Thomas Aschacher

Characteristics of TAV- and BAV-associated thoracic aortic aneurysms—Smooth muscle cell biology, expression profiling, and histological analyses

OBJECTIVE Past studies on the pathogenesis of thoracic aortic aneurysms have, by concentrating on histological and total tissue analyses, revealed several disease-relevant processes. Despite these studies, there is still a significant lack in the understanding of aneurysmal cell biology today. Hence, it was the goal of this study to assess differences between aneurysmal and healthy aortic smooth muscle cells (SMCs) on a broad - screening-like - basis, allowing us to formulate new hypotheses on the role of SMCs in thoracic aneurysm formation. METHODS AND RESULTS After histological characterization of a total of 16 samples from healthy aortas and thoracic aortic aneurysms (TAA) of patients with bicuspid (BAV) and tricuspid (TAV) aortic valves, we isolated aortic SMCs and subjected them to cell biological and gene expression analyses. The data obtained indicate that aneurysmal SMCs exert reduced proliferation and migration rates compared to controls. BAV TAA SMCs have significantly shorter telomeres, whereas TAV TAA SMCs showed a reduced metabolic activity. In BAV TAA SMCs osteopontin (OPN) expression was significantly elevated, and TAV TAA SMCs showed decreased expression of tissue inhibitor of metalloproteinase 3 (TIMP3). CONCLUSION Our study provides evidence that TAA-associated aortic wall disintegration in BAV and TAV TAAs shows similarities, but also significant differences. BAV and TAV TAAs differ with regard to medial elastic fiber mass and the occurrence of fibroblasts, SMC telomere length, metabolism, and gene expression. This study may form the basis for future in-depth analyses on the relevance of these findings in the pathophysiology of BAV and TAV TAAs.

LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

PICSO: from myocardial salvage to tissue regeneration

Despite advances in primary percutaneous interventions (PPCI), management of microvascular obstructions in reperfused myocardial tissue remains challenging and is a high-risk procedure. This has led to renewed interest in the coronary venous system as an alternative route of access to the myocardium. This article reviews historical data describing therapeutic options via cardiac veins as well as discussing the clinical potential and limitations of a catheter intervention: pressure controlled intermittent coronary sinus occlusion (PICSO). Collected experimental and clinical information suggest that PICSO also offers the potential for tissue regeneration beyond myocardial salvage. A meta-analysis of observer controlled pICSO application in animal studies showed a dose dependent reduction in infarct size of 29.3% (p < 0.001). Additionally, a 4-fold increase of hemeoxygenase-1 gene expression (p < 0.001) in the center of infarction and a 2.5 fold increase of vascular endothelial growth factor (VEGF) (p < 0.002) in border zones suggest that molecular pathways are initiating structural maintenance. Early clinical evidence confirmed significant salvage and event free survival in patients with acute myocardial infarction and risk reduction for event free survival 5 years after the acute event (p < 0.0001). This experimental and clinical evidence was recently corroborated using modern PICSO technology in PPCI showing a significant reduction of infarct size, when compared to matched controls (p < 0.04). PICSO enhances redistribution of flow towards deprived zones, clearing microvascular obstruction and leading to myocardial protection. Beyond salvage, augmentation of molecular regenerative networks suggests a second mechanism of PICSO involving the activation of vascular cells in cardiac veins, thus enhancing structural integrity and recovery.

IL-24 sensitizes tumor cells to TLR3-mediated apoptosis.

Interleukin-24 (IL-24), a member of the IL-10 cytokine family whose physiological function remains largely unknown, has been shown to induce apoptosis when expressed in an adenoviral background. It is yet little understood, why IL-24 alone induced apoptosis only in a limited number of tumor cell lines. Analyzing an influenza A virus vector expressing IL-24 for its oncolytic potential revealed enhanced pro-apoptotic activity of the chimeric virus compared with virus or IL-24 alone. Interestingly, IL-24-mediated enhancement of influenza-A-induced apoptosis did not require viral replication but critically depended on toll-like receptor 3 (TLR3) and caspase-8. Immunoprecipitation of TLR3 showed that infection by influenza A virus induced formation of a TLR3-associated signaling complex containing TRIF, RIP1, FADD, cFLIP and pro-caspase-8. Co-administration of IL-24 decreased the presence of cFLIP in the TLR3-associated complex, converting it into an atypical, TLR3-associated death-inducing signaling complex (TLR3 DISC) that induced apoptosis by enabling caspase-8 activation at this complex. The sensitizing effect of IL-24 on TLR3-induced apoptosis, mediated by influenza A virus or the TLR3-specific agonist poly(I:C), was also evident on tumor spheroids. In conclusion, rather than acting as an apoptosis inducer itself, IL-24 sensitizes cancer cells to TLR-mediated apoptosis by enabling the formation of an atypical DISC which, in the case of influenza A virus or poly(I:C), is associated with TLR3.

Targeting an Oncolytic Influenza A Virus to Tumor Tissue by Elastase

Oncolytic viruses are currently established as a novel type of immunotherapy. The challenge is to safely target oncolytic viruses to tumors. Previously, we have generated influenza A viruses (IAVs) containing deletions in the viral interferon antagonist. Those deletions have attenuated the virus in normal tissue but allowed replication in tumor cells. IAV entry is mediated by hemagglutinin (HA), which needs to be activated by a serine protease, for example, through trypsin. To further target the IAV to tumors, we have changed the trypsin cleavage site to an elastase cleavage site. We chose this cleavage site because elastase is expressed in the tumor microenvironment. Moreover, the exchange of the cleavage site previously has been shown to attenuate viral growth in lungs. Newly generated elastase-activated influenza viruses (AE viruses) grew to similar titers in tumor cells as the trypsin-activated counterparts (AT viruses). Intratumoral injection of AE viruses into syngeneic B16f1 melanoma-derived tumors in mice reduced tumor growth similar to AT viruses and had a better therapeutic effect in heterologous human PANC-1-derived tumors. Therefore, the introduction of the attenuation marker “elastase cleavage site” in viral HA allows for safe, effective oncolytic virus therapy.

Tibiodistal vein bypass in critical limb ischemia and its role after unsuccessful tibial angioplasty

Objective: Technical progress in angioplasty expanded its application to very distal arterial lesions of the lower extremity. In cases of unsuccessful angioplasty tibiodistal bypass surgery may be required for limb salvage. We investigated the long‐term outcome of this technique in patients with critical limb ischemia. The purpose of this study was to evaluate whether tibiodistal bypasses done after unsuccessful tibial angioplasty had inferior patency, limb salvage, or survival rates compared with primary tibiodistal bypasses. Methods: This single‐center, retrospective data analysis included all distal bypass procedures originating from a tibial artery. Primary study end points were primary patency, secondary patency, and limb salvage. Secondary end points included survival, wound healing, and systemic and local complications. Society for Vascular Surgery reporting standards were applied. Results: There were 61 tibiodistal vein bypasses for critical limb ischemia performed in 23 years. Indications for tibiodistal bypass was Rutherford category 5 in 41 cases (67%) and category 6 in 20 cases (33%). Procedures were allocated to group A (primary bypass; n = 28) and group B (bypass after unsuccessful tibial angioplasty; n = 33). Primary patency was 55% versus 53% at 1 year and 47% versus 44% at 3 years (P = .58). Secondary patency was 59% versus 64% at 1 year and 52% versus 55% at 3 years (P = .36). Limb salvage was 96% versus 90% at 1 year and 91% versus 85% at 3 years (P = .44). Overall survival rates were 91% versus 97% at 1 year and 85% versus 92% at 3 years (P = .76). The median follow‐up was 4.0 years in group A and 4.9 years in group B. In multivariate analyses for loss of primary patency and limb loss, no significant predictors could be identified. Conclusions: This study showed that tibiodistal vein bypass is a feasible, efficient, and safe technique in patients with critical limb ischemia. It provides acceptable primary and secondary patency rates to prevent major amputation and ensure survival. Previous unsuccessful tibial angioplasty had no significant impact on tibiodistal vein bypass outcome. This technique should be part of the armamentarium of vascular surgeons.

Telomere Biology and Thoracic Aortic Aneurysm

Ascending aortic aneurysms are mostly asymptomatic and present a great risk of aortic dissection or perforation. Consequently, ascending aortic aneurysms are a source of lethality with increased age. Biological aging results in progressive attrition of telomeres, which are the repetitive DNA sequences at the end of chromosomes. These telomeres play an important role in protection of genomic DNA from end-to-end fusions. Telomere maintenance and telomere attrition-associated senescence of endothelial and smooth muscle cells have been indicated to be part of the pathogenesis of degenerative vascular diseases. This systematic review provides an overview of telomeres, telomere-associated proteins and telomerase to the formation and progression of aneurysms of the thoracic ascending aorta. A better understanding of telomere regulation in the vascular pathology might provide new therapeutic approaches. Measurements of telomere length and telomerase activity could be potential prognostic biomarkers for increased risk of death in elderly patients suffering from an aortic aneurysm.

The megaaortic syndrome: Progression of ascending aortic aneurysm or a disease of distinct origin?

BACKGROUND Thoracic aortic aneurysm (TAA) is an often asymptomatic disease with fatal outcome, such as dissection or rupture. The megaaortic syndrome (MAS) is an extensive dilatation of the whole aorta with low incidence but high lethal outcome with unknown pathophysiology so far. METHODS AND RESULTS We compared aortic tissue of patients with sporadic TAAs and MAS of the ascending aorta with non-aneurysmal control tissues. Specimens of MAS patients showed a significantly reduced thickness of the media but an increased thickness of the intima compared to control tissue and TAAs with moderate dilatation. Advanced media degeneration however was detectable in both, TAAs with enhanced luminal diameter and MAS specimens, accompanied by reduced medial smooth muscle cell-density. Further specimens of MAS were characterized by massive atherosclerotic lesions in contrast to specimens of sporadic TAA patients. Infiltrations of macrophages in atherosclerotic lesions but also in the media adjacent to the adventitia were significantly elevated in tissue of TAAs with dilatation ≤6cm. Of note, atherosclerotic plaque-associated macrophages as well as those in the external media produce huge amounts of MMP-9 which is possibly involved in media degeneration and tissue destruction. CONCLUSIONS Taken together these results demonstrate that the pathology of MAS shows similarities with that of TAAs but pathological differences in the ascending aorta, suggesting that MAS might be a disease of different origin.

STIMULATING ANGIOGENESIS WITH A TRANS-CORONARY SINUS CATHETER INTERVENTION IN AN ISCHEMIA/REPERFUSION PORCINE ANIMAL MODEL

We examined the potential of a trans-coronary sinus catheter intervention activating endothelium to induce angiogenesis and the potential of temporal coronary venous pressure elevation (PICSO) to initiate cardiac repair in an ischemia/reperfusion model. 32 open chest pigs were divided: sham-

Abstract LB-084: A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres

LINE-1 elements (L1s, long interspersed nuclear elements-1) are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Here we show the involvement of L1 in telomere maintenance in cells using the less characterized alternative lengthening of telomeres (ALT) mechanism. Interestingly, ALT cells show significantly higher expression levels of L1 when compared to TA cells. Knock-down (KD) of L1 in ALT cell lines was associated with reduced length of telomeres, an increase in telomere dysfunction foci, a reduced expression of ALT characteristics such as c-circles and ALT associated PML bodies, and a decreased growth. On the other hand, overexpression of L1 in ALT cell lines lead to a higher rate of c-circles and increased length of telomeres. LINE-1 not only bound to the C-strand of telomeric DNA but also to telomere-repeat-containing RNA (TERRA), a multifunctional component of human telomeres that is highly expressed in ALT cells and has been described to be involved in telomere stabilization. Whereas L1-KD decreased overall TERRA, L1 overexpression increased this RNA. Moreover, L1 KD abrogated the nuclear retention of TERRA. Moreover, the L1-ribonucleoprotein can use the polyadenylated form of TERRA as a template to generate telomere-specific DNA. Thus, L1 appears to contribute to telomere maintenance by a mechanism involving TERRA, either by ensuring its nuclear localization or by using this RNA as a template for the generation of telomere-specific DNA. Our findings now render L1 proteins a promising target in cancer therapy by interfering with telomere lengthening in ALT cells. Citation Format: Thomas Aschacher, Brigitte Wolf, Philip Kienzl, Florian Enzmann, Barbara Messner, Klaus Holzmann, Michael M. Bergmann. A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-084. doi:10.1158/1538-7445.AM2015-LB-084