The Role of Polymerase Delta Interacting Protein 2 in Neointima Formation Open Access

Harousseau, Mark Eric Philippe (2012)

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The process of restenosis, or renarrowing of an injured artery, results in the proliferation and migration of vascular smooth muscle cells (VSMCs) into the intima, forming the neointima. The pathological mechanism of neointima formation is complex and is believed to involve multiple cellular mechanisms including migration, proliferation, apoptosis, and extracellular matrix (ECM) formation. Polymerase delta interacting protein 2 (Poldip2, also known as PDIP38 and Mitogenin I) is a multifunctional protein that regulates DNA repair, cytoskeletal remodeling, proliferation, and the generation of reactive oxygen species, products of oxygen metabolism in all aerobic systems, via its interaction with the NADPH oxidase Nox4. This latter function may be particularly relevant to vascular pathology, as Nox4 regulates phenotypic differentiation and migratory function of VSMCs. Little is known about the functionality of Poldip2. Here, using a Poldip2 (+/-) gene trap animal model and a transluminal wire injury model with rapid onset of medial cell apoptosis and replicable neointimal hyperplasia ~3-4 weeks post injury, we demonstrate that Poldip2 heterozygosity results in reduced neointima area, an increased deposition of an ECM component within the neointima and a trend of decreased proliferation in injured femoral arteries of Poldip2 (+/-) mice compared to wildtype (WT) mice. We also demonstrate that Poldip2 expression does not affect apoptosis, which mediates the initial vessel response to injury. Our findings implicate Poldip2 as an important regulator of neointima formation, and demonstrate that it exerts its effects via multiple cellular mechanisms. Future studies should be designed to delve further into these mechanisms in order to design therapeutic agents to treat the clinically important problem of restenosis.

Table of Contents

Introduction 1-10

Materials and methods 11-18

a. Animals 11

b. Transluminal wire Injury 12

c. Tissue collection and slide preparation 13

d. Collagen Staining 13

e. Apoptosis 14

f. Proliferation detected by immunofluorescent histology 14

g. Smooth muscle α-actin immunolabeling 15

h. In vitro studies 15

i. Microscopy 16

j. Image analysis and calculations 16

k. Statistical analysis 17

Results 19-35

a. Poldip2 (+/-) heterozygosity and neointimal reduction 19-21

b. In vitro ECM protein studies 21-22

c. In vivo characterization of collagen 22-24

d. Medial Layer Apoptosis 25-26

e. siPoldip2 limits proliferation in vitro 27

f. Optimizing PCNA Immunolabeling 27-30

g. Proliferation within the medial layer and neointima following wire injury 30-35

h. Cellular counts within the medial layer and neointima 35-36

i. Vascular smooth muscle cell localization 36-37

Discussion 38-46

References 47-50

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