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The 3 PUFA, EPA and DHA, down-regulated human endothelial adhesion molecules in the presence of an inflammatory stimulus, DHA exerted more pronounced effects than EPA. The 6 PUFA, in contrast, exerted limited control over endothelial adhesion molecule expression under the conditions employed in this study. This indicates that the specific structure of the 3 PUFA, possibly combined with their degree of unsaturation, may be critical to their regulation of endothelial function. The antioxidant, quercetin, down-regulated some cytokine-induced endothelial adhesion molecules. In addition, quercetin acted synergistically with EPA and AA to decrease TNF--induced ICAM-1 or E-selectin protein, respectively. This may reflect regulation of eicosanoid production from these PUFA by quercetin, since this antioxidant inhibits enzymes critical to these metabolic pathways (i.e. cyclooxygenase and lipoxygenase). Hence, quercetin may mediate its inhibitory effects independently of its antioxidant properties. In contrast, -tocopheryl acetate up-regulated IL-1-induced E-selectin proteins levels. This antioxidant also, at least partially, blocked most of the inhibitory actions of the PUFA investigated. Recent data in the literature indicate that PUFA may exert their inhibitory effects on endothelial function through their oxidised derivatives. This provides a putative pathway via which -tocopheryl acetate may block the PUFA effects. The strength and type of inflammatory stimulus determined the sensitivity of the activated endothelial cells to each of the agents investigated. Inhibition of leukocyte adhesion to activated HUVEC, in response to EPA, DHA, or AA in the presence of quercetin, demonstrated a direct effect of these combined agents on endothelial function. The individual agents did not significantly reduce leukocyte adhesion.

Background: Cardiovascular health disparities across subpopulations and geographies have been well-documented in urban areas. Evidence suggests that racial minorities and low-socioeconomic groups have high risks of developing cardiovascular diseases (CVD). Residents of the Appalachia also exhibit high rates of CVD, but little is known about the relationships between cardiovascular risk factors, spatial disadvantage, and cardiovascular health outcomes in this region. Thus, this study aimed to examine the independent association between neighborhood factors and subclinical atherosclerosis in an asymptomatic population from central Appalachia. Methods: Community-dwelling asymptomatic individuals (n=210) were screened for Coronary Artery Calcium (CAC), a subclinical marker for coronary atherosclerosis, from January 2010 to January 2014. Based on the standard Agatston Scale, participants were grouped into 4 CAC scores: zero (CAC = 0), mild (CAC = 1-99), moderate (CAC = 100-399) and severe (CAC ≥ 400) to determine the severity of coronary artery disease (CAD). Demographic information (e.g., age, gender, race, and marital status), cardiovascular risk factors (e.g., hypertension, hypercholesterolemia, obesity, smoking, and family history of CAD), and neighborhood level characteristics (racial and socioeconomic characteristics of the population) were used in ordinal logistic regression analyses performed in Stata 14.1. Results: Of the 210 participants, over three-fourths (79%) had a CAC score greater than 1. While 67% of the participants were hypertensive, 80% had hypercholesterolemia, 75% were overweight or obese, 52% had a history of smoking, and 55% had a family history of CAD. There were significant differences in the socioeconomic environment of these residents. Specifically, zip-code median household income was higher for individuals with zero CAC score. Additionally, the zip-code household poverty percentage was higher for those with CAC scores ≥ 1. Although all the neighborhood factors increased the odds of having higher CAC score, none of them were statistically significant. Conclusion: The positive, albeit statistically non-significant, association of adverse neighborhood factors with higher CAC scores suggests the need for larger studies for further understanding of this association. Finally, achieving the Healthy People 2020 goal of reducing or eliminating disparities requires risk factor screening and control in high prevalent areas such as central Appalachia, and understanding the neighborhood level dynamics for CVD.

The nuclear hormone receptor peroxisome proliferator-activated receptor Γ (PPARΓ) is a ligand-dependent transcription factor of increasing importance in cardiovascular physiology. Treatment of type II diabetes patients with thiazolidinediones (TZD), synthetic ligands of PPARΓ, improves insulin sensitivity and also lowers blood pressure despite increased water and salt retention by the kidneys. In 1999, Stephen O'Rahilly's group reported that patients carrying mutations in PPARΓ exhibit severe type II diabetes and early-onset hypertension. The missense mutations in PPARΓ (e.g. V290M, P467L) affect the ligand-binding domain and render the transcription factor dominant negative (DN). These findings suggested that the PPARΓ activation plays a vital role in cardiovascular regulation but they did not differentiate whether the cardiovascular protective effects of TZDs result from systemic metabolic changes or direct actions of PPARΓ in the vasculature. To answer this, our group generated transgenic mice that express DN PPARΓ specifically in vascular endothelial or vascular smooth muscle cell types. Herein we are reporting the molecular and physiological mechanism linking mutation of PPARΓ to impaired vascular function leading to hypertension. Smooth muscle-specific expression of DN PPARΓ in transgenic mice causes increased arterial pressure and enhanced agonist-mediated contraction and blunting of nitric oxide-mediated relaxation in aorta via a RhoA/Rho-kinase-dependent mechanism. Our results demonstrate that interference with PPARΓ in smooth muscle impairs Cullin-3 RING E3 ubiquitin ligase-mediated regulation of RhoA/Rho-kinase signaling and identify Cullin-3 as a novel regulator of vascular function. Hypertension, insulin resistance and atherosclerosis are major targets for therapeutic intervention against morbidity and mortality caused by coronary artery disease. In addition to the beneficial blood-pressure lowering and insulin-sensitizing effects of treatment with TZD PPARΓ agonists, they also have potent inhibitory effects on atherosclerosis progression. Given the concerns over TZD use including weight gain and edema, it is essential to understand the fundamental mechanisms by which vascular PPARΓ affects atherosclerosis lesion development. We tested this by crossing our transgenic mice onto the apolipoprotein E-deficient (ApoE-/-) mouse model of hypercholesterolemia. Either endothelial- or smooth muscle-specific expression of DN PPARΓ on the ApoE-/- background led to enhanced atherosclerotic lesion formation in aorta without altering levels of plasma cholesterol and triglycerides. Furthermore, endothelial- or smooth muscle-specific DN PPARΓ induced distinct alterations in the signature of genes related to atherogenesis when comparing aortic tissue from either model with its respective non-transgenic control. Our results obtained using PPARΓ-interfering mutations provide novel mechanistic insight into pathways critical to the pathogenesis of cardiovascular diseases.

The multigene family of Ca2+-binding S100 proteins comprises 22 members that have various important intra- and extracellular roles. The three inflammation-associated members of this family???S100A8, S100A9 and S100A12 (collectively termed &quotcalgranulins&quot)???are constitutive neutrophil and monocyte proteins also expressed by macrophages within acute and chronic inflammatory lesions, but not in tissue macrophages. They are expressed in human/murine wounds and by appropriately activated macrophages, microvascular endothelial cells and keratinocytes in vitro. The &quot calgranulins&quot are implicated in leukocyte activation/deactivation, fatty acid transport, leukocyte/fibroblast chemotaxis, transmigration and adhesion, embryogenesis, wound healing, protection against oxidants and antibacterial defence. Chapter 3 of this thesis explores growth-factor- and cytokine-mediated regulation and expression of S100A8 and S100A9 in fibroblasts, and demonstrates spatio-temporal expression of S100A8 in rat dermal wounds. Fibroblasts are stromal resident cells with important regulatory immune-inflammatory functions in wound healing, tissue remodelling and fibrosis. Fibroblast migration, proliferation, differentiation and their synthetic repertoire are modulated by various factors including extracellular matrix components, growth factors, prostaglandins, reactive oxygen species and cytokines. Fibroblast growth factor-2 (FGF-2), interleukin-1?(IL-1? and platelet-derived growth factor (PDGF) are potent fibroblast mitogens; PDGF and transforming growth factor-? (TGF-? are fibroblast chemoattractants. FGF-2 and IL-1?promote fibroblast proliferation, whereas TGF-?promotes myofibroblast differentiation and collagen production. Lipopolysaccharide (LPS), interferon ?(IFN?, tumour-necrosis factor ? (TNF?, TGF-?and PDGF did not induce the S100A8 gene in fibroblasts whereas FGF-2 (25 ng/ml) maximally induced mRNA 12 hr. after stimulation and this declined over 36 hr. The FGF-2 response was strongly enhanced and prolonged by optimal levels of heparin (1-10 IU/ml), maximally at 18 hr. post-stimulation. FGF-2/heparin-induced responses depended on cell-cell contact in vitro. IL-1?(10 U/ml) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 and primary fibroblasts. Dexamethasone (10???6 M) enhanced LPS- and FGF-2/-IL-1?induced responses. S100A9 mRNA was not induced by any of these mediators. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblast-like cells by real-time reverse-transcriptase polymerase chain-reaction. FGF-2-heparin- and IL-1?induced mRNA expression depended on de-novo protein synthesis and was partially mediated by the mitogenactivated protein kinase pathway of activation. Preliminary promoter deletion analyses indicated that FGF-2-responsive elements in the gene promoter were distinct from those responsive to IL-1? TGF-?(2 ng/ml) significantly suppressed gene induction mediated by FGF-2 ?heparin/LPS/dexamethasone, but not by IL-1? TGF-?may compromise mRNA stability. Protein levels in FGF-2-heparin-IL-1?stimulated fibroblasts correlated well with mRNA levels and expression was mainly cytoplasmic. Immunohistochemistry indicated S100A8 associated with keratinocytes, neutrophils, macrophage-like cells and some hair follicles in wounded rat skin. Rat wounds also contained numerous S100A8- positive fibroblast-like cells 2 and 4 days post-injury; numbers declined by 7 days. Upregulation of S100A8 by FGF-2/IL-1? down-regulation by TGF-? and time-dependent expression of S100A8 in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair. Intracellular fibroblast-derived S100A8 may also regulate intracellular redox equilibrium and antioxidant defence. Atherosclerosis is a progressive chronic disease with complex aetiology and pathogenesis. S100A1 and S100B are associated with dendritic cells and lymphocytes in experimental rodent and human atherosclerotic lesions. Monocytes and macrophages in plaques of ApoE???/??? mice express S100A9 but not S100A8. Myeloperoxidase and HOClmediated oxidative mechanisms are fundamental in the pathogenesis of atherosclerosis and S100A8 is exquisitely sensitive to HOCl oxidation which generates sulphinamide bonds, novel non-reducible cysteine-lysine covalent bonds. Chapter 4 of this thesis presents novel evidence that, in contrast to the murine ApoE???/??? model, the three human &quot calgranulins&quot were expressed in human atherosclerotic plaques, but not in normal arteries. High levels of S100A8, S100A9 and S100A12 were evident in macrophages and foam cells. Some neovessels were anti-S100A8-/anti-S100A9-immunoreactive; S100A9 staining was also evident on the extracellular matrix. Patterns of expression of S100A8, S100A9 and S100A12 were overlapping in serial sections, except that only smooth muscle cells were S100A12-positive. S100A8 and S100A9 mRNA were also expressed by macrophages, foam cells and endothelial cells, indicating gene up-regulation rather than passive protein uptake. Western blotting of plaque extracts revealed monomeric S100A8, S100A9 and S100A12 and larger complexes. Some were resistant to reduction, suggesting non-disulfide covalent cross-linking, possibly via sulphinamide bonds. Stable S100A8-S100A9 complexes were also detected after immunoaffinity purification. In an in-vitro system, molar ratios of HOCl of &gt1 generated stable complexes of S100A8 and S100A9 whereas ~800 and ~100-fold excess HOCl oxidises apolipoprotein B-100 and BSA, respectively. S100A8 and S100A9 protected low-density lipoprotein (LDL) against HOCl oxidation in a thiol-independent manner. Because HOCl-oxidised S100s did not contain epitopes recognised by an antibody used to detect HOCl-oxidised proteins in plaque, levels of oxidised proteins in plaque are likely to be significantly greater than described. S100A8 and S100A9 may protect LDL by functioning as HOCl-scavengers. However, chronic oxidative cross-linking of S100A8 and S100A9 with other proteins and extracellular matrix components may contribute to plaque pathogenesis. These studies support key roles for the &quot calgranulins&quot in chronic inflammation, wound healing and atherogenesis possibly by regulating cellular differentiation, activation and modulation of redox-dependent mechanisms.

The multigene family of Ca2+-binding S100 proteins comprises 22 members that have various important intra- and extracellular roles. The three inflammation-associated members of this family???S100A8, S100A9 and S100A12 (collectively termed &quotcalgranulins&quot)???are constitutive neutrophil and monocyte proteins also expressed by macrophages within acute and chronic inflammatory lesions, but not in tissue macrophages. They are expressed in human/murine wounds and by appropriately activated macrophages, microvascular endothelial cells and keratinocytes in vitro. The &quot calgranulins&quot are implicated in leukocyte activation/deactivation, fatty acid transport, leukocyte/fibroblast chemotaxis, transmigration and adhesion, embryogenesis, wound healing, protection against oxidants and antibacterial defence. Chapter 3 of this thesis explores growth-factor- and cytokine-mediated regulation and expression of S100A8 and S100A9 in fibroblasts, and demonstrates spatio-temporal expression of S100A8 in rat dermal wounds. Fibroblasts are stromal resident cells with important regulatory immune-inflammatory functions in wound healing, tissue remodelling and fibrosis. Fibroblast migration, proliferation, differentiation and their synthetic repertoire are modulated by various factors including extracellular matrix components, growth factors, prostaglandins, reactive oxygen species and cytokines. Fibroblast growth factor-2 (FGF-2), interleukin-1?(IL-1? and platelet-derived growth factor (PDGF) are potent fibroblast mitogens; PDGF and transforming growth factor-? (TGF-? are fibroblast chemoattractants. FGF-2 and IL-1?promote fibroblast proliferation, whereas TGF-?promotes myofibroblast differentiation and collagen production. Lipopolysaccharide (LPS), interferon ?(IFN?, tumour-necrosis factor ? (TNF?, TGF-?and PDGF did not induce the S100A8 gene in fibroblasts whereas FGF-2 (25 ng/ml) maximally induced mRNA 12 hr. after stimulation and this declined over 36 hr. The FGF-2 response was strongly enhanced and prolonged by optimal levels of heparin (1-10 IU/ml), maximally at 18 hr. post-stimulation. FGF-2/heparin-induced responses depended on cell-cell contact in vitro. IL-1?(10 U/ml) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 and primary fibroblasts. Dexamethasone (10???6 M) enhanced LPS- and FGF-2/-IL-1?induced responses. S100A9 mRNA was not induced by any of these mediators. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblast-like cells by real-time reverse-transcriptase polymerase chain-reaction. FGF-2-heparin- and IL-1?induced mRNA expression depended on de-novo protein synthesis and was partially mediated by the mitogenactivated protein kinase pathway of activation. Preliminary promoter deletion analyses indicated that FGF-2-responsive elements in the gene promoter were distinct from those responsive to IL-1? TGF-?(2 ng/ml) significantly suppressed gene induction mediated by FGF-2 ?heparin/LPS/dexamethasone, but not by IL-1? TGF-?may compromise mRNA stability. Protein levels in FGF-2-heparin-IL-1?stimulated fibroblasts correlated well with mRNA levels and expression was mainly cytoplasmic. Immunohistochemistry indicated S100A8 associated with keratinocytes, neutrophils, macrophage-like cells and some hair follicles in wounded rat skin. Rat wounds also contained numerous S100A8- positive fibroblast-like cells 2 and 4 days post-injury; numbers declined by 7 days. Upregulation of S100A8 by FGF-2/IL-1? down-regulation by TGF-? and time-dependent expression of S100A8 in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair. Intracellular fibroblast-derived S100A8 may also regulate intracellular redox equilibrium and antioxidant defence. Atherosclerosis is a progressive chronic disease with complex aetiology and pathogenesis. S100A1 and S100B are associated with dendritic cells and lymphocytes in experimental rodent and human atherosclerotic lesions. Monocytes and macrophages in plaques of ApoE???/??? mice express S100A9 but not S100A8. Myeloperoxidase and HOClmediated oxidative mechanisms are fundamental in the pathogenesis of atherosclerosis and S100A8 is exquisitely sensitive to HOCl oxidation which generates sulphinamide bonds, novel non-reducible cysteine-lysine covalent bonds. Chapter 4 of this thesis presents novel evidence that, in contrast to the murine ApoE???/??? model, the three human &quot calgranulins&quot were expressed in human atherosclerotic plaques, but not in normal arteries. High levels of S100A8, S100A9 and S100A12 were evident in macrophages and foam cells. Some neovessels were anti-S100A8-/anti-S100A9-immunoreactive; S100A9 staining was also evident on the extracellular matrix. Patterns of expression of S100A8, S100A9 and S100A12 were overlapping in serial sections, except that only smooth muscle cells were S100A12-positive. S100A8 and S100A9 mRNA were also expressed by macrophages, foam cells and endothelial cells, indicating gene up-regulation rather than passive protein uptake. Western blotting of plaque extracts revealed monomeric S100A8, S100A9 and S100A12 and larger complexes. Some were resistant to reduction, suggesting non-disulfide covalent cross-linking, possibly via sulphinamide bonds. Stable S100A8-S100A9 complexes were also detected after immunoaffinity purification. In an in-vitro system, molar ratios of HOCl of &gt1 generated stable complexes of S100A8 and S100A9 whereas ~800 and ~100-fold excess HOCl oxidises apolipoprotein B-100 and BSA, respectively. S100A8 and S100A9 protected low-density lipoprotein (LDL) against HOCl oxidation in a thiol-independent manner. Because HOCl-oxidised S100s did not contain epitopes recognised by an antibody used to detect HOCl-oxidised proteins in plaque, levels of oxidised proteins in plaque are likely to be significantly greater than described. S100A8 and S100A9 may protect LDL by functioning as HOCl-scavengers. However, chronic oxidative cross-linking of S100A8 and S100A9 with other proteins and extracellular matrix components may contribute to plaque pathogenesis. These studies support key roles for the &quot calgranulins&quot in chronic inflammation, wound healing and atherogenesis possibly by regulating cellular differentiation, activation and modulation of redox-dependent mechanisms.

The multigene family of Ca2+-binding S100 proteins comprises 22 members that have various important intra- and extracellular roles. The three inflammation-associated members of this family???S100A8, S100A9 and S100A12 (collectively termed &quotcalgranulins&quot)???are constitutive neutrophil and monocyte proteins also expressed by macrophages within acute and chronic inflammatory lesions, but not in tissue macrophages. They are expressed in human/murine wounds and by appropriately activated macrophages, microvascular endothelial cells and keratinocytes in vitro. The &quot calgranulins&quot are implicated in leukocyte activation/deactivation, fatty acid transport, leukocyte/fibroblast chemotaxis, transmigration and adhesion, embryogenesis, wound healing, protection against oxidants and antibacterial defence. Chapter 3 of this thesis explores growth-factor- and cytokine-mediated regulation and expression of S100A8 and S100A9 in fibroblasts, and demonstrates spatio-temporal expression of S100A8 in rat dermal wounds. Fibroblasts are stromal resident cells with important regulatory immune-inflammatory functions in wound healing, tissue remodelling and fibrosis. Fibroblast migration, proliferation, differentiation and their synthetic repertoire are modulated by various factors including extracellular matrix components, growth factors, prostaglandins, reactive oxygen species and cytokines. Fibroblast growth factor-2 (FGF-2), interleukin-1?(IL-1? and platelet-derived growth factor (PDGF) are potent fibroblast mitogens; PDGF and transforming growth factor-? (TGF-? are fibroblast chemoattractants. FGF-2 and IL-1?promote fibroblast proliferation, whereas TGF-?promotes myofibroblast differentiation and collagen production. Lipopolysaccharide (LPS), interferon ?(IFN?, tumour-necrosis factor ? (TNF?, TGF-?and PDGF did not induce the S100A8 gene in fibroblasts whereas FGF-2 (25 ng/ml) maximally induced mRNA 12 hr. after stimulation and this declined over 36 hr. The FGF-2 response was strongly enhanced and prolonged by optimal levels of heparin (1-10 IU/ml), maximally at 18 hr. post-stimulation. FGF-2/heparin-induced responses depended on cell-cell contact in vitro. IL-1?(10 U/ml) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 and primary fibroblasts. Dexamethasone (10???6 M) enhanced LPS- and FGF-2/-IL-1?induced responses. S100A9 mRNA was not induced by any of these mediators. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblast-like cells by real-time reverse-transcriptase polymerase chain-reaction. FGF-2-heparin- and IL-1?induced mRNA expression depended on de-novo protein synthesis and was partially mediated by the mitogenactivated protein kinase pathway of activation. Preliminary promoter deletion analyses indicated that FGF-2-responsive elements in the gene promoter were distinct from those responsive to IL-1? TGF-?(2 ng/ml) significantly suppressed gene induction mediated by FGF-2 ?heparin/LPS/dexamethasone, but not by IL-1? TGF-?may compromise mRNA stability. Protein levels in FGF-2-heparin-IL-1?stimulated fibroblasts correlated well with mRNA levels and expression was mainly cytoplasmic. Immunohistochemistry indicated S100A8 associated with keratinocytes, neutrophils, macrophage-like cells and some hair follicles in wounded rat skin. Rat wounds also contained numerous S100A8- positive fibroblast-like cells 2 and 4 days post-injury; numbers declined by 7 days. Upregulation of S100A8 by FGF-2/IL-1? down-regulation by TGF-? and time-dependent expression of S100A8 in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair. Intracellular fibroblast-derived S100A8 may also regulate intracellular redox equilibrium and antioxidant defence. Atherosclerosis is a progressive chronic disease with complex aetiology and pathogenesis. S100A1 and S100B are associated with dendritic cells and lymphocytes in experimental rodent and human atherosclerotic lesions. Monocytes and macrophages in plaques of ApoE???/??? mice express S100A9 but not S100A8. Myeloperoxidase and HOClmediated oxidative mechanisms are fundamental in the pathogenesis of atherosclerosis and S100A8 is exquisitely sensitive to HOCl oxidation which generates sulphinamide bonds, novel non-reducible cysteine-lysine covalent bonds. Chapter 4 of this thesis presents novel evidence that, in contrast to the murine ApoE???/??? model, the three human &quot calgranulins&quot were expressed in human atherosclerotic plaques, but not in normal arteries. High levels of S100A8, S100A9 and S100A12 were evident in macrophages and foam cells. Some neovessels were anti-S100A8-/anti-S100A9-immunoreactive; S100A9 staining was also evident on the extracellular matrix. Patterns of expression of S100A8, S100A9 and S100A12 were overlapping in serial sections, except that only smooth muscle cells were S100A12-positive. S100A8 and S100A9 mRNA were also expressed by macrophages, foam cells and endothelial cells, indicating gene up-regulation rather than passive protein uptake. Western blotting of plaque extracts revealed monomeric S100A8, S100A9 and S100A12 and larger complexes. Some were resistant to reduction, suggesting non-disulfide covalent cross-linking, possibly via sulphinamide bonds. Stable S100A8-S100A9 complexes were also detected after immunoaffinity purification. In an in-vitro system, molar ratios of HOCl of &gt1 generated stable complexes of S100A8 and S100A9 whereas ~800 and ~100-fold excess HOCl oxidises apolipoprotein B-100 and BSA, respectively. S100A8 and S100A9 protected low-density lipoprotein (LDL) against HOCl oxidation in a thiol-independent manner. Because HOCl-oxidised S100s did not contain epitopes recognised by an antibody used to detect HOCl-oxidised proteins in plaque, levels of oxidised proteins in plaque are likely to be significantly greater than described. S100A8 and S100A9 may protect LDL by functioning as HOCl-scavengers. However, chronic oxidative cross-linking of S100A8 and S100A9 with other proteins and extracellular matrix components may contribute to plaque pathogenesis. These studies support key roles for the &quot calgranulins&quot in chronic inflammation, wound healing and atherogenesis possibly by regulating cellular differentiation, activation and modulation of redox-dependent mechanisms.