Abnormal vascular smooth muscle cell (VSMC) proliferation is thought to play an important role in the pathogenesis of atherosclerosis. Adipocytes produce several paracrine bioactive substances that can affect VSMC growth and migration. Our study focused on the ability of epicardial adipocytes to produce bioactive substances together with studying of direct effect of these substances on the VSMC proliferation rate. The gene expression of human cytokines (IL-6, IL-8, IL-18, RANTES and MCP-1) and adipokines (leptin and adiponectin) was measured in primary cell lines of epicardial and visceral adipocytes, both in undifferentiated and mature statuses. Moreover, adipokine production (IL-6, IL-8, MCP-1, VEGF and adiponectin) in conditioned media obtained from above mentioned primary cell cultures of adipocytes was measured by a Luminex assay. The VSMC proliferation rate was measured after co-culturing with CM obtained from primary cell cultures of adipocytes. The epicardial preadipocytes showed an increased expression of IL-8 (3,25-fold, p<0,05) compared with visceral preadipocytes. The expression of the adiponectin in epicardial preadipocytes was markedly decreased in comparison of the expression in visceral preadipocytes (p< 0,0001). Moreover, the gene expression was dependent on the differentiation...

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The mechanical behavior of the arterial wall is determined by the composition and structure of its internal constituents as well as the applied traction-forces, such as pressure and axial stretch. The purpose of this work is to develop new theoretical frameworks and experimental methodologies to further the understanding of arterial mechanics and role of the various intrinsic and extrinsic mechanically motivating factors. Specifically, residual deformation, matrix organization, and perivascular support are investigated in the context of their effects on the overall and local mechanical behavior of the artery. We propose new kinematic frameworks to determine the displacement field due to residual deformations previously unknown, which include longitudinal and shearing residual deformations. This allows for improved predictions of the local, intramural stresses of the artery. We found distinct microstructural differences between the femoral and carotid arteries from non-human primates. These arteries are functionally and mechanically different, but are geometrically and compositionally similar, thereby suggesting differences in their microstructural alignments, particularly of their collagen fibers. Finally, we quantified the mechanical constraint of perivascular support on the coronary artery by mechanically testing the artery in-situ before and after surgical exposure.

Ο λιπώδης ιστός θεωρείται πλέον ενδοκρινές όργανο που παράγει πληθώρα βιολογικά δραστικών πεπτιδίων, που καλούνται λιποκίνες. Ανάλογα με την ανατομική τους εντόπιση, οι διαφορετικές αποθήκες λίπους έχουν και διαφορετική ικανότητα παραγωγής λιποκινών και επίδρασης σε φυσιολογικές λειτουργίες. Οι λιποκίνες που παράγονται από τον περιαγγειακό λιπώδη ιστό εμπλέκονται στην παθογένεια αγγειακών νόσων, συμπεριλαμβανόμενης της αθηροσκλήρωσης. Είναι γνωστό πως η αντιπονεκτίνη ασκεί αντιαθηρογόνες δράσεις, ενώ ο ρόλος της Τ-καντχερίνης ως υποδοχέα της αντιπονεκτίνης δεν έχει πλήρως διαλευκανθεί. Το απελινεργικό σύστημα, αποτελούμενο από την απελίνη και τον υποδοχέα της APJ, αποτελεί μεσολαβητή ποικίλων καρδιαγγειακών λειτουργιών και ενδέχεται να συμμετέχει και στην αθηροσκληρωτική διαδικασία. Η χεμερίνη είναι λιποκίνη με γνωστό ρόλο στην ανοσία, στη λειτουργία του λιπώδους ιστού και στο μεταβολισμό, δρώντας κυρίως μέσω του υποδοχέα της CMKLR1. Μελετήσαμε την πρωτεϊνική έκφραση της αντιπονεκτίνης και της Τ-καντχερίνης, της απελίνης και του APJ, της χεμερίνης και του CMKLR1 σε ανθρώπινες αορτές, στεφανιαίες αρτηρίες και στον αντίστοιχο περιαγγειακό λιπώδη ιστό και συσχετίσαμε την έκφρασή τους με την παρουσία αθηροσκλήρωσης και με κλινικές παραμέτρους. Εφαρμόστηκε ανοσοϊστοχημική χρώση για την αντιπονεκτίνη, την Τ-καντχερίνη, την απελίνη, τον APJ, τη χεμερίνη και τον CMKLR1 σε δείγματα ανθρώπινων αορτών και στεφανιαίων αρτηριών, περιλαμβανόμενου και του περιαγγειακού λίπους. Οι αορτικές και στεφανιαίες αθηρωματικές βλάβες αξιολογήθηκαν με βάση την κατάταξη του AHA. Ανοσοϊστοχημική χρώση, ποικίλης έντασης, για την αντιπονεκτίνη ανιχνεύθηκε μόνο στα λιποκύτταρα, ενώ η Τ-καντχερίνη εντοπίστηκε στα αγγειακά λεία μυικά κύτταρα (ΑΛΜΚ) και στα ενδοθηλιακά κύτταρα. Ανοσοϊστοχημική χρώση για την απελίνη ανιχνεύθηκε σε λιποκύτταρα, ΑΛΜΚ, ενδοθηλιακά κύτταρα και μακροφάγα-αφρώδη κύτταρα των αθηρωματικών βλαβών, ενώ ο APJ εντοπίστηκε στα ΑΛΜΚ και στο ενδοθήλιο των αγγείων. Ανοσοθετικότητα για τη χεμερίνη παρατηρήθηκε και στις δύο αποθήκες λίπους ,στα ΑΛΜΚ και σε αφρώδη κύτταρα των αθηρωματικών βλαβών. Ο CMKLR1 εκφράστηκε σε ΑΛΜΚ και σε αφρώδη κύτταρα αορτών και στεφανιαίων αγγείων με αθηρωματικές βλάβες. Η έκφραση αντιπονεκτίνης στον περιαγγειακό λιπώδη ιστό και η έκφραση Τ-καντχερίνης στα ΑΛΜΚ συσχετίστηκαν αρνητικά με την αθηροσκλήρωση και στις δύο εντοπίσεις, όπως και η έκφραση απελίνης στα ΑΛΜΚ. Η έκφραση χεμερίνης στις περιαγγειακές αποθήκες λίπους και στα αφρώδη κύτταρα συσχετίστηκε στατιστικά σημαντικά με τη βαρύτητα της αθηροσκλήρωσης και στις δύο εντοπίσεις. Πολλές ακόμα – ειδικές για την εντόπιση – συσχετίσεις παρατηρήθηκαν. Τα αποτελέσματά μας υποδεικνύουν πιθανό ρόλο της Τ-καντχερίνης ως μεσολαβητή των αντιαθηρογόνων δράσεων της αντιπονεκτίνης, ενώ υποστηρίζουν το ενδεχόμενο αντιαθηρογόνο προφίλ της απελίνης και του υποδοχέα της APJ στις ανθρώπινες αρτηρίες. Ενισχύουν, ακόμα, τον υποτιθέμενο ρόλο της χεμερίνης στην εξέλιξη των αθηρωματικών βλαβών, πιθανότατα δρώντας μέσω του CMKLR1 υποδοχέα της. Περαιτέρω έρευνα είναι αναγκαία για να αποσαφηνιστεί ο ρόλος της τοπικά παραγόμενης αντιπονεκτίνης, απελίνης και χεμερίνης και της σηματοδότησης μέσω των αντίστοιχων υποδοχέων τους – T-cadherin, APJ και CMKLR1 – στην παθογένεια της αθηροσκλήρωσης στον άνθρωπο. / Adipose tissue is considered an endocrine organ, producing numerous bioactive peptides, called adipokines. Depending on their anatomical location, different fat depots have a different capacity to produce adipokines and influence physiological functions. Adipokines produced by periadventitial fat have been implicated in the pathogenesis of vascular disease, including atherosclerosis. Adiponectin has established anti-atherogenic actions, while the role of T-cadherin as an adiponectin receptor is not fully elucidated. The apelinergic system, consisting of apelin and its APJ receptor, is a mediator of various cardiovascular functions and may also be involved in the atherosclerotic process. Chemerin is an adipokine with an established role in immunity, adipose tissue function and metabolism, acting, mainly through its CMKLR1 receptor. We investigated the protein expression of adiponectin and T-cadherin, apelin and APJ, chemerin and CMKLR1 in human aortas, coronary vessels and the respective periadventitial adipose tissue and correlated their expression with the presence of atherosclerosis and clinical parameters. Immunohistochemistry for adiponectin, T-cadherin, apelin, APJ, chemerin and CMKLR1 was performed on human aortic and coronary artery samples including the periadventitial adipose tissue. Aortic and coronary atherosclerotic lesions were assessed using the AHA classification. Adiponectin immunostaining, of varied intensity, was detected only in adipocytes, while T-cadherin was localized to vascular smooth muscle cells (VSMCs) and endothelial cells. Apelin immunostaining was detected in adipocytes, VSMCs, endothelial cells and foam cells in atherosclerotic lesions, while APJ was found in VSMCs and endothelia. Chemerin immunopositivity was noticed in both periadventitial fat depots, in VSMCs and foam cells in atherosclerotic lesions. CMKLR1 was expressed in VSMCs and foam cells in aortic and coronary vessels with atherosclerotic lesions. Periadventitial adiponectin and VSMC T-cadherin expression were negatively correlated with atherosclerosis in both sites, as was VSMC apelin expression. Chemerin expression in periadventitial fat depots and foam cells was statistically significantly correlated with the severity of atherosclerosis in both locations. Several other – depot specific – associations were observed. Our results suggest a possible role for T-cadherin as a mediator of anti-atherogenic adiponectin actions, while they support the putative anti-atherogenic profile for apelin and its APJ receptor in human arteries. They also lend some support to a presumable role of chemerin in the progression of atherosclerotic lesions, possibly acting through its CMKLR1 receptor. Further research is necessary to elucidate the role of locally produced adiponectin, apelin and chemerin and signaling through their respective receptors – T-cadherin, APJ and CMKLR1 – in the pathogenesis of human atherosclerosis.

Les troubles métaboliques caractéristiques d’une alimentation de type « Western diet », sont à l’origine de pathologies cardiovasculaires, première cause de mortalité dans le monde. Il apparait nécessaire d’améliorer la compréhension des mécanismes impliqués dans l’installation des dysfonctions cardiovasculaires afin de pouvoir proposer des stratégies thérapeutiques ou préventives adaptées. Ainsi, le premier objectif de la thèse a été d’évaluer les effets d’une boisson sucrée sur la fonction vasculaire macro- et micro-circulatoire chez des sujets sains, par une approche translationnelle allant de la clinique humaine à un modèle expérimental de rongeur. Nos résultats montrent une altération de la fonction endothéliale en réponse à une prise de boisson sucrée, dans l’ensemble des lits vasculaires. L’exploration des mécanismes sous-jacents ces altérations nous a permis d’identifier l’implication du couple stress-oxydant/voie du NO. Un second objectif de thèse, a été d’étudier l’impact d’un stress métabolique chronique sur la fonction vasculaire et son incidence sur la régulation de la pression artérielle. Comme observé chez certains sujets souffrant de syndrome métabolique, notre modèle de rat ne présentait pas d’hypertension artérielle, malgré une hyperactivité du système sympathique. Ceci semble être expliqué par une compensation endothéliale eNOS-dépendant, qui permet de garantir le maintien d’une pression artérielle normale en dépit de l’effet vasopresseur adrénergique élevé. Le troisième objectif de thèse a porté sur un nouvel élément participant au maintien de l’homéostasie vasculaire et impacté par les situations pathologiques : le tissu adipeux périvasculaire (PVAT). Nos travaux démontrent dans le contexte du SMet, une altération de la voie adiponectine/eNOS dans le PVAT, en parallèle d’une augmentation de la production d’espèces oxygénées réactives.La pratique régulière d’un exercice physique est aujourd’hui reconnue comme une stratégie non-pharmacologique permettant d’impacter à la fois les désordres métaboliques et cardiovasculaires, notamment via une amélioration de la voie du NO. Nos résultats démontrent une limitation de l’apparition des dysfonctions endothéliales causée par une hyperglycémie aigue lorsqu’un protocole d’exercice physique chronique est réalisé. Enfin, l’exercice physique permet également de prévenir les modifications des propriétés vaso-actives du PVAT dans un modèle de rat SMet. Ce phénomène pourrait être expliqué par une amélioration du statut oxydant de la paroi artérielle, et à une potentialisation de la voie adiponectine/eNOS par l’exercice physique / The globalization of the western diet has mediated prevalence in cardiovascular disease related mortality, the single leading cause of death worldwide. Considering this, it is imperative that the underlying mechanisms of cardiovascular dysfunctions are continually investigated to establish a greater understanding of its pathogenesis from a healthy state to the presence of cardiometabolic diseases; and to improve upon current treatment and preventative strategies. Therefore, the first aim of this research was to identify vascular impact of acute hyperglycaemic stress induced by sweet sugar beverage consumption, with a translational approach. The results of this study demonstrated that consumption of a single commercially available sugar-sweetened beverage (SSB) induced transient micro- and macrovascular endothelial dysfunction, even in a healthy population. Further exploration into the underlying mechanisms of SSB-mediated endothelial dysfunction indicated that an increase in oxidative stress disrupts normal function of the nitric oxide pathway. Although disturbances in cardiovascular function may initially be transient, repetitive acute metabolic stress may translate to chronic cardiometabolic disease. Therefore, the second aim of this research was to assess the impact of a chronic metabolic disorder, metabolic syndrome (MetS), on vascular function in a rat model. Despite increasing sympathetic activity, the MetS rats didn’t present elevated arterial pressure. Such findings may be explained by a compensatory adaptation of endothelial function that increases production of nitric oxide in response to α-adrenergic agonist and, thus, regulates arterial pressure despite sympathetic hyperactivity. Considering this, the third aim of this research evaluated the impact of perivascular adipose tissue (PVAT) on vascular fucntion in MetS rats; demonstrating that MetS altered the adiponectin-endothelial nitric oxide synthase pathway in PVAT, in an oxidative stress-dependant manner.Exercise training is well recognized as a non-pharmacological strategy that has a beneficial impact on both metabolic and cardiovascular disorders via an improvement in function of the nitric oxide pathway. Considering this, research also assessed the efficacy of this approach to prevent vascular injury induced by acute hyperglycaemia in a healthy population and by PVAT in those with MetS. It was demonstrated that exercise attenuated acute hyperglycemia-mediated endothelial dysfunction; and restored endothelium-dependent vascular reactivity in rats with MetS, due to an improvement in the biocommunication between PVAT and arterial tissue and a notable enhancement of the adiponectine-endothelial nitric oxide synthase pathway.

<p>Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells [3, 4]. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / The epidemic of obesity and associated cardiovascular complications continues to grow at an alarming rate. Currently, obesity is thought to initiate a state of chronic inflammation, which if unresolved potentially causes cardiovascular dysfunction and disease. Although poorly understood, release of inflammatory mediators and other cytokines from adipose tissue (adipocytokines) has been proposed to be the molecular link between obesity and coronary artery disease. Furthermore, the anatomic location of adipose has been increasingly recognized as a potential contributor to vascular disease. Importantly, the development of coronary atherosclerosis, a key component of heart disease, is typically found in segments of coronary arteries surrounded by perivascular adipose tissue. Accordingly, the goal of this project was to determine how perivascular adipose tissue affects coronary artery function and elucidate the critical mechanisms involved. Initial studies assessing arterial function were conducted with and without perivascular adipose tissue. Preliminary results demonstrated that factors released by perivascular adipose tissue effectively impaired coronary endothelial function both in vitro and in vivo. This observation was determined to be caused by direct inhibition of nitric oxide synthase (NOS), a critical enzyme for the production nitric oxide. Attenuation of endothelium-dependent vasodilation was independent of changes in superoxide production, smooth muscle response, or peroxide-mediated vasodilation. Additional studies revealed that perivascular adipose-induced impairment of NOS was due to increased inhibitory regulation by the β isoform of protein kinase C (PKC-β). Specifically, perivascular adipose-derived factors caused site specific phosphorylation of nitric oxide synthase at Thr-495. Additional experiments investigated how perivascular adipose-derived factors contributed to coronary artery disease in an animal model of obesity. Results from these studies indicated that perivascular adipose-derived leptin markedly exacerbated underlying endothelial dysfunction, and significantly contributed to coronary endothelial dysfunction through a PKC-β dependent mechanism. Findings from this project confirm epicardial perivascular adipose tissue as a local source of harmful adipocytokines. In addition, perivascular adipose-derived leptin was demonstrated to be a critical mediator of coronary vascular dysfunction in obesity. Together, the results strongly suggest that perivascular adipose tissue is a key contributor to coronary artery disease in obesity.