disorder that affects connective tissue

Renal Safety of Hydroxyethyl starch 130/0.42 After Cardiac Surgery: A Retrospective Cohort Analysis

Introduction!#!The risk for renal complications from hydroxyethyl starch 130/0.42 (HES) impacts treatment decisions in patients after cardiac surgery.!##!Objective!#!The objective of this study was to determine the impact of postoperatively administered HES on renal function and 90-day mortality compared to sole crystalloid administration in patients after elective cardiac surgery.!##!Methods!#!Using electronic health records from a university hospital, confounding-adjusted models analyzed the associations between postoperative HES administration and the occurrence of postoperative acute kidney injury. In addition, 90-day mortality was evaluated. The impact of HES dosage and timing on renal function on trajectories of estimated glomerular filtration rates over the postoperative period was investigated using linear mixed-effects models.!##!Results!#!Overall 1009 patients (45.0%) experienced acute kidney injury. Less acute kidney injury occurred in patients receiving HES compared with patients receiving only crystalloids for fluid resuscitation (43.7% vs 51.2%, p = 0.008). In multivariate acute kidney injury models, HES had a protective association (odds ratio: 0.89; 95% confidence interval 0.82-0.96). Crystalloids were not as protective as HES (odds ratio: 0.98; 95% confidence interval 0.95-1.00). There was no association between HES and 90-day mortality (odds ratio: 1.05; 95% confidence interval 0.88-1.25). Renal function trajectories were dose dependent and biphasic, HES appeared to slow down the late postoperative decline.!##!Conclusions!#!This study showed no association between HES and the postoperative occurrence of acute kidney injury and thus further closes the evidence gap on HES safety in cardiac surgery patients. Although this was a retrospective cohort study, the results indicated that HES might be safely administered to cardiac surgery patients with regard to renal outcomes, especially if it was administered early and dosed appropriately

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy.

OBJECTIVES:Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. METHODS:We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). RESULTS:IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.β-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space. CONCLUSIONS:Murine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome

Ischemic Colitis after Cardiac Surgery: Can We Foresee the Threat?

Ischemic colitis (IC) remains a great threat after cardiac surgery with use of extracorporeal circulation. We aimed to identify predictive risk factors and influence of early catecholamine therapy for this disease.We prospectively collected and analyzed data of 224 patients, who underwent laparotomy due to IC after initial cardiac surgery with use of extracorporeal circulation during 2002 and 2014. For further comparability 58 patients were identified, who underwent bypass surgery, aortic valve replacement or combination of both. Age ±5 years, sex, BMI ± 5, left ventricular function, peripheral arterial disease, diabetes and urgency status were used for match-pair analysis (1:1) to compare outcome and detect predictive risk factors. Highest catecholamine doses during 1 POD were compared for possible predictive potential.Patients' baseline characteristics showed no significant differences. In-hospital mortality of the IC group with a mean age of 71 years (14% female) was significantly higher than the control group with a mean age of 70 (14% female) (67% vs. 16%, p<0.001). Despite significantly longer bypass time in the IC group (133 ± 68 vs. 101 ± 42, p = 0.003), cross-clamp time remained comparable (64 ± 33 vs. 56 ± 25 p = 0.150). The majority of the IC group suffered low-output syndrome (71% vs. 14%, p<0.001) leading to significant higher lactate values within first 24h after operation (55 ± 46 mg/dl vs. 31 ± 30 mg/dl, p = 0.002). Logistic regression revealed elevated lactate values to be significant predictor for colectomy during the postoperative course (HR 1.008, CI 95% 1.003-1.014, p = 0.003). However, Receiver Operating Characteristic Curve calculates a cut-off value for lactate of 22.5 mg/dl (sensitivity 73% and specificity 57%). Furthermore, multivariate analysis showed low-output syndrome (HR 4.301, CI 95% 2.108-8.776, p<0.001) and vasopressin therapy (HR 1.108, CI 95% 1.012-1.213, p = 0.027) significantly influencing necessity of laparotomy.Patients who undergo laparotomy for IC after initial cardiac surgery have a substantial in-hospital mortality risk. Early postoperative catecholamine levels do not influence the development of an IC except vasopressin. Elevated lactate remains merely a vague predictive risk factor

Alginate hydrogel polymers enable efficient delivery of a vascular-targeted AAV vector into aortic tissue

Gene therapeutic approaches to aortic diseases require efficient vectors and delivery systems for transduction of endothelial cells (ECs) and smooth muscle cells (SMCs). Here, we developed a novel strategy to efficiently deliver a previously described vascular-specific adeno-associated viral (AAV) vector to the abdominal aorta by application of alginate hydrogels. To efficiently transduce ECs and SMCs, we used AAV9 vectors with a modified capsid (AAV9SLR) encoding enhanced green fluorescent protein (EGFP), as wild-type AAV vectors do not transduce ECs and SMCs well. AAV9SLR vectors were embedded into a solution containing sodium alginate and polymerized into hydrogels. Gels were surgically implanted around the adventitia of the infrarenal abdominal aorta of adult mice. Three weeks after surgery, an almost complete transduction of both the endothelium and tunica media adjacent to the gel was demonstrated in tissue sections. Hydrogel-mediated delivery resulted in induction of neutralizing antibodies but did not cause inflammatory responses in serum or the aortic wall. To further determine the translational potential, aortic tissue from patients was embedded ex vivo into AAV9SLR-containing hydrogel, and efficient transduction could be confirmed. These findings demonstrate that alginate hydrogel harboring a vascular-targeting AAV9SLR vector allows efficient local transduction of the aortic wall

AP-1 Oligodeoxynucleotides Reduce Aortic Elastolysis in a Murine Model of Marfan Syndrome

Marfan syndrome is characterized by high expression of matrix metalloproteinases (MMPs) in aortic smooth muscle cells (AoSMCs) associated with medial elastolysis and aortic root aneurysm. We aimed to reduce aortic elastolysis through decrease of MMP expression with decoy oligodeoxynucleotides (dODNs) neutralizing the transcription factor activating factor-1 (AP-1). AP-1 abundance in nuclear extracts as well as MMP-2 and MMP-9 expression were significantly increased in isolated mAoSMC of mgR/mgR Marfan mice compared to wild-type cells. Exposure to AP-1 neutralizing dODNs resulted in a significant reduction of basal and interleukin-1β-stimulated MMP expression and activity in mAoSMCs. Moreover, increased migration and formation of superoxide radical anions was substantially decreased in mAoSMCs by AP-1 dODN treatment. Aortic grafts from donor Marfan mice were treated with AP-1- dODN ex vivo and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Pretreatment of aortic grafts with AP-1 dODN led to reduced elastolysis, macrophage infiltration, and MMP activity. Permeability of the endothelial monolayer was increased for dODN in mgR/mgR aortae with observed loss of tight junction proteins ZO-1 and occludin, enabling dODN to reach the tunica media. Targeting AP-1 activity offers a new potential strategy to treat the vascular phenotype associated with Marfan syndrome

Intimal Hyperplasia in mgR/mgR Marfan Grafts after Adenoviral Transduction.

<p>A-E: Van Gieson stain of 30-day old mouse aortic grafts after heterotopic infrarenal transplantation (40x magnification). WT grafts point out almost no (A, NI = 1.24%) and native mgR/mgR Marfan grafts just a minor stenosis of the lumen (NI = 5.7%). Marfan graft after treatment with adenoviral vectors coding for β-galactosidase (C, NI = 23.05%) and hTIMP-1 (D, 43,45%) display intimal hyperplasia narrowing the lumen (p = 0.038). In contrast, wildtype vessels do not present intimal hyperplasia after transduction with Ad.hTIMP-1 (E, NI = 0.00%). F: Mean of neointimal index in 30-day old transplanted aortas (n = 7). Compared to native mgR/mgR grafts NI is significant greater in Ad.hTIMP-1 (p = 0.026) and borderline significant in Ad.β-Gal. transduced mgR/mgR grafts (p = 0.053). WT, wildtype aorta; mgR/mgR, Marfan aorta; Ad.β-Gal, treated with adenovirus coding for β-galactosidase; Ad.hTIMP-1, treated with adenovirus coding for human TIMP-1; NI, neointimal index, neointima / (neointima + lumen). *p-value ≤0.05; ** p-value ≤0.001, calculated using Mann-Whitney-U-Test (n = 7).</p

Overexpression of hTIMP-1 after Ad.hTIMP-1 Transduction.

<p>A: Western blot analysis for proof of hTIMP-1 overexpression after transdudtion with Ad.hTIMP-1 in HEK-293 cells. Ad.hTIMP-1 transduced cells (HEK+) demonstrated a strong signal for hTIMP-1 above the 25kDa lane (left), whereas untreated native HEK-293 (HEK-) showed weaker lane for hTIMP-1 (right). B: Immunohistochemistry of hTIMP-1 in aortic grafts after transduction with Ad.hTIMP-1 (100x magnification). Even 30 days after transduction sufficient transgene expression is detected in all layers of the aorta (left). Untreated marfan aorta was used as negative control (right). C: <i>In situ</i> zymography (100x magnification) of untretead (upper row) and Ad.hTIMP-1 treated mgR/mgR marfan grafts (lower row). Increase of light emission represents conversion of collagen substrate during incubation (24h) by Matrix-Metalloproteinases (left: pre incubation). After Ad.TIMP-1 transduction and incubation (down, right) we saw less MMP activity than in untreated aortas (up, right).</p

Neointimal index as parameter for stenosis within aortic grafts 30 days after transplantation.

<p>Neointimal index as parameter for stenosis within aortic grafts 30 days after transplantation.</p