Atherosclerosis is the number one cause of death in the United States and one of the most common treatments is the implantation of a stent. In order to eliminate the two most common complications - restenosis and thrombosis - a novel covered stent is investigated. A covered stent membrane should be able to undergo large stretch, prevent restenosis, and be relatively non-thrombogenic. Polyvinyl alcohol (PVA) cryogels are examined as a candidate material for covered stent membranes. Mechanical testing included uniaxial tensile testing, puncture testing, and the fabrication and expansion of PVA cryogel covered stents. Uniaxial testing showed PVA cryogels to have sufficient ultimate stretch which was similar to bare metal stents during deployment. Puncture testing revealed that PVA cryogels are not likely to puncture in vivo. No tears were seen in the PVA cryogel membrane after expansion of the covered stents. Finite element analysis was used to determine a PVA cryogel membrane's effect on artery wall stress. PVA cryogel covered stents reduced both artery wall stress and tissue prolapse when compared to equivalent uncovered stents. Migration assays were used to determine if PVA cryogels are able to block the smooth muscle cell migration seen during restenosis. PVA cryogels significantly reduced cellular migration in modified Boyden chambers - suggesting that they would be able to prevent restenosis in vivo. Thrombogenicity was tested in vitro with a gravity-fed flow loop using porcine blood and in vivo with a sheep model. PVA cryogels were found to be less thrombogenic than polyester controls with the flow loop system. The sheep study demonstrated the feasibility of implanting PVA cryogel covered stents and good early patency. After explantation, the PVA cryogel membranes were intact - providing in vivo evidence for the durability of PVA cryogel covered stents. Overall, this work provides evidence that covered stents made with PVA cryogels are a feasible device in terms of their mechanics, ability to prevent restenosis, and low thrombogenicity. This work represents a major advancement in the development of PVA cryogel covered stents and provides necessary safety and feasibility data for future clinical trials.

<p>Today’s rapid development of powerful tools for geneexpression analysis provides unprecedented resources forelucidating complex molecular events.</p><p>The objective of this workhas been to apply, combine andevaluate tools for analysis of differential gene expressionusing atherosclerosis as a model system. First, an optimisedsolid-phase protocol for representational difference analysis(RDA) was applied to two<i>in vitro</i>model systems. Initially, The RDA enrichmentprocedure was investigated by shotgun cloning and sequencing ofsuccessive difference products. In the subsequent steps,combinations of RDA and microarray analysis were used tocombine the selectivity and sensitivity of RDA with thehigh-throughput nature of microarrays. This was achieved byimmobilization of RDA clones onto microarrays dedicated forgene expression analysis in atherosclerosis as well ashybridisation of labelled RDA products onto global microarrayscontaining more than 32,000 human clones. Finally, RDA wasapplied for the investigation of the focal localisation ofatherosclerotic plaques in mice using<i>in vivo</i>tissue samples as starting material.</p><p>A large number of differentially expressed clones wereisolated and confirmed by real time PCR. A very diverse rangeof gene fragments was identified in the RDA products especiallywhen they were screened with global microarrays. However, themicroarray data also seem to contain some noise which is ageneral problem using microarrays and should be compensated forby careful verification of the results.</p><p>Quite a large number of candidate genes related to theatherosclerotic process were found by these studies. Inparticular several nuclear receptors with altered expression inresponse to oxidized LDL were identified and deserve furtherinvestigation. Extended functional annotation does not liewithin the scope of this thesis but raw data in the form ofnovel sequences and accession numbers of known sequences havebeen made publicly available in GenBank. Parts of the data arealso available for interactive exploration on-line through aninteractive software tool. The data generated thus constitute abase for new hypotheses to be tested in the field ofatherosclerosis.</p><p><b>Keywords:</b>representational difference analysis, geneexpression profiling, microarray analysis, atherosclerosis,foam cell formation</p>

Atherosclerosis is an inflammatory disease of the arterial walls and is the major cause of heart attack and stroke. Atherosclerosis is localized to curves or branches in the vasculature where disturbed blood flow alters endothelial cell (EC) gene expression and induces EC dysfunction. Epigenetics controls aberrant gene expression in many diseases, but the mechanism of flow-induced epigenetic gene regulation in ECs via DNA methylation has not been well studied until very recently. The goal of this project was to determine how the DNA methylome responds to flow, causes altered gene expression, and regulates atherosclerosis development. Here, we found that d-flow increases DNA Methyltransferase 1 (DNMT1) expression in ECs, and we hypothesized that this causes a shift in the EC methylome and transcriptome towards a pro-inflammatory, pro-atherosclerotic gene expression program, and further that this leads to atherosclerosis development. To test this hypothesis, we employed both in vitro and in vivo experimental approaches combined with genome-wide studies of the transcriptome and DNA methylome according to the following three specific aims: 1) to elucidate the role of DNA Methyltransferase 1 in EC function, 2) to uncover the DNA methylation-dependent EC gene expression response to flow, and 3) to discover and examine master regulators of EC function that are controlled by DNA methylation. The work presented here has resulted in new knowledge about the epigenetic EC shear response, details the previously unstudied EC methylome, and implicates specific loci within the genome for additional studies on their role in EC biology and atherosclerosis. This work provides a foundation for novel and more targeted therapeutic strategies for CVD.

The persistence of high global mortality rates directly attributable to cardiovascular disease drives ongoing research into novel approaches for improved diagnosis and treatment of its primary underlying cause, atherosclerosis. Combined intravascular ultrasound and photoacoustic (IVUS/IVPA) imaging is one such modality, actively being developed as a tool for improved characterization of high-risk atherosclerotic plaques. The pathophysiology associated with progression and destabilization of atherosclerotic plaques leads to characteristic changes in arterial morphology and composition. IVUS/IVPA imaging seeks to expand upon the ability of clinically utilized intravascular ultrasound (IVUS) imaging to assess vessel anatomy by adding improved sensitivity to image the underlying cellular and molecular composition through intravascular photoacoustic (IVPA) imaging of either endogenous chromophores (e.g. lipid) or exogenously delivered contrast agents. This dissertation focuses on the expansion of IVUS/IVPA imaging using exogenous contrast agents to enable the detection and subsequent optically-triggered therapy of atherosclerotic plaques. The passive extravasation and aggregation of systemically injected plasmonic gold nanorods absorbing within the near infrared tissue optical window within plaques of atherosclerotic rabbit models is first demonstrated, along with the ability to localize the contrast agents using ex vivo IVUS/IVPA imaging. The motivation for nanoparticle labeling of atherosclerosis is then expanded from that of purely image contrast agents to vehicles for image-guided, dual-modality phototherapy. The integrated IVUS/IVPA imaging catheter is utilized for photothermal delivery with simultaneous IVPA temperature monitoring using the high optical absorption of gold nanorod contrast agents to enable localized heating. Subsequently, the potential role for IVUS/IVPA-guided phototherapy is further expanded through the characterization and in vitro assessment of novel multifunctional theranostic nanoparticles comprised of a gold nanorod core with a degradable, photosensitizer-doped silica shell. Together, the results presented within this dissertation provide a framework for ongoing research into the expansion of IVUS/IVPA imaging as a platform for complimentary diagnosis and local treatment of atherosclerotic plaques using multifunctional theranostic nanoparticle contrast agents. / text

Atherosclerosis (Heart Disease) is an inflammatory disease caused by the formation of plaque within the arterial wall. In response to inflammation, monocytes enter the artery wall, differentiate into macrophages and take up altered low-density-lipoprotein (such as oxidised-LDL). This oxLDL is taken up into the phagocytotic macrophages via the action of the scavenger receptors. If more oxLDL is engulfed than the cell can process, they further differentiate into lipid-loaded foam cells. These are the main cell type found in atherosclerotic plaques. The scavenger receptor CD36 is responsible for 70% of oxLDL uptake by macrophages. Previous studies show that CD36 expression can be down regulated by the antioxidant, 7,8-dihydroneopterin. This research focuses on the effect of CD36 down regulation by 7,8-dihydroneopterin on foam cell formation. Human macrophages prepared from monocytes purified from human blood were incubated with copper oxidised LDL for up to 48 hours. Macrophage accumulation of the sterols was measured using a high performance chromatograph (HPLC) method developed as part of this project. The HPLC analysis measured: cholesterol, cholesteryl-oleate and -palmitate and 7-ketocholesterol accumulation within human macrophages. A flow cytometry procedure was developed where the strongly adherent macrophages could be lifted from the tissue culture plates before immuno staining for CD36. Effect of incubating macrophages with 7,8-dihydroneopterin on the formation of foam cells was studied by measuring the lipid content by HPLC and flow cytometry measurement of CD36. HPLC analysis showed non-cytotoxic levels of oxLDL produced a large accumulation of cholesterol and cholesteryl esters in the macrophages. Cholesterol, 7-ketocholesterol and cholesteryl-oleate and -palmitate concentrations in the cells rose significantly over the first 24 hours and stayed at a steady level for the following 24 hours. CD36 levels was further analysed on human macrophages. This study shows that foam cell formation can be measured using human macrophages. 7,8-Dihydroneopterin treatment resulted in a reduction of cholesterol and oxysterol uptake back to basal levels. It also reduced CD36 cell surface expression by a third. These results suggest that even a small reduction in CD36 cell surface expression may have a large effect on foam cell formation. This is another mechanism by which 7,8-dihydroneopterin protects against atherosclerosis developing.

Background: Glycated hemoglobin is the indicator of long-term diabetes control and a value below 7 percent is recommended by the American Diabetes Association (ADA) to reduce cardiovascular complications. Diabetic patients have a two- to four-fold risk of cardiovascular disease and approximately two-thirds of diabetic patients die as a result of cardiovascular complications. Three large prospective randomized controlled long-term trials within the last decade reported no significant reduction in cardiovascular complications in type 2 diabetic patients by intensive glycemic control. To the author's knowledge, no known retrospective studies have examined the association between mean serial glycated hemoglobin and coronary atherosclerosis (CA) or acute coronary syndromes (ACS). Objective: This study was designed to determine the association between mean serial glycated hemoglobin with incident CA or ACS in type 2 diabetic patients after controlling for age, gender, hypertension, low density lipoprotein cholesterol (LDL-C), microalbuminuria, aspirin use, statin use, insulin use, tobacco use, and body mass index (BMI). Methods: The study was a retrospective cohort database analysis using the Austin Travis County CommUnityCare[trademark] clinics' electronic medical record for the time period between October 1, 2004 and September 30, 2009. The primary outcome of the study was the incidence of CA or ACS and the primary independent variable was glycated hemoglobin (<7% vs. [greater than or equal to]7%). The study subjects included type 2 diabetic patients aged 30 to 80 years with at least one glycated hemoglobin value per year for a minimum of two consecutive years. Study subjects were excluded if CA or ACS occurred within six months of the index date (i.e., first glycated hemoglobin). Logistic regression analysis was used to address the study objective. Results: Overall, 3069 subjects met the study inclusion criteria with a mean follow-up period of approximately two years. Two percent (N=62) of the subjects had incident CA or ACS. After controlling for age, gender, hypertension diagnosis, LDL-C, microalbuminuria, aspirin use, statin use, insulin use, tobacco use and BMI, there was no significant association (OR=1.026, 95% CI=0.589-1.785, p=0.9289) between mean serial glycated hemoglobin and the incident diagnosis of CA or ACS. Increasing age (OR=1.051, 95% CI=1.025-1.077, p<0.0001), male gender (OR=1.855, 95% CI=1.105-3.115, p=0.0195) and normal weight (normal or underweight compared to obese: OR=0.122, 95% CI=0.017-0.895, p=0.0438) were significantly associated with incident CA or ACS. Conclusions: Mean serial glycated hemoglobin (comparing [greater than or equal to]7% to <7%) was not significantly associated with CA or ACS over a mean follow-up period of approximately two years. Until more evidence becomes available, clinicians and diabetic patients should target glycated hemoglobin level below or close to 7 percent as recommended by the ADA soon after diagnosis while concomitantly controlling nonglycemic risk factors of cardiovascular disease (statin use, aspirin use, blood pressure control, smoking cessation and life style modification), to reduce their long-term risk of incident CA or ACS. / text

The ability of sub 100 nm nanoparticles to target and modulate the biology of cells will enable major advancements in cellular imaging and therapy in cancer and atherosclerosis. A key challenge is to load an extremely high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. A general mechanism is presented for thin autocatalytic growth on nanoparticle substrates (TAGS), as demonstrated for a homologous series of < 5 nm textured Au coatings on < 42 nm iron oxide cluster cores. Very low Au supersaturation levels are utilized to prevent commonly encountered excessive autocatalytic growth that otherwise produce thick shells. The degree of separation of nucleation to form the seeds from growth is utilized to control the morphology and uniformity of the thin Au coatings. The thin and asymmetric Au shells produce strong near infrared (NIR) absorbance with a cross section of ~10⁻¹⁴ m², whereas the high magnetic content per particles provides strong r2 spin-spin magnetic relaxivity of 200 mM⁻¹s⁻¹. TAGS may be generalized to a wide variety of substrates and high energy coatings to form core-shell nanoparticles of interest in a variety of applications as diverse as catalysis and bionanotechnology. High uptake of the nanoclusters by macrophages is facilitated by the dextran coating, producing intense NIR contrast both in cell culture and an in vivo rabbit model of atherosclerosis. A novel conjugation technique further allows covalent binding of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (Ab) to the nanoclusters for highly selective targeting to EGFR over expressing cancer cells. AlexaFluor 488 tagged Ab nanocluster conjugates were prepared to correlate the number of conjugated Abs with the hydrodynamic diameter. The high targeting efficacy was evaluated by dark field reflectance imaging and atomic absorbance spectrometry (AAS). Colocalization of the nanoparticles by dual mode in-vitro imaging with dark field and fluorescence microscopy demonstrates the Abs remained attached to the Au surfaces. The extremely high curvature of the Au shells with features below 5 nm influence the spacing and orientations of the Abs on the surface, which has the potential to have a marked effect on biological pathways within cells. These targeted small multifunctional nanoclusters may solve some key molecular imaging challenges for cancer and atherosclerosis. / text

Rupture of atherosclerotic plaque is closely related to plaque composition. Currently, plaque composition cannot be clinically characterized by any imaging modality. The objective of this dissertation is to use a recently developed imaging modality – ultrasound-guided intravascular photoacoustic (IVPA) imaging – to detect the distribution of two critical components in atherosclerotic plaques: lipid and phagocytically active macrophages. Under the guidance of intravascular ultrasound imaging, spectroscopic IVPA imaging is capable of detecting the spatially resolving optical absorption property inside a vessel wall. In this study, contrast in spectroscopic IVPA imaging was provided by either the endogenous optical property of lipid or optically absorbing contrast agent such as gold nanoparticles (Au NPs). Using a rabbit model of atherosclerosis, this dissertation demonstrated that ultrasound guided spectroscopic IVPA imaging could simultaneously image lipid deposits as well as macrophages labeled in vivo with Au NPs. Information of macrophage activity around lipid rich plaques may help to identify rupture-prone or vulnerable plaques. The results show that ultrasound guided IVPA imaging is promising for detecting plaque composition in vivo. Clinical use of ultrasound guided IVPA imaging may significantly improve the accuracy of diagnosis and lead to more effective treatments of atherosclerosis. / text

Τα αγγειακά λεία μυικά κύτταρα (ΑΛΜΚ) αποτελούν το κυρίαρχο στοιχείο του μέσου χιτώνα των αιμοφόρων αγγείων, ενώ συμμετέχουν ενεργά και στο σχηματισμό και την ωρίμανση του καρδιαγγειακού συστήματος. Η δομή τους εξυπηρετεί την εκτέλεση της σημαντικότερης λειτουργίας τους, που είναι η συστολή. Αξιοσημείωτο χαρακτηριστικό των αγγειακών λείων μυικών κυττάρων αποτελεί η φαινοτυπική τους πλαστικότητα, δηλαδή η ικανότητα στροφής από το συσταλτικό σε έναν περισσότερο συνθετικό φαινότυπο, που λαμβάνει χώρα υπό προϋποθέσεις. Οι αλληλεπιδράσεις με τα υπόλοιπα κυτταρικά στοιχεία του τοιχώματος των αρτηριών και των έμμορφων συστατικών του αίματος, αλλά και η φαινοτυπική πλαστικότητα καθιστούν καθοριστικό το ρόλο των αγγειακών λείων μυικών κυττάρων στην παθογένεια της αθηροσκλήρωσης. / Vascular smooth muscle cells (VSMCs) comprise the main element of the tunica media of blood vessels, while they actively participate in the formation and maturation of the cardiovascular system. Their structure serves their basic function, which is contraction. An interesting feature of vascular smooth muscle cells is their phenotypic plasticity, the ability to shift from a contractile to a more synthetic phenotype, under certain conditions. The interaction with other cellular elements within the vascular wall or in the bloodstream, as well as their phenotypic plasticity, give vascular smooth muscle cells a decisive role in the pathogenesis of atherosclerosis.

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