Ekspresi PDGF-B dan SCUBE 1 pada Arteri Karotis Mencit yang Diligasi dan Tidak Diligasi

AbstrakDiffuse Intimal Thickening (DIT)terjadi sebagai adaptasi fisologis terhadap shear stress yang dapat terbentuk melalui ligasi arteri karotis. Ligasi akan menyebabkan terbentuknya penebalan tunika intima pada pembuluh darah. Penebalan tunika intima terjadi melalui proses inflamasi yang melibatkan sitokin seperti PDGF-Bdan SCUBE 1 yang terdapat di dalam trombosit dan endotel. Tujuan penelitian adalah mengetahui ekspresi PDGF-B dan SCUBE 1 pada arteri karotis mencit yang diligasi dan tidak diligasi. Desain penelitian adalah eksperimental dengan menggunakan 5 ekor mencit yang dilakukan ligasi pada arteri karotis kiri sedangkan arteri karotis kanan tidak diligasi. Kemudian, arteri karotis tersebut dikumpulkan dan diperiksa kadar PDGF-B dan SCUBE 1 dengan menggunakan PCR. Uji hipotesis yang digunakan adalah independent t-test. Hasil penelitian adalah tidak terdapat perbedaan bermakna PDGF-B pada arteri karotis mencit yang diligasi dan tidak diligasi (p = 0,66; p>0,05) dan tidak terdapat perbedaan bermakna SCUBE 1 pada arteri karotis mencit yang diligasi dan tidak diligasi (p = 0,33; p>0,05).Kata kunci: diffuse intimal thickening, DIT, PDGF-B, SCUBE 1, PCR. AbstractDiffuse Intimal Thickening (DIT) develops as physiological adaptive process of shear stress which can be performed by carotid artery ligation in mice. Ligation will cause an intimal thickening in vessel. Intimal thickening occurs through an inflammatory process that involves cytokines such as PDGF-B and SCUBE 1 that are found in platelet and endothelial cells.The objective of this study was to determine the expression of PDGF-B and SCUBE 1 in ligated and non ligated carotid artery of mice.The design study was experimental. This study used 5 mouse that were ligated in left carotid artery and non ligated in right carotid artery. Then, those carotid arteries were collected and examined PDGF-B and SCUBE 1 levels using PCR. The hypothesis test of this study was Independent t-test.The result was no significant difference between PDGF-B in ligated and non ligated carotid artery of mice (p = 0.66; p>0.05). The same result was also found in SCUBE 1 as well (p = 0.33; p>0.05).Keywords: diffuse intimal thickening, DIT, PDGF-B, SCUBE 1, PCR.

Abstract 192: A Hypothesis on Initiation of Coronary Atherosclerosis: The Role of Intimal Neovascularization and Proliferation. Do Lipoproteins Invade Coronary Tunica Intima from the Opposite Site?

Objectives Tremendous success of statins in coronary atherosclerosis (CA) prevention offered great expectations for extended protection and effective therapeutics. However, stalled progress in pharmaceutical treatment gives a good reason to review whether the hypothesis underlining our efforts is consistent with undoubted facts on coronary artery in normal and diseased forms. Analysis An accepted hypothesis states that CA is initiated by endothelial dysfunction due to inflammation and high levels of LDL, followed by lipids and macrophage penetration into arterial intima and plaque formation. It is crucial to highlight that normal coronary intima is not a single-layer endothelium covering thin acellular compartment, as is commonly claimed in most publications, but always appears as a multi-layer cellular compartment, or diffuse intimal thickening (DIT), where cells are arranged in a few dozens layers. Since it is unanimously agreed that LDL invade DIT from lumen, the initial depositions ought to be most proximal to blood, i.e. in inner DIT layers. The facts show that the opposite is true, and lipids are deposited in the outer DIT. This contradiction is resolved by noting that normal DIT is always avascular, receiving oxygen and nutrients by diffusion from lumen, whereas in CA outer DIT is always neovascularized from adventitial vasa vasorum . Proteoglycan biglycan, confined to outer DIT of normal and diseased coronary, has high binding capacity for LDL. However, normal DIT is avascular, whereas in CA biglycan of outer DIT layers appears in direct contact with blood and extract lipoproteins. These facts explain patterns and mechanisms of CA initiation, which is not unique: normally avascular cornea accumulates lipoproteins after neovascularization, resulting in lipid keratopathy. The author offers a hypothesis on neovascularization. Cells in coronary DIT possess high proliferative capacity. Excessive cell replication increases DIT thickness, impairs diffusion, resulting in hypoxia of outer DIT. Hypoxia induces neovascularization of outer DIT layers, where biglycan extracts LDL from newly formed capillary bed, initiating CA. Conclusion Controls of cell proliferation and neovascularization in coronary DIT should be a priority of our research.

Abstract 593: Hypoxia is Present in the Macrophage-rich Center of Human Carotid Atherosclerotic Plaques

Intraplaque neovascularization is linked to plaque instability and thought to be stimulated by hypoxia. However, hypoxia has not been demonstrated yet in human atherosclerosis. The hypoxia marker pimonidazole was administrated intravenously 2 hours prior to carotid endarterectomy in 6 symptomatic patients to evaluate the presence of hypoxia. Subsequent immunohistochemistry of the operatively removed atherosclerotic plaques demonstrated the presence of hypoxia, especially in the macrophage-rich center of the lesions. Notably, two hypoxic gradients were observed: hypoxia was very strong in the center of the plaque, but almost absent close to the main artery lumen and in the media. hypoxia was most intense in segments with advanced atheroma and almost absent in segments containing only diffuse intimal thickening. Hypoxia strongly correlated with CD68 immunoreactivity (ρ= 0.7, p=0.000), neovascularization (ρ= 0.6, p=0.000) and the presence of a thrombus (ρ= 0.4, p=0.009). In addition, hypoxia co-localized with expression of HIF1α and VEGF . To exclude that pimonidazole immunoreactivity in the atherosclerotic plaque was the result of surgery-induced ischemia, arterial wall segments were collected at two time-points: directly after incision of the carotid artery and directly following excision of the plaque. Pimonidazole immunoreactivity in these two pieces was not different, suggesting that hypoxia and pimonidazole adducts were already present in the plaques before surgery . To show that pimonidazole reactivity was hypoxia-specific and independent of reactive oxygen species, human THP-1 macrophages were exposed to normoxia (20% O 2 ), hypoxia (0.2% O 2 ) and/or H 2 O 2 (100 μM) in the presence of pimonidazole. Indeed, flow cytometry only showed pimonidazole-positive cells after hypoxic exposure. This is the first study proving direct evidence of the existence of hypoxia in advanced human atherosclerotic lesions, most prominently in the macrophage-rich center. Also, hypoxia was associated with the expression of HIF1α, VEGF and intraplaque microvessels, suggesting its involvement in the regulation of human intraplaque neovascularization.