Whether innate immune cells may be adapted into potential memory states has becoming an important question in the field of immunity. Although previous conceptual paradigm failed to acknowledge this important question, emerging clinical and basic observations have started to shed intriguing clues to shake the previous dogma regarding innate immunity of being "simple", "raw", "first-line defense with no memory". We have aimed to further address this fundamental issue in this dissertation work, under the close guidance of Dr. Liwu Li. We have chosen to use the model system of Toll-Like-Receptor (TLR) signaling networks within primary monocytes. TLRs play fundamental roles in sensing pathogen-associated molecular patterns (PAMPs) and modulation of innate immunity. Lipopolysaccharide (LPS), an endotoxin found on the cell membrane of gram-negative bacteria, is the ligand of TLR4 and induces a range of inflammatory as well as anti-inflammatory responses. Higher dosages of LPS were known to cause robust yet transient expression of pro-inflammatory mediators. On the other hand, the effects of super-low dose LPS, commonly manifested in humans with adverse health conditions, have been largely ignored in the basic research field. Super-low dose LPS may skew host immune environment into a mild non-resolving pro-inflammatory state, which is a risk factor for inflammatory diseases such as atherosclerosis, compromised wound healing, and elevated risks for sepsis. Our central hypothesize is that monocytes may be adapted by super-low dose LPS into a non-resolving low-grade inflammatory state conducive for the pathogenesis of inflammatory diseases. We have employed both in vitro cell culture system as well as in vivo disease models to test this hypothesis. For the in vitro system, we have cultured primary murine monocytes with increasing signal strength of LPS. Monocyte phenotypes such as the expression of key inflammatory mediators including cytokines, chemokines, and cellular surface markers were studied. Potential molecular and cellular mechanisms were examined. We revealed a novel low-grade inflammatory monocyte phenotype termed ML adapted by super-low dose LPS, mediated through IRF5. For the in vivo system, we have employed both acute and chronic models of inflammation. For the chronic model, we have tested the effects of super-low dose LPS on monocyte polarization in vivo, as well as its contribution to the pathogenesis of atherosclerosis. Furthermore, we have tested the effects of programmed monocytes on wound healing. For the acute model, we have tested the effects of pre-conditioning with super-low dose LPS on the subsequence risks of sepsis elicited by cecal ligation and puncture. We have demonstrated aggravated atherosclerosis, compromised wound healing, and increased sepsis mortality in mice pre-conditioned with super-low dose LPS. Taken together, our findings reveal that monocytes can be differentially programmed into distinct states, depending on the signal strength of LPS. The differential programming and adaptation of monocytes can occur both in vitro and in vivo, and may bear profound pathological consequences. / Ph. D.

Sphingosine-1-phosphate receptor type 1 (S1PR1), one of the five S1PRs that signals in response to bioactive lysosphingolipid S1P, regulates several fundamental processes in distinct cell types and is implicated in atherosclerosis. Using the cre-lox recombination system, previous studies identified that knocking out S1PR1 in myeloid and endothelial cells promotes plaque development in atherogenic mouse models. In the process of generating S1pr1lox/lox; ApoEKO/KO control mice, we unexpectedly noticed that S1pr1lox/lox mutation alone, in the absence of cre recombinase, reduces high-fat (HF) diet-induced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice compared to S1pr1WT/WT; ApoEKO/KO mice. Although S1pr1lox/lox allele partially suppressed S1pr1 levels in macrophages and vascular smooth muscle cells (VSMC), the presence of this mutation in a non-BM derived cell type was responsible for this reduced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice. We speculated that it could be VSMCs due to their abundance in the vascular wall and their role in foam cell formation. In this thesis, we directly tested the effects of inactivating S1PR1 in smooth muscle cells (Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice) on atherosclerosis. Our results demonstrated that deleting S1PR1 in smooth muscle cells drastically reduces atherosclerosis in apoE-deficient mice. The aortic SMCs isolated from these mice also exhibited reduced cell proliferation and lipid droplet formation in response to S1PR1 agonist SEW2871 compared to S1PR1-WT VSMCs. Furthermore, we also tested the effects of directly inhibiting S1PR1 with S1PR1 selective antagonist Ex26 at a dosage of 0.1 mg/kg/hr in S1pr1WT/WT; ApoEKO/KO mice and Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice. The prolonged exposure to Ex26 substantially reduced atherosclerotic plaque development in apoE KO mice on an HFD compared to DMSO-treated apoE KO mice. However, this protection was completely lost in mice that lack the S1pr1 gene in VSMCs. Overall, our results suggest that knocking out S1PR1 in VSMCs results in atheroprotection that surpasses the effects of inactivating S1PR1 in macrophages and endothelial cells which are known to promote atherosclerosis. / Dissertation / Doctor of Philosophy (PhD)

No / Vascular inflammation is implicated in the pathogenesis of atherosclerosis and abdominal aortic aneurysm (AAA), and is thought to involve reactive species such as the nitric oxide-derived oxidant peroxynitrite. In the present study nitrotyrosine was measured as a stable marker of peroxynitrite production in vivo. Perioperative blood samples were obtained from patients undergoing elective open or endovascular repair of an AAA and from patients with intermittent claudication, smoking aged-matched controls, non-smoking aged-matched controls and non-smoking young healthy controls. Plasma nitrotyrosine was measured by an enzyme-linked immunosorbent assay. The median plasma nitrotyrosine concentration in patients with an AAA (0·46 nmol nitrated bovine serum albumin equivalents per mg protein) was significantly higher than that in patients with intermittent claudication (0·35 nmol; P = 0·002), smoking controls (0·36 nmol; P = 0·001), non-smoking controls (0·35 nmol; P = 0·002) and young healthy controls (0·27 nmol; P < 0·001). Nitrotyrosine concentrations increased during early reperfusion in open AAA repair, but not during endovascular repair. AAA exclusion from the circulation reduced levels to control values (P = 0·001). Patients with an AAA had raised levels of circulating nitrated proteins compared with patients with claudication and controls, suggesting a greater degree of ongoing inflammation that was not related to smoking. Copyright

No / The expression and potential role of platelet membrane CD154 and sCD154 in atherosclerosis was investigated in patients with peripheral arterial disease. This prospective observational study measured the expression of platelet-bound CD154 and soluble CD154 (sCD154) in 39 patients with critical limb ischaemia (CLI, n = 15), stable intermittent claudication (SIC, n = 12) and age-matched controls (AMC, n = 12). Basal and agonist-stimulated CD154, P-selectin expression and fibrinogen binding was measured by whole blood flow cytometry, while sCD154 was measured in paired plasma samples by ELISA. Basal expression of CD154 on the platelet surface was enhanced in both groups of patients with peripheral arterial disease. However, the critical limb ischaemics showed the highest level of basal expression 0.7 ± 0.3 [median ± IQR] and was significantly increased compared to both stable intermittent claudicants and age-matched controls (P < 0.001). On agonist stimulation with either ADP or thrombin critical limb ischaemics demonstrated greater platelet reactivity and propensity to express CD154 compared to age-matched controls (P < 0.05). Confirmation of the cellular expression of CD154 results was obtained by measuring sCD154 concentrations in autologous plasma samples. Here plasma levels of sCD154 in critical limb ischaemics were significantly greater than both stable intermittent claudicants and age-matched controls (P < 0.005).

Yes / Vascular calcification is a complex biological process that is a hallmark of atherosclerosis. While macrocalcification confers plaque stability, microcalcification is a key feature of highrisk atheroma and is associated with increased morbidity and mortality. Positron emission tomography and X-ray computed tomography (PET/CT) imaging of atherosclerosis using 18F-sodium fluoride (18F-NaF) has the potential to identify pathologically high-risk nascent microcalcification. However, the precise molecular mechanism of 18F-NaF vascular uptake is still unknown. Here we use electron microscopy, autoradiography, histology and preclinical and clinical PET/CT to analyse 18F-NaF binding. We show that 18F-NaF adsorbs to calcified deposits within plaque with high affinity and is selective and specific. 18F-NaF PET/CT imaging can distinguish between areas of macro- and microcalcification. This is the only currently available clinical imaging platform that can non-invasively detect microcalcification in active unstable atherosclerosis. The use of 18F-NaF may foster new approaches to developing treatments for vascular calcification.

Atherosclerosis, the underlying cause of cardiovascular diseases such as coronary artery disease, is driven by inflammation and cell death of leukocytes, particularly macrophages. These processes contribute significantly to plaque destabilization, leading to the development of vulnerable plaques prone to rupture and thrombotic events. Understanding the mechanisms underlying leukocyte inflammation and cell death in atherosclerosis is crucial for identifying novel therapeutic targets to stabilize plaques and reduce the risk of cardiovascular events. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a key player in atherosclerosis, with its role in lipid metabolism and plaque development being extensively studied. While PCSK9 is predominantly produced in hepatocytes, emerging evidence suggests its expression in leukocytes and macrophages may have distinct effects on atherosclerosis, beyond its role in lipid regulation. In this thesis, the role of leukocyte PCSK9 in atherosclerosis was investigated by restoring normal PCSK9 expression in leukocytes of PCSK9 and ApoE-deficient mice. Contrary to expectations, restoring leukocyte PCSK9 expression did not significantly alter plaque size, macrophage or smooth muscle cell content, pro-inflammatory cytokine expression, or apoptosis within plaques. These findings suggest that leukocyte PCSK9 expression may not significantly impact atherosclerotic plaque development or the local plaque environment in this mouse model. In this thesis, we also explored the role of ApoA1 deficiency and Bim-mediated apoptosis in atherosclerotic plaque development. ApoA1 deficiency increased plaque size, necrotic core size, and apoptosis in macrophages within plaques, potentially through upregulation of Bim protein. Inhibiting Bim in all bone marrow-derived cells and myeloid-specific cells reduced plaque apoptosis, necrotic core sizes, and plaque sizes, highlighting the significance of Bim in atherosclerosis progression, and suggesting a specific role of myeloid cell-derived Bim in plaque stability. These findings provide insights for future PCSK9 research and contribute to our understanding of HDL's protective effects and a potential therapeutic target for apoptotic cell death in atherosclerosis. / Thesis / Doctor of Philosophy (Medical Science)

The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis, abdominal aortic aneurysms, and heart valve disease in regions exposed to disturbed flow leading to low or oscillatory shear stress at the wall of the blood vessel or the surface of the valve leaflet. The novel and significant finding of this study is that mouse aortic endothelial cell exposure to pro-atherogenic oscillatory shear stress (OS) (+/- 5 dynes/cm2) increased their production of cathepsins, the family of lysosomal cysteine proteases that are potent elastases and collagenases leading to protease degradation and remodeling of the extracellular matrix structural components. Conversely, atheroprotective unidirectional laminar shear stress (LS) (15 dynes/cm2) decreased elastase and gelatinase activities of endothelial cells through a shear stress mediated reduction in cathepsins K, L, and S activity. Their endogenous inhibitor, cystatin C, was found to be inversely regulated by shear stress; LS increased its secretion by endothelial cells while OS decreased it. Binding of free cystatin C in the conditioned media to carboxymethylated papain coated agarose beads led to an increase in cathepsin activity since the available cathepsin was not inhibited. To verify these findings in human samples, immunohistochemical analysis of cystatin C and cathepsin K was performed on human coronary arteries. Cathepsin K stained strongly in the endothelial layer of vessels with degraded internal elastic lamina while cystatin C staining intensity was strongest overlying minimally diseased vessels. Additional roles for cathepsins K, L, and S were found in endothelial cell alignment in response to unidirectional laminar shear stress, endothelial cell migration, and programmed cell death. We conclude that there is an inverse regulation of cathepsins and cystatin C in endothelial cells by LS and OS and identify the cathepsin family of proteases as potential targets for therapeutic intervention of cardiovascular disease development at sites of disturbed flow.

Thesis (M.Sc.), Memorial University of Newfoundland, 2000. / Restricted until June 2003. Bibliography: leaves 98-117.

Background: Atherosclerosis is an important cause of stroke. Ultrasound offers the convenience of real-time and detailed assessment of carotid plaque features as well as arterial wall thickening and composition. Evaluation of these features is important for determining patients’ risk of suffering vascular events and also contributes to selecting the best treatment strategy. Methods: Using ultrasound data analysis we have determined plaque features in the bifurcation and internal carotid artery (ICA), including: surface plaque irregularities, calcification, echogenicity (grey scale median-GSM) and other textural plaque features (Juxtaluminal black area, entropy, coarseness). In addition, intima media thickens (IMT) and its grey scale median (IM-GSM) was measured in common carotid artery (CCA). Using Cone Beam CT (CBCT) we have quantified calcification volume of the carotid plaques extracted after carotid endarterectomy procedure. For the meta-analysis we have used comprehensive meta-analysis software version 3. Study I: We have included 39 patients and we compared carotid plaque features of the contralateral arteries with those located ipsilateral to symptomatic side and arteries of asymptomatic patients. Study II: The accuracy of US to detect atherosclerosis calcification was assessed against CBCT in 88 patients. Study III: Based on the previous vascular events in coronary, carotid and lower extremity arterial system, 87 patients were divided into three groups: asymptomatic, symptoms in one vascular system and symptoms in more that one vascular system. IMT, IM-GSM and plaque features were compared between groups. Study IV: We have meta-analyzed ten cohort prospective studies evaluating carotid plaque echogenicity for cerebrovascular symptoms prediction. Results: Study I. Plaques of the contralateral to symptomatic arteries had similar features to those in symptomatic and more vulnerable than asymptomatic arteries. Study II. Carotid ultrasound was accurate in detecting calcification volumes of ≥8mm3 with very high sensitivity but it was less accurate in detecting lower calcification volumes (&lt;8mm3). Carotid calcification was not different between symptomatic and asymptomatic patients. Study III. Echogenicity of the intima-media complex (IM-GSM), but not its thickness (IMT), was significantly decreased with increasing number of arterial systems affected by atherosclerosis. IM-GSM was lower in patients with prior myocardial infarction and stroke. Study IV. Carotid plaque echogenicity evaluated by US could predict future cerebrovascular events in patients with asymptomatic, relative risk RR 2.72 (95% CI, 1.86 to 3.96), and recurrent symptoms in symptomatic patients, RR 2.97 (95% CI, 1.85-4.78). Conclusion: Plaques located in the contralateral to symptomatic arteries have similar features as symptomatic side and more vulnerable than asymptomatic arteries. Carotid ultrasound could accurately detect larger but not smaller carotid plaque calcification volumes (&lt;8 mm3). Low IM- GSM could identify patients with multi-system atherosclerosis disease, suggesting a better marker for determining systemic atherosclerosis disease burden compared to conventional IMT. Finally, carotid plaque echogenicity predicts future cerebrovascular events in patients with symptomatic and asymptomatic carotid stenosis.

Atherosclerosis is an inflammatory disease, occurring preferentially in arterial regions with disturbed flow. We have shown that disturbed flow induces inflammation in endothelial cells (ECs) by producing bone morphogenic protein-4 (BMP4). Moreover, chronic BMP4 infusion induces endothelial dysfunction and systemic hypertension in mice. Here, we examined which BMP receptors (BMPR) mediate BMP4 action in ECs. Western blot, immunostaining and RT-PCR studies using human and bovine ECs, mouse aortas and human coronary arteries (HCA) showed that BMPRI (ALK2 and 6) and BMP-RII were expressed in ECs. As a functional test, ECs were treated with a BMPRII siRNA to knockdown expression. BMPRII knockdown blocked a well-known BMP4 response - smad1/5/8 phosphorylation, as expected. Unexpectedly, BMPRII knockdown itself significantly stimulated ICAM-1 and VCAM-1 expression and monocyte adhesion in a BMP4-independent manner. Inflammatory responses caused by BMPRII knockdown were blocked by inhibitors of NADPH oxidase and NFκ B. From these results, we hypothesized that BMP-RII knockdown in ECs would cause inflammation, which is a critical event in atherosclerosis initiation and progression. Genetic mutations of BMPRII have been linked to primary pulmonary hypertension. However, it is not known whether BMP-RII is regulated by atherosclerotic conditions and plays a role in non-pulmonary vessels causing inflammation and atherosclerosis. We examined BMPRII levels in HCA by immunostaining. While non-diseased arteries showed intense staining of BMPRII, the expression decreased as lesions became more advanced. BMPRII was virtually undetectable in the most advanced lesions. These findings suggested a potential link between pro-atherosclerotic conditions and BMP-RII levels. We tested this hypothesis by treating ECs with pro-inflammatory cytokines found in atheromas: TNFα decreased BMPRII by 2-fold. In contrast, statins increased BMPRII by 4-fold. In summary, we demonstrate for the first time that BMPRII can be down- or up-regulated by pro- or anti-atherogenic conditions, respectively, and it is dramatically decreased in HCA with advanced plaques. Moreover, BMPRII knockdown in ECs induces inflammation, a critical atherogenic step. We propose that focal inflammation initiated by disturbed flow, together with circulating pro-atherogenic risk factors, may lead to a vicious cycle of BMPRII down-regulation causing secondary inflammation and atheroma progression.