Technological insights into a histopathological and protein composition analysis of aortic aneurysms in Marfan syndrome

[eng] The aorta is the main artery of the body, whose function is to collect the blood ejected by the heart and distribute it to all tissues. The elastic degree of the aortic wall necessary to withstand blood pressure is mainly provided by the distribution of collagen fibres and elastic lamellae. Fibrilin-1 is an essential protein of the elastic lamellae, which is mutated in Marfan syndrome (MFS), a connective tissue disorder characterized by the formation of aortic aneurysms with a high risk of mortality. Multiphoton microscopy and the microCT, among other approaches, have been used to evaluate the structural characteristics of the aortic tissue, the but there is currently little information about the three-dimensional histopathological structure of the MFS aorta. Besides, the differences in the protein content with respect to the healthy aorta have been analysed by means of studies that used conventional cell culture, neglecting the effect of the mechanical forces that the vascular smooth muscle cells (VSMC) constantly perceive in vivo due to blood pressure. In this context, we have developed four innovative technologies that have allowed us to generate new data on the histopathological structure and the protein content of the aorta MFS in comparison to healthy samples: - A multiphoton microscopy and image processing methodology was applied to MFS mouse aortae to visualise and analyse the microscale morphology of the elastic lamellae. Bigger and more abundant fenestrae were observed in the aorta of MFS compared to those of the WT. With this results, fenestrae become potential markers of lamellar damage in MFS. - A microCT and image processing protocol to evaluate the histological integrity of the aortic wall. This technology was applied to MFS and WT mice aortae, and an increase in the area and the thickness of the tunica media and other histological parameters was observed in relation to age progression (3, 6, and 9 months (mo)). This increase was parallel between WT and MFS, but the SMF 9mo showed values significantly higher than the WT 9m. Therefore, we hypothesise that the MFS aorta may undergo an accelerated middle-ageing process. - A bioreactor of mechanical stretching to examine the protein content of VSMCs of healthy human aorta and MFS cultivated under in vivo-like mechanical conditions. The MFS cells showed an altered reaction to the administration of stretch, not related to the content or distribution of phenotypic markers. Therefore, the MFS VSMCs show altered cell-matrix communication not linked to the phenotype. - Finally, by means of the systematic review of the scientific literature, the most complete human aortic tissue proteome to date was generated. It is divided into a healthy and an aneurysmal databases, containing 919 and 724 different proteins, respectively. The application of these technologies has provided new knowledge in the field of vascular biology, especially with regard to Marfan syndrome.[cat] L'aorta és la principal artèria del cos, la funció de la qual és recollir la sang expulsada pel cor i distribuir-la cap a tots els teixits. El grau elàstic de la paret aòrtica necessari per a suportar la pressió sanguínia ve donat principalment per la distribució de fibres de col·lagen i lamel·les elàstiques. La fibrilina-1 és una proteïna essencial de les lamel·les elàstiques, la qual es troba mutada en la síndrome de Marfan (SMF), un trastorn del teixit connectiu caracteritzat per la formació d’aneurismes d’aorta amb alt risc de mortalitat. Per a avaluar les característiques estructurals del teixit aòrtic s’han utilitzat, entre d’altres, la microscòpia multifotó i el microCT, però avui encara hi ha poca informació sobre l'estructura histopatològica tridimensional de l'aorta SMF. D'altra banda, s’han analitzat les diferències en el contingut proteic respecte l’aorta sana mitjançant estudis que empraven cultiu cel·lular convencional, descuidant l'efecte de les forces mecàniques que les cèl·lules musculars llises vasculars (VSMC) perceben constantment in vivo a causa de la pressió arterial. En aquest context, hem desenvolupat quatre tecnologies innovadores que han permès generar noves dades sobre l'estructura histopatològica i el contingut proteic de l'aorta SMF en comparació a mostres sanes: - Una metodologia de microscòpia multifotó i de processament d'imatges es va aplicar a aortes de ratolins SMF per a visualitzar y analitzar la morfologia microscala de les lamel·les elàstiques. Es van observar fenestres més grans i abundants en l'aorta de SMF en comparació amb les del tipus WT; esdevenint les fenestres potencials marcadors del dany lamel·lar en la SMF. - Un protocol de microCT i processament d'imatges per a avaluar la integritat histològica de la paret aòrtica. Aquesta tecnologia es va aplicar a aortes de ratolins SMF i WT, i es va observar un increment de l'àrea i el gruix de la túnica media i d’altres paràmetres histològics en relació amb la progressió en edat (3, 6 i 9 mesos (mo)). Aquest increment fou paral·lel entre WT i SMF, però el SMF 9mo mostrà valors significativament més alts que els WT 9mo. Per tant, plantegem la hipòtesi que l'aorta SMF deu patir un procés accelerat d'envelliment a edat adulta mitjana. - Un biorreactor d'estirament mecànic per a examinar el contingut proteic de VSMCs d’aorta humana sana i SMF cultivades sota un ambient mecànic similar al real. Les cèl·lules SMF van mostrar una reacció alterada a l'administració de l'estirament, no relacionada amb el contingut ni distribució de marcadors fenotípics. Per tant, les VSMCs de SMF mostren una comunicació cèl·lula-matriu alterada no associada al fenotip. - Finalment, mitjançant la revisió sistemàtica de la literatura científica es va generar el proteoma de teixit aòrtic humà més complet de l’actualitat, dividit entre les bases de dades sana i aneurismàtica, que contenen 919 i 724 proteïnes diferents, respectivament. L'aplicació d'aquestes tecnologies ha proporcionat nous coneixements al camp de la biologia vascular, especialment pel que fa a la síndrome de Marfan

Early detection and treatment strategies for vulnerable atherosclerotic plaques

Atherosclerotic plaque ruptures have been determined as the most common underlying cause of acute coronary syndromes and stroke. Currently, the standard of care for plaque rupture risk is based on the amount of luminal stenosis presented in a particular vessel; however, X-ray angiographic studies have shown that plaques at risk of rupture generally show <50% luminal narrowing. These findings explicate the need for other, more accurate methods of identifying problem lesions prior to the rupture event. Unfortunately, the study of thrombotic events and vulnerable plaque lesions in humans is difficult due to the spontaneity of rupture and the lengthy time course of disease progression. To further the understanding of plaque rupture risk in light of vulnerability detection, a rabbit model of atherothrombosis was used in conjunction with magnetic resonance imaging (MRI). MRI has been validated as a suitable imaging modality for in vivo, non-invasive detection of atherosclerosis and has provided quantitative predictors of plaques at risk of rupture. Additionally, the rabbit model has been shown, histologically, to present 6 of the 8 human plaque types classified by the American Heart Association. The first portion of this dissertation work focuses on using MRI to serially image rabbits undergoing the atherosclerotic protocol in order to assess rupture risk at the various time points. Previous work has determined that an increase in the vessel remodeling ratio (which hides a large plaque in the vessel wall) and contrast uptake (which indicates inflammation) are both characteristics of increased rupture risk. By obtaining these parameters at various time points in the disease progression, it was possible to determine when a certain plaque displays a heightened risk of rupture. The second portion of this work tested the efficacy of a pro-resolving molecule, lipoxin (an endogenous molecule), in reducing atherosclerotic disease state, specifically rupture with a luminal thrombus. Using chronic administration of this molecule in the same rabbit model of atherosclerosis yielded a faint reduction in atherosclerotic severity based on the parameters of decreased vessel lipid content and decreased thrombotic events presented in the treated group

Assessment of abdominal aortic aneurysm biology using magnetic resonance imaging and positron emission tomography-computed tomography.

Background Although abdominal aortic aneurysm (AAA) growth is non-linear, serial measurements of aneurysm diameter are the mainstay of aneurysm surveillance and contribute to decisions on timing of intervention. Aneurysm biology plays a key part in disease evolution but is not currently routinely assessed in clinical practice. Magnetic Resonance Imaging (MRI) and Positron Emission Tomography-Computed Tomography (PET-CT) provide insight into disease processes on a cellular or molecular level, and represent exciting new imaging biomarkers of disease activity. Macrophage-mediated inflammation may be assessed using ultrasmall superparamagnetic particles of iron oxide (USPIO) MRI and the PET radiotracer 18FSodium Fluoride (18F-NaF) identifies microcalcification which is a response to underlying necrotic inflammation. The central aim of this thesis was to investigate these imaging modalities in patients with AAA. Methods and Results USPIO MRI: MULTI-CENTRE STUDY In a prospective multi-centre observational cohort study, 342 patients (85.4% male, mean age 73.1±7.2 years, mean AAA diameter 49.6±7.7mm) with asymptomatic AAA ≥4 cm anteroposterior diameter underwent MRI before and 24-36 hours after intravenous administration of USPIO. Colour maps (depicting the change in T2* caused by USPIO) were used to classify aneurysms on the basis of the presence of USPIO uptake in the aneurysm wall, representing mural inflammation. Intra- and inter-observer agreement were found to be very good, with proportional agreement of 0.91 (kappa 0.82) and 0.83 (kappa 0.66), respectively. At 1 year, there was 29.3% discordant classification of aneurysms on repeated USPIO MRI and at 2 years, discordance was 65%, suggesting that inflammation evolves over time. In the observational study, after a mean of 1005±280 days of follow up, there were 126 (36.8%) aneurysm repairs and 17 (5.0%) ruptures. Participants with USPIO enhancement (42.7%) had increased aneurysm expansion rates (3·1±2·5 versus 2·5±2·4 mm/year; difference 0·6 [95% confidence intervals (CI), 0·02 to 1·2] mm/year, p=0·0424) and had higher rates of aneurysm rupture or repair (69/146=47·3% versus 68/191=35·6%; difference 11·7%, 95% CI 1·1 to 22·2%, p=0·0308). USPIO MRI was therefore shown to predict AAA expansion and the composite of rupture or repair, however this was not independent of aneurysm diameter (c-statistic, 0·7924 to 0·7926; unconditional net reclassification -13·5%, 95% confidence intervals -36·4% to 9·3%). 18F-NaF PET-CT: SINGLE-CENTRE STUDY A sub-group of 76 patients also underwent 18F-NaF PET-CT, which was evaluated using the maximum tissue-to-background ratio (TBRmax) in the most diseased segment (MDS), a technique that showed very good intra- (ICC 0.70-0.89) and inter-observer (ICC 0.637-0.856) agreement. Aneurysm tracer uptake was compared firstly in a case-control study, with 20 patients matched to 20 control patients for age, sex and smoking status. 18F-NaF uptake was higher in aneurysm when compared to control aorta (log2TBRmax 1.712±0.560 vs. 1.314±0.489; difference 0.398 (95% CI 0.057, 0.739), p=0.023), or to non-aneurysmal aorta in patients with AAA (log2TBRmax 1.647±0.537 vs. 1.332±0.497; difference 0.314 (95% CI 0.0685, 0.560), p=0.004). An ex vivo study was performed on aneurysm and control tissue, which demonstrated that 18F-NaF uptake on microPET-CT was higher in the aneurysm hotspots and higher in aneurysm tissue compared to control tissue. Histological analysis suggested that 18F-NaF was highest in areas of focal calcification and necrosis. In an observational cohort study, aneurysms were stratified by tertiles of TBRmax in the MDS and followed up for 510±196 days, with 6 monthly serial ultrasound measurements of diameter. Those in the highest tertile of tracer uptake expanded more than 2.5 times more rapidly than those in the lowest tertile (3.10 [3.58] mm/year vs. 1.24 [2.41] mm/year, p=0.008) and were also more likely to experience repair or rupture (15.3% vs. 5.6%, log-rank p=0.043). In multivariable analyses, 18F-NaF uptake on PET-CT emerged as an independent predictor of AAA expansion (p=0.042) and rupture or repair (HR 2.49, 95% CI1.07, 5.78; p=0.034), even when adjusted for age, sex, body mass index, systolic blood pressure, current smoking and, crucially, aneurysm diameter. Conclusion These are the largest USPIO MRI and PET-CT studies in AAA disease to date and the first to investigate 18F-NaF. Both USPIO MRI and 18F-NaF PET-CT are able to predict AAA expansion and the composite of rupture and repair, with 18F-NaF PETCT emerging as the first imaging biomarker that independently predicts expansion and AAA events, even after adjustment for aneurysm diameter. This represents an exciting new predictor of disease progression that adds incremental value to standard clinical assessments. Feasibility and randomised clinical trials are now required to assess the potential of this technique to change the management and outcome of patients with AAA

Multimodality imaging of coronary artery bypass grafts

This thesis describes multiple imaging modalities to examine coronary artery bypass grafts, and the research which was performed to further develop noninvasive imaging techniques to detect stenoses in native coronary arteries and bypass grafts in patients who experienced recurrent chest pain after coronary artery bypass grafting (CABG).UBL - phd migration 201

Multimodality Imaging of Anatomy and Function in Coronary Artery Disease

Various modalities are available in the diagnostic and prognostic evaluation of patients presenting with known or suspected coronary artery disease (CAD). A rapidly expanding technique is noninvasive coronary angiography with Multi-Slice Computed Tomography (MSCT), which allows accurate detection of significant stenoses. The main value of the technique lies in the noninvasive exclusion of CAD in patients with intermediate pre-test likelihood. Although imaging in populations such as patients with previous stent placement appears to be more challenging, promising results have been obtained in these populations as well. However, it remains important to realize that the presence of coronary atherosclerosis with luminal obstruction does not invariably imply the presence of ischemia. Accordingly, a noninvasive angiographic imaging technique as MSCT cannot be used to predict the hemodynamical importance of lesions. In patients with borderline stenosis, therefore, functional testing (which can be performed by nuclear imaging, stress echocardiography or MRI) will remain necessary to determine management. Nonetheless, detection of CAD at a far earlier stage than functional imaging is an important advantage of MSCT. Initial investigations suggest that MSCT may distinguish different plaque characteristics between various presentations. Potentially, this information could be useful for risk stratification. Finally, additional non-coronary information can be derived as well. LV function can be evaluated with high accuracy while also information on the cardiac venous system can be obtained.LEI Universiteit LeidenNederlandse Hartstichting, ICIN Toshiba Medical Systems BV, Vital Images BV, Biotronik BV, Stichting EMEX, Foundation Imago, J.E. Jurriaanse Stichting, Medtronic BV, Astellas Pharma BV, St Jude Medical BV, Tyco Healthcare BV, Amgen BV (Breda), Boehringer Ingelheim BV, GE Healthcare Medical Diagnostics (Eindhoven), Pfizer BV, Siemens BV, Bristol-Myers Squibb, Boston Scientific Benelux BV, Merck Sharp & Dohme BV.Vasculaire biologie en interventi

Studies on the role of GPR55 in cardiovascular physiology and pathophysiology.

Atherosclerosis is a multifactorial, chronic inflammatory condition characterised by endothelial dysfunction, hyperlipidaemia and the accumulation of fatty deposits within the tunica intima of medium-to-large sized muscular arteries. This disease can prove fatal with patients suffering lethal myocardial infarction or stroke. Recently, two studies investigating the role of G-protein-coupled receptor 55 (GPR55) in atherosclerosis reported conflicting results; one reported a pro-atherogenic role for GPR55 and the other, an anti-atherogenic role for this receptor. Interestingly, another study demonstrated that the activation of GPR55 by lysophosphatidylinositol (LPI) in cultured rat neonatal ventricular cardiomyocytes provokes distinct cellular functions that are dependent on the location of GPR55, leading to suggestions that GPR55 may regulate cardiomyocyte function at two cellular sites and be a potential therapeutic target for cardiac disorders. While it has been demonstrated that GPR55 is important in the maintenance of cardiac function of healthy mice, what is currently unknown is if GPR55 has a role in the cardiovascular remodelling and cardiac function of atherosclerosis prone mice. To address this, the present studies were conducted to investigate 1) the role of GPR55 in atherogenesis, 2) if GPR55 has a role in the cardiac function of mice suffering from atherosclerosis, 3) the signalling pathway by which LPI activates cardiomyocytes, 4) the impact of GPR55 activation on the outcome of myocardial ischaemia/reperfusion (I/R) injury and, 5) the signalling mechanisms by which GPR55 elicits any observed effects on the myocardium in response to such injury. Using C57BL/6 (wildtype; WT), apolipoprotein E knockout (ApoE-/-; mouse model of atherosclerosis), GPR55 knockout (GPR55-/-) and novel ApoE-/-/GPR55-/- mice, this study has established that in the presence of high fat feeding (to accelerate atherosclerosis), GPR55 has a complex role whereby it both regulates risk factors associated with atherosclerosis (i.e. body weight and fat mass) yet promotes the development of fatty streaks within the vasculature, via a lipid independent mechanism. In terms of cardiac function, GPR55 exerted a protective role by maintaining the systolic function of high fat fed ApoE-/- mice, yet negatively affected the contractile reserve of these mice. With regard to infarct size, the present study established that LPI-induced activation of GPR55 (pre-global ischaemia) exacerbates myocardial tissue injury via a Rho-associated protein kinase (ROCK) dependent mechanism. Finally, this study established that LPI signals through the same signalling pathway as it did in the isolated heart, in both mouse and human-induced pluripotent stem cell-derived cardiomyocytes thus suggesting a translational role for GPR55 in the human heart. In conclusion, despite further research being required, the data presented within this thesis provides evidence that GPR55 may have the potential to be targeted for therapeutic gains in atherosclerosis and myocardial I/R injury

Image and Signal Processing in Intravascular Ultrasound

Intravascular ultrasound (rvUS) is a new imaging mOdality providing real-time, crosssectional, high-resolution images of the arterial lumen and vessel wall. In contrast to conventional x-ray angiography that only displays silhouette views of the vessel lumen, IVUS imaging permits visualization of lesion morphology and accurate measurements of arterial cross-sectional dimensions in patients. These unique capabilities have led to many important clinical applications including quantitative assessment of the severity, restenosis, progression of atherosclerosis, selection and guidance of catheterbased therapeutic procedures and short- and long-term evaluation of the outcome of an intravascular intervention. Like the progress of other medial imaging modalities, the advent of IVUS techniques has brought in new challenges in the field of signal and image processing. Quantitative analysis of IVUS images requires the identification of arterial structures such as the lumen and plaque within an image. Manual contour tracing is well known to be time consuming and subjective. Development of an automated contour detection method may improve the reproducibility of quantitative IVUS and avoid a tedious manual procedure. Computerized three-dimensional (3D) reconstruction of an IVUS image series may extend the tomographic data to a more powerful volumetric assessment of the vessel segment. Obviously, this could not be achieved without the advance of 3D image processing techniques. Furthermore, it is demonstrated that processing of the original radio frequency (RF) echo signals provides an efficient means to improve the IVUS image quality as well as a new approach to extract volumetric flow information. The goals of the studies reported in this thesis are therefore directed toward development of video image and RF signal processing techniques for image enhancement, automated contour detection, 3D reconstruction and flow imaging. In this chapter several IVUS scanning mechanisms and some background information about ultrasonic imaging are briefly introduced. The principles of different video-based contour detection approaches and examples of contour detection in echocardiograms are discussed. Subsequently, applications of RF analysis in IVUS images are reviewed, followed by the scope of this thesis in the final part

Novel applications of positron emission tomography in the non-invasive assessment of cardiovascular disease

Introduction. Fused Positron Emission Tomography and Computed Tomography (PET/CT) is an emerging investigative tool in cardiovascular disease that provides an imaging-based quantification of pathophysiological processes of interest. The purpose of this thesis was to study the application of PET to identify fundamental pathophysiological processes driving 3 forms of cardiovascular disease: aortic stenosis, myocardial infarction, and atherosclerosis. Methods. Aortic Stenosis. Patients with a spectrum of calcific aortic valve disease (n=121) who underwent PET-CT imaging for the identification of valvular calcification (18Ffluoride) and inflammation (18F-fluorodeoxyglucose, 18F-FDG) underwent serial imaging and clinical follow-up over 2 years. Baseline imaging findings were compared with echocardiographic and CT markers of disease progression and clinical outcome. Myocardial Infarction. Patients underwent PET-CT imaging with 18F-fluciclatide (a novel αvβ3-selective radiotracer highlighting active angiogenesis, inflammation and fibrosis) after ST-segment elevation MI (n=21), alongside stable patients with chronic total occlusion (CTO) of a major coronary vessel (n=7), and healthy volunteers (n=9). Myocardial radiotracer uptake was compared with clinical and cardiac magnetic resonance imaging (CMR) markers of infarction and remodeling. Atherosclerosis. Patients with a spectrum of atherosclerotic disease categorized as stable or unstable (recent MI) underwent PET/CT imaging with 18F-fluciclatide (n=46). Thoracic aortic 18F-fluciclatide uptake was compared with aortic atherosclerotic burden quantified by CT plaque thickness, plaque volume and calcium scoring. Histological validation. Tissue from the aortic valve, myocardium and carotid arteries of study subjects was acquired and examined ex vivo using histology and autoradiography. Results. Aortic Stenosis. Baseline valvular 18F-fluoride uptake correlated strongly with the rate of progression in AVC (r=0.80, p<0.001) and with haemodynamic progression (mean aortic valve gradient r=0.32, p=0.001). It emerged as independently associated with clinical outcome after age and sex-adjustment (HR 1.55 [1.33-1.81], p<0.001). 18F-FDG demonstrated moderate correlations with disease progression as assessed by CT (r=0.43, p=0.001) and echocardiography (18F-FDG r=0.30, p=0.001), and was associated with clinical outcomes independent of age and sex (HR 1.35 [1.16-1.58], p<0.001). Valvular 18F-fluoride uptake correlated with immunohistochemical markers of calcification activity. There was no correlation between 18F-FDG uptake and inflammation. Myocardial Infarction. 18F-Fluciclatide binding was demonstrated in ex vivo peri-infarct myocardium and uptake was increased in vivo at sites of acute infarction compared to remote myocardium (tissue-to-background ratio (TBRmean) 1.34±0.22 vs 0.85±0.17 respectively, p<0.001) and myocardium of healthy volunteers (TBRmean 1.34±0.22 vs 0.70±0.03; p<0.001). There was no 18F-fluciclatide uptake at sites of established prior infarction in patients with CTO, with myocardial activity similar to healthy volunteers (TBRmean 0.71±0.06 vs. 0.70±0.03,p=0.83). 18F-Fluciclatide uptake occurred at sites of regional wall hypokinesia (wall motion index ≥1 vs 0; TBRmean 0.93±0.31 vs 0.80±0.26 respectively, p<0.001), was increased in segments displaying functional recovery (TBRmean 0.95±0.33 vs 0.81±0.27, p=0.002) and associated with increase in probability of regional recovery. Atherosclerosis. 18F-Fluciclatide vascular binding ex vivo co-localised with regions of increased αvβ3 integrin expression, and markers of inflammation and angiogenesis. 18F-Fluciclatide uptake in vivo correlated with measures of aortic atherosclerotic burden: plaque thickness (r=0.57, p=0.001), total plaque volume (r=0.56, p=0.001) and the CT aortic calcium score (r=0.37, p=0.01). Patients with recent MI had greater aortic 18F-fluciclatide uptake than those with stable disease (TBRmax 1.33 vs 1.21, p=0.01). Conclusions. In a range of cardiovascular diseases, PET-CT can provide insights into key pathophysiological processes, guide patient risk stratification and prognosis, and identify important biomarkers of disease activity that can be used for the development of future therapeutic interventions