Maggie Folkesson

Presence of NGAL/MMP-9 complexes in human abdominal aortic aneurysms

It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA enlargement and rupture. The growth of the AAA is dependent on proteolytic degradation of elastin. Here, we analysed whether the neutrophil gelatinase-associated lipocalin (NGAL) is expressed within the thrombus and the aneurysm wall. NGAL can bind to metalloproteinase-9 (MMP-9) and inhibit its degradation, thereby preserving enzymatic activity. Biopsies were obtained from thrombus-free and thrombus-covered aneurysm wall and the intraluminal thrombus from patients undergoing elective surgery for AAA. Immunohistochemistry and real-time PCR were used to study NGAL and MMP-9 expression. Immunoprecipitation, gel zymography, Western blot and ELISA were used to detect and quantify NGAL/MMP-9 complexes. NGAL was detected in the thrombus, the interface between the thrombus and the underlying wall and in the wall itself. Double staining showed that neutrophils are the major source of NGAL expression. Immunoprecipitation of MMP-9 with antibody against NGAL showed that complexes of NGAL and active MMP-9 were present in thrombus, the interface fluid and the aneurysm wall. Western blot analyses using non-reducing conditions and gel zymography demonstrated that high-molecular-weight complexes of NGAL/MMP-9 were present within the different regions. The concentration of the NGAL/MMP-9 complex was highest in the luminal part of the thrombus. In conclusion, NGAL in complex with activated MMP-9 is present in AAA wall and thrombus. Neutrophil-derived NGAL could enhance the proteolytic activity associated with AAA, but the importance of this mechanism for aneurysm growth remains to be shown.

Mast cells associate with neovessels in the media and adventitia of abdominal aortic aneurysms

OBJECTIVE Mast cells (MCs) are inflammatory cells present in atherosclerotic lesions and neovascularized tissues. Recently, MCs were shown to modulate abdominal aortic aneurysm (AAA) formation in a mouse model. Progression of aneurysmatic disease process may also depend on intraluminal thrombus and neovascularization of the aneurysm wall. Here we investigated the relationship between MCs and inflammation, neovascularization, and the presence of intraluminal thrombus in human AAA. METHODS AND RESULTS Specimens from AAAs and normal control aortas were analyzed with basic histology, immunohistochemical staining, and quantitative real-time polymerase chain reaction (PCR). Double immunostainings with endothelial cell markers CD31/CD34 and MC tryptase showed that, in contrast to histologically normal aorta, MCs in AAA were abundant in the media, but absent from the intima. Medial MCs and (CD31/CD34)(+) neovessels increased significantly in AAA compared with normal aorta (P < .0001 for both), and the highest densities of neovessels and MCs were observed in the media of thrombus-covered AAA samples. Also, the proportional thickness of aortic wall penetrated by the neovessels was significantly higher in the AAA samples (P < .0001), and the neovascularized area correlated with the density of medial MCs (P < .0001). In histologic analysis, the medial MCs were mainly located adjacent to the stem cell factor (SCF)(+) medial neovessels. Real-time PCR analysis also showed that mRNA levels of genes associated with neovascularization (vascular endothelial growth factor [VEGF], FLT1, VE-cadherin, CD31), and MCs (tryptase, chymase, cathepsin G) were higher in AAA samples than in controls. Demonstration of adhered platelets by CD42b staining and lack of endothelial cell (CD31/CD34) staining in the luminal surface of AAA specimens suggest endothelial erosion of the aneurysm walls. CONCLUSIONS The results support participation of MCs in the pathogenesis of AAA, particularly regarding neovascularization of aortic wall.

Perforin-Independent Extracellular Granzyme B Activity Contributes to Abdominal Aortic Aneurysm

Granzyme B (GZMB) is a serine protease that is abundantly expressed in advanced human atherosclerotic lesions and may contribute to plaque instability. Perforin is a pore-forming protein that facilitates GZMB internalization and the induction of apoptosis. Recently a perforin-independent, extracellular role for GZMB has been proposed. In the current study, the role of GZMB in abdominal aortic aneurysm (AAA) was assessed. Apolipoprotein E (APOE)(-/-) x GZMB(-/-) and APOE(-/-) x perforin(-/-) double knockout (GDKO, PDKO) mice were generated to test whether GZMB exerted a causative role in aneurysm formation. To induce aneurysm, mice were given angiotensin II (1000 ng/kg/min) for 28 days. GZMB was found to be abundant in both murine and human AAA specimens. GZMB deficiency was associated with a decrease in AAA and increased survival compared with APOE-KO and PDKO mice. Although AAA rupture was observed frequently in APOE-KO (46.7%; n = 15) and PDKO (43.3%; n = 16) mice, rupture was rarely observed in GDKO (7.1%; n = 14) mice. APOE-KO mice exhibited reduced fibrillin-1 staining compared with GDKO mice, whereas in vitro protease assays demonstrated that fibrillin-1 is a substrate of GZMB. As perforin deficiency did not affect the outcome, our results suggest that GZMB contributes to AAA pathogenesis via a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity.

Protease activity in the multi-layered intra-luminal thrombus of abdominal aortic aneurysms

INTRODUCTION Rupture of an abdominal aortic aneurysm (AAA) is the cause of death in approximately 2% of men above 65 years. Most AAAs contain an intra-luminal thrombus (ILT), which is a potential source of proteases capable of degrading the underlying aneurysm wall. The AAA wall covered by a thick ILT shows more signs of matrix degradation compared to the wall free from ILT. The purpose of the present study was to evaluate the presence of protease activity in the ILT. MATERIALS AND METHODS ILT and peripheral blood from 32 patients undergoing elective surgery were collected. The ILT was divided into abluminal, luminal, and a middle layer in between. Collagenases, gelatinases, elastase, and their inhibitors were measured using ELISA in protein extracts from these layers. Immunohistochemistry was used for identification of cells. RESULTS Neutrophil leukocytes and platelets were mostly detected in the luminal layer of the ILT. MMP-9 and neutrophil elastase were also abundant in this layer but with low activity. High concentrations of TIMP-1 and PAI-1 were detected in the abluminal layer, while alpha 1 antitrypsin was mostly found in the luminal layer of the ILT. CONCLUSIONS In AAA thick ILTs with multiple layers contain substantial amounts of proteases, but their activity is limited to the luminal layer. Proteases in the abluminal layer are mostly inactive, probably due to excess amounts of inhibitors and are consequently unable to directly participate in the pathogenesis of AAA.

Proteolytically active ADAM10 and ADAM17 carried on membrane microvesicles in human abdominal aortic aneurysms

The intraluminal thrombus (ILT) of human abdominal aortic aneurysm (AAA) has been suggested to damage the underlying aortic wall, but previous work found scant activity of soluble proteases in the abluminal layer of the ILT, adjacent to the aneurysm. We hypothesised that transmembrane proteases carried by membrane microvesicles (MV) from dying cells remain active in the abluminal ILT. ILTs and AAA segments collected from 21 patients during surgical repair were assayed for two major transmembrane proteases, ADAM10 (a disintegrin and metalloprotease-10) and ADAM17. We also exposed cultured cells to tobacco smoke and assessed ADAM10 and ADAM17 expression and release on MVs. Immunohistochemistry showed abundant ADAM10 and ADAM17 protein in the ILT and underlying aneurysmal aorta. Domain-specific antibodies indicated both transmembrane and shed ADAM17. Importantly, ADAM10 and ADAM 17 in the abluminal ILT were enzymatically active. Electron microscopy of abluminal ILT and aortic wall showed MVs with ADAM10 and ADAM17. By flow cytometry, ADAM-positive microvesicles from abluminal ILT carried the neutrophil marker CD66, but not the platelet marker CD61. Cultured HL60 neutrophils exposed to tobacco smoke extract showed increased ADAM10 and ADAM17 content, cleavage of these molecules into active forms, and release of MVs carrying mature ADAM10 and detectable ADAM17. In conclusion, our results implicate persistent, enzymatically active ADAMs on MVs in the abluminal ILT, adjacent to the aneurysmal wall. The production of ADAM10- and ADAM17-positive MVs from smoke-exposed neutrophils provides a novel molecular mechanism for the vastly accelerated risk of AAA in smokers.

Differences in cardiovascular toxicities associated with cigarette smoking and snuff use revealed using novel zebrafish models

ABSTRACT Tobacco use is strongly associated with cardiovascular disease and the only avoidable risk factor associated with development of aortic aneurysm. While smoking is the most common form of tobacco use, snuff and other oral tobacco products are gaining popularity, but research on potentially toxic effects of oral tobacco use has not kept pace with the increase in its use. Here, we demonstrate that cigarette smoke and snuff extracts are highly toxic to developing zebrafish embryos. Exposure to such extracts led to a palette of toxic effects including early embryonic mortality, developmental delay, cerebral hemorrhages, defects in lymphatics development and ventricular function, and aneurysm development. Both cigarette smoke and snuff were more toxic than pure nicotine, indicating that other compounds in these products are also associated with toxicity. While some toxicities were found following exposure to both types of tobacco product, other toxicities, including developmental delay and aneurysm development, were specifically observed in the snuff extract group, whereas cerebral hemorrhages were only found in the group exposed to cigarette smoke extract. These findings deepen our understanding of the pathogenic effects of cigarette smoking and snuff use on the cardiovascular system and illustrate the benefits of using zebrafish to study mechanisms involved in aneurysm development. Summary: We analyze common and distinct developmental and cardiovascular toxicities caused by cigarette smoke and snuff, and include a new zebrafish embryo model for studying abdominal aortic aneurysm.

Inflammatory cells, ceramides, and expression of proteases in perivascular adipose tissue adjacent to human abdominal aortic aneurysms

Background: Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study. Methods and Results: Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T‐cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T‐cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T‐cells in the PVAT. Conclusions: Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage. Clinical Relevance: Abdominal aortic aneurysm is an inflammatory disease. This study shows that adipocytes surrounding the aorta may be a great source of inflammatory leukocytes that are attracted by adipocytes undergoing necrosis and by proinflammatory ceramides. Future strategies preventing the formation of perivascular adipose tissue and targeting inflammation from the adventitial side must be taken into consideration.

Altered PPARγ Coactivator-1 Alpha Expression in Abdominal Aortic Aneurysm: Possible Effects on Mitochondrial Biogenesis.

Introduction: Abdominal aortic aneurysm (AAA) is a complex and deadly vascular disorder. The pathogenesis of AAA includes destruction and phenotypic alterations of the vascular smooth muscle cells (VSMCs) and aortic tissues. PPARγ coactivator-1 alpha (PGC1α) regulates VSMC migration and matrix formation and is a major inducer of mitochondrial biogenesis and function, including oxidative metabolism. Methods: Protein and gene expression of PGC1α and markers for mitochondria biogenesis and cell type-specificity were analysed in AAA aortas from humans and mice and compared against control aortas. Results: Gene expression of PPARGC1A was decreased in human AAA and angiotensin (Ang) II-induced AAA in mice when compared to control vessels. However, high expression of PGC1α was detected in regions of neovascularisation in the adventitia layer. In contrast, the intima/media layer of AAA vessel exhibited defective mitochondrial biogenesis as indicated by low expression of PPARGC1A, VDAC, ATP synthase and citrate synthase. Conclusion: Our results suggest that mitochondrial biogenesis is impaired in AAA in synthetic SMCs in the media, with the exception of newly formed supporting vessels in the adventitia where the mitochondrial markers seem to be intact. To our knowledge, this is the first study investigating PGC1α and mitochondria biogenesis in AAA.

ADAMTS-1 in abdominal aortic aneurysm

Introduction Extracellular matrix degradation is a hallmark of abdominal aortic aneurysm (AAA). Among proteases that are capable of degrading extracellular matrix are a disintegrin and metalloproteases with thrombospondin motifs (ADAMTS). Pathogenesis of these proteases in AAA has not been investigated until date. Methods and results Human aneurysmal and control aortas were collected and analyzed with RT-PCR measuring the ADAMTS-1, 4,5,6,8,9,10,13,17 and ADAMTSL-1. Expression of a majority of the investigated ADAMTS members on mRNA level was decreased in aneurysm compared to control aorta. ADAMTS-1 was one of the members that was reduced most. Protein analysis using immunohistochemistry and western blot for localization and expression of ADAMTS-1 revealed that ADAMTS-1 was present predominantly in areas of SMCs and macrophages in aneurysmal aorta and higher expressed in AAA compared to control aortas. The role of ADAMTS-1 in AAA disease was further examined using ADAMTS-1 transgenic/apoE-/- mice with the experimental angiotensin II induced aneurysmal model. Transgenic mice overexpressing ADAMTS-1 showed to be similar to ADAMTS-1 wild type mice pertaining collagen, elastin content and aortic diameter. Conclusion Several of the ADAMTS members, and especially ADAMTS-1, are down regulated at mRNA level in AAA, due to unknown mechanisms, at the same time ADAMTS-1 protein is induced. The cleavage of its substrates, don’t seem to be crucial for the pathogenesis of AAA but rather more important in the development of thoracic aortic aneurysm and atherosclerosis as shown in previous studies.