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Objectives: We investigated the association of 47 candidate proteomic markers in pathways of inflammation, thrombosis, lipoprotein metabolism, vascular remodeling, and vasoregulation, with the ankle-brachial index (ABI) and peripheral arterial disease (PAD) in a bi-ethnic cohort of African Americans (AA) and non-Hispanic Whites (NHW). Methods: Multiple assay platforms were used to measure 47 candidate proteomic markers in 1324 AA and 1237 NHW participants belonging to hypertensive sibships. ABI was measured using a standard protocol, with values >1.4 excluded and PAD defined as ABI <0.9. Multivariable regression analyses (with generalized estimating equations) were employed to identify markers independently associated with ABI and PAD after adjusting for age, BMI, smoking, hypertension, diabetes, history of heart attack and stroke, medication use, total and HDL cholesterol, and estimated GFR as well as physical activity, alcohol consumption and education level. Results: Higher circulating levels of CRP, myeloperoxidase, NT-proBNP, osteoprotegerin, D-Dimer and fibrinogen were each associated with lower ABI in both AA and NHW. Additional markers significantly associated with lower ABI were; in AA - serum amyloid A, ICAM, IL-6, TNFR-I, TNFR-II, MCP-1, Lp(a), pro-atrial natriuretic peptide, pro-arginine vasopressin, pro-adrenomedullin, pro-endothelin, osteonectin, Factor VIII; and in NHW- Hsp27, ox-LDL and resistin. Markers associated with presence of PAD were: in AA - CRP, NT-proBNP, Lp(a), pro-arginine vasopressin and osteoprotegerin; in NHW -CRP, NT-proBNP, Hsp27, myeloperoxidase, oxidized-LDL, Factor VIII, and D-Dimer. When the significant markers were included in a final model, the R 2 for ABI increased from 15.9 to 19.4 for AA and from 18.1 to 22.1 for NHW; the c-statistic for PAD increased from 0.791 to 0.833 inAA and from 0.841 to 0.866 in NHW. Conclusions: Novel protein markers significantly add to prediction of ABI and PAD in both AA and NHW and may have clinical utility in diagnosis and prognosis of PAD as well as in identifying new therapies for atherosclerotic vascular disease.

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the tissue factor/factor VIIa complex. Systemic genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. To define the tissue specific roles of TFPI we utilized a Cre-lox strategy and generated mice with flanking loxP sites surrounding exon 4 (encodes for the K1 domain). Homozygous floxed mice (TFPI Flox ) were crossed with the Tie2 Cre + or LysM Cre + mice and subsequently inbred to create homozygously floxed mice expressing Cre (TFPI Tie2 or TFPI LysM ). Methods and Results: The presence and efficiency of the deleted mRNA product was determined by RT-PCR. TFPI Tie2 and TFPI LysM mice were phenotypically similar to TFPI Flox littermates including weight, blood counts, and prothrombin and activated partial thromboplastin times. TFPI Tie2 mice have complete deletion in endothelial cells, blood cells and bone marrow cells. TFPI LysM mice show partial deletion in blood cells and complete deletion in macrophages. TFPI Tie2 mice had a 60% reduction (P < 0.0001) in circulating TFPI activity compared to TFPI Flox littermate controls. TFPI LysM mice did not have a significant reduction in circulating TFPI activity levels compared to their TFPI Flox counterparts. Neither TFPI Tie2 nor TFPI LysM mice had significant changes in hemostasis compared to TFPI Flox mice as determined by a tail bleeding assay. In an ADP-induced model of thromboembolism there were no significant changes in mortality between the TFPI Tie2 or TFPI LysM and their TFPI Flox littermates. In a ferric chloride model of arterial thrombosis, TFPI Tie2 mice had a significant reduction (P < 0.05) in time to occlusion while TFPI LysM mice did not differ compared to TFPI Flox mice. Conclusions: Deletion of TFPI from cells expressing Tie2 or LysM is consistent with normal development and hemostasis. TFPI Tie2 mice have decreased circulating levels of TFPI and an enhanced thrombotic response to ferric chloride. These data support an important regulatory role for EC TFPI in arterial thrombosis.

Background: B-type natriuretic peptide (BNP1–32) is a 32- amino acid cardiac peptide secreted in response to cardiac stress and plays an important compensatory role in cardiorenal homeostasis. Studies have recently reported that a mRNA encoding an alternatively spliced variant of BNP (ASBNP) exists in the human heart and like BNP1–32 is increased in the failing heart. While ASBNP and BNP1–32 have identical ring structures and amino termini, ASBNP and BNP1–32 differ in their carboxy terminus (34 vs. 6 amino acids, respectively). Studies have also reported that ASBNP activates cGMP, the second messenger of BNP1–32, in isolated glomeruli but not in endothelial cells. Also, unlike BNP1–32 ASBNP does not vasorelax isolated arteries or veins. The cardiorenal actions in vivo of ASBNP are unknown. We hypothesized that ASBNP in vivo would have selective renal actions yet as suggested by studies in isolated vessels would be devoid of blood pressure effects. Methods: We therefore determined for the first time the cardiorenal actions of three subsequent intravenous doses of ASBNP (10, 50, and 150 pmol/kg/min) in 5 healthy canines in an acute study under anesthesia in 30-minute clearances at baseline, during, and following infusion. Values provided are mean±SEM for baseline vs. 150 pmol/kg/min. *p<0.05. Results: ASBNP dose-dependently increased plasma levels of cGMP (8.6±1.0 vs. 11.5±1.3* pmol/mL), and the same was true for urinary cGMP excretion (878±191 vs. 2028±551* pmol/min). Mean arterial pressure (p=0.83) and heart rate (p=0.43) remained unchanged. Urine flow increased (0.38±0.05 vs. 1.32±0.20* mL/min) as did urinary sodium excretion (38±15 vs. 133±38* μ Eq/min). Renal blood flow (p=0.51) and glomerular filtration rate (p=0.99) remained unchanged. BNP-immunoreactivity as measured by the Biosite BNP assay increased (5±4 vs. 3807±573* pg/mL). Conclusion: ASBNP, an alternatively spliced form of BNP produced in the human heart, activates cGMP and has diuretic and natriuretic actions but is without blood pressure lowering actions. Thus, ASBNP represents a novel renal selective, non-hypotensive, natriuretic, and diuretic peptide.

Background: C-type natriuretic peptide (CNP) is of endothelial cell origin and represents the most potent anti-fibrotic peptide of the natriuretic peptide family and is important for extracellular matrix homeostasis including mediating bone growth in the young. Cardiorenal aging is associated with altered cardiac and renal structure and function that may contribute to the development of heart and kidney failure, particularly in the elderly. The impact of aging on circulating CNP and associated changes in cardiorenal structure and function are undefined. Hypothesis: We hypothesized that a decrease in endogenous plasma CNP occurs with aging and is associated with an increase in cardiorenal fibrosis and altered left ventricular (LV) and renal structure and function. Methods: Studies were performed in 2, 11 and 20 month old male Fischer rats (n = 8 –10/group). Echo was used to assess LV structure and function. Blood pressure (BP), urinary protein excretion, plasma B-type natriuretic peptide (BNP) and CNP were measured. Kidneys and LV were harvested for gross and histology analysis. Mean±SE, *p<0.05. Results: Aging from 2 to 20 months was associated with progressive reductions in plasma CNP (30±3 to 21±1* to 9±1*pg/ml). Significant and progressive cardiac and renal fibrosis were observed with aging (LV: 9±0.3 to 15±0.7* to 21±1*% and kidney: 1±0.3 to 8±2* to 17±1* %). In contrast to CNP, plasma BNP was modestly increased from the 2 to 20 month old group (21±2 to 26±1*pg/ml). The reduction in CNP and increase in BNP, the latter a biomarker for LV dysfunction, were associated with decreased EF (88±1 to 80±1*%) and increased LVEDd (6.61±0.09 to 7.48±0.09*mm). BP and urinary protein excretion were mildly elevated with aging (91±1 to 101±3* mmHg and 8±0.3 to 20±3* mg/day, respectively). Conclusion: We report for the first time that aging is associated with a progressive decline in circulating CNP and progressive cardiorenal fibrosis, systolic dysfunction and proteinuria. Further studies are warranted to explore the hypothesis that a mechanism of cardiorenal aging with altered cardiorenal structure and function may include a decrease in the bioavailability of CNP.

Background: We have found that the vascular large conductance Ca 2+ -activated K + channels (BK) are targeted to caveolae. The goal of this study is to determine the role of caveolae in BK regulation by angiotensin II (AII) in normal and diabetic rats. Results: In coronary arterial smooth muscle cells (CASMC) from control rats, AII (2 μ M) inhibited BK by 40.5% from 227±26 pA/pF to 135±14 pA/pF (HP −60 mV, TP 150 mV, n=7, p<0.05). However, AII had no effects on BK in CASMC transfected with caveolin-1 siRNA (vs 50% inhibition in cells transfected with control siRNA) or in cells from Cav1−/− mice, suggesting the AII effects are caveolae-dependent. Density gradient analysis of rat vessel homogenates showed that caveolin-1, BK, Gq, cSrc, and NOX-1 all reside in the caveolae-rich fraction, where AT 1 R was absent unless the animal was pre-treated with AII, suggesting receptor translocation upon agonist activation. However, CASMC from streptozotocin-induced diabetic rats showed significant reduction in BK (61.5±10.6 pA/pF) with no response to AII (61.2±15.7 pA/pF, n=8). Vascular BK, caveolin-1, AT 1 R, cSrc, and NOX-1 expression in caveolae were all upregulated by 1.6- to 3.1-fold in diabetes. Density gradient analysis of diabetic vessels showed the presence of AT 1 R in the caveolae-rich fraction without AII pretreatment, suggesting that the AT 1 R signaling pathway might be constitutively active in diabetes. This was accompanied by a 2-fold increase in BK tyrosine phosphorylation and BK tyrosine nitration. The AII effects on BK function in diabetic CASMC were abolished by pretreatment (1 hr) with PKC inhibitor or by LavA (cSrc inhibitor) and can be mimicked by incubation (2 hr) of control CASMC with peroxynitrite. Conclusion: AT 1 R-BK-enzyme complex in the caveolae microdomain is critical in vascular BK regulation. Enhanced AII-signaling facilitates BK post-translational modification, resulting in BK dysfunction in diabetes.

Background and Aim: Cardioembolic stroke from non-valvular atrial fibrillation (NVAF) begins with the development of left atrial appendage thrombus (LAAT). Although nearly 15% of patients with NVAF have LAAT by transesophageal echocardiography, the annual stroke rate is 5% on average. The overall aim of this project is to identify variables associated with embolic propensity of LAAT. Materials and Methods: LAAT specimens were obtained during cardiac surgery (MAZE procedure; n=26). Embolized thrombi (n=51) were obtained during surgical embolectomy from lower (n=36) and upper extremities (n=11) and mesenteric arteries (n=4). Platelet-rich regions within sectioned thrombi were sampled by punch biopsy from which total RNA was extracted and analyzed for expression of 5 platelet specific genes: A1 domain of factor XIII, (F13A1); integrin α 2b β 3 (ITGA2B); glycoprotein IX (GP9), platelet factor 4 (PF4), glycoprotein Ib (GP1BB). Gene expression was quantified using TaqMan MGB-probe based quantitative real-time polymerase chain reaction normalized to β 2 macroglobulin using cDNAs reverse transcribed from total RNA. Results: Factor XIII gene expression (Figure ) was significantly lower in embolic (mean fold change 0.08) compared to left atrial thrombi in situ (mean fold change 0.28; p=0.03). Expression of other genes did not show significant change by embolic status. Conclusions: Variable Factor XIII gene expression in thrombi generated during NVAF may in part explain the propensity to embolization. Whereas Factor XIII stabilizes thrombi by crosslinking fibrin without influencing atrial attachment, this data suggests that embolization occurs through fragmentation.

Background: Patients suffering post operative myocardial infarction (PMI) have higher mortality and morbidity rates than observed with non-PMI subsets. Little is known about the true incidence of PMI and how PMI differs from non-PMI with regard to clinical presentation, management and outcome. Methods: We examined the incidence and outcomes of patients with PMI in 1212 subjects (≥65 years) undergoing hip fracture surgery from 1988 – 2002 using a population-based retrospective cohort study from the Rochester Epidemiology Project in Olmstead County, Minnesota. PMI was defined on the basis of cardiac biomarker and/or electrocardiographic evidence of definitive MI within 7 days post operatively. Results: PMI occurred in 170 (14.0%) patients yet only 36 (21.2 %) patients had one or more clinical symptoms suggesting coronary ischemia. One or more classic symptoms of myocardial infarction were present in 26 (15.3%) including shortness of breath (SOB) in 18 (10.6%), chest/arm pain in 11 (6.5%), nausea/vomiting in 8 (4.7%) and diaphoresis in 1 (0.6%). Delirium was present in 15 (8.8%) patients. Median hospital length of stay was longer (10.0 vs. 8 days, p<0.01) and the in-hospital mortality rate was significantly higher (14.1% vs. 1.6%, p<0.001) among patients who had PMI within 7 days. Conclusion: The clinical presentation of PMI differs from non-PMI in the elderly undergoing hip fracture surgery. Most patients were asymptomatic or had atypical clinical presentations. We observed a significantly higher rate of hospital mortality among those experiencing a PMI. Further studies are necessary to better understand the presentation, optimal management and prevention of PMI. Table 1. Study Population Table 2. Clinical Presentation and Outcomes in Patients with and without Post Operative MI This research has received full or partial funding support from the American Heart Association, National Center.

Background Sustained Acting Atrial Natriuretic Peptide (SA-ANP) is a 40 AA peptide consisting of native ANP (1–28) with a 12 AA C-terminus extension which we synthesized based upon a familial ANP gene frameshift mutation. SA-ANP possesses markedly greater and more sustained mean arterial pressure (MAP) lowering, diuretic/natriuretic, angiotensin-aldosterone suppressing, and cGMP activating properties compared to ANP in normal dogs consistent with a super agonist of the ANP receptor (NPR-A) ( JACC in press ). Here we evaluated SA-ANP in a model of acute hypertension with the hypothesis that SA-ANP would have potent MAP lowering and renal enhancing properties. Methods Integrated physiological studies were performed in a canine model of ANG induced hypertension (20 ng/kg/min continuous infusion) before, during, and up to 3 hours following a 45 minute infusion of SA-ANP (30 pmol/kg/min) (n=6) or vehicle (n=6). Results MAP is shown in Figure 1 . SVR significantly decreased and despite a significant reduction in MAP both renal blood flow (RBF) and GFR were significantly increased following SA-ANP infusion. There were significant reductions in proximal and distal tubule sodium reabsorption resulting in a significant increase (>50 fold) in diuresis and natriuresis with SA-ANP. These renal actions were associated with significant increases in plasma and urine cGMP and a significant inhibition of aldosterone activation. Discussion We report for the first time in an acute model of hypertension that SA-ANP reduced MAP while enhancing RBF, GFR, and sodium/water excretion and thus may serve as a potential therapeutic agent in the treatment of hypertension in the setting of volume overload.

Increased oxidative stress has been suggested to play an important role in the biology of mesenchymal stem cells (MSCs). However, little is known of the effect of increased oxidative stress in-vivo, partly due to the lack of appropriate imaging technologies for monitoring of stem cell biology in the living subject. Recent developments in molecular imaging permit, for the first time, the longitudinal monitoring of stem cell biology in vivo. We hypothesize that changes in oxidative stress can be monitored using a reporter gene strategy (molecular imaging). Methods: We have developed a novel oxidative stress sensor, using a two-step transcriptional amplification strategy to enhance the expression of firefly luciferase (Fluc), driven by the pro-oxidant enzyme NAD(P)H promoter, and used to monitor oxidative stress in rMSCs. rMSCs was induced by hypoxia (1% O2 for 24 hours) and serum deprivation (SD) and confirmed using a dichlorofluorescein colorimetric assay (measures the production of hydrogen peroxide-H2O2-) and NAD(P)H protein expression. Results: Both serum deprivation and hypoxia led to a pro-oxidant state (Fig. A–B ). Most importantly, increased oxidative stress led to increased activity of the novel reporter gene sensor (Fig. C ) which was decreased in the presence of antioxidants (superoxide dismuatse mimetic Tempol, 5mM, SD+T, Fig D ). Results were reproduced in a different cell type (rat cardiomyoblast, data not shown). Conclusion: Changes in oxidative stress in MSCs can be monitored using molecular imaging. Reporter Gene-based Molecular Imaging, a strategy that is clinically applicable, provides the opportunity to monitor stem cell survival in the living subject.

GTP-Cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis, an essential co-factor required for enzymatic activity of endothelial nitric oxide synthase (eNOS). Recent studies have suggested that endothelium-targeted overexpression of GTPCH I has vasoprotective effects. Therefore, we hypothesized that in endothelium GTPCH I localization in caveolae protects eNOS against oxidative stress. Human umbilical vein endothelial cells (HUVECs) were incubated with recombinant adenovirus encoding the human GTPCH I gene (Ad-GTPCH I, 100 MOI) or control recombinant adenoviral vector (Ad-ΔE1) for 12 hours in serum-free media. Cells were then fed with growth medium for 48 hours prior to analysis. Using sucrose gradient ultracentrifugation we detected both protein expression and enzymatic activity of GTPCH I in caveolar microdomains of HUVECs. Treatment of Ad-ΔE1 transduced endothelial cells for 24 hours with LY83583 (1 μ M), a superoxide anion generator, significantly decreased enzymatic activity of GTPCH I in caveolae-enriched membranes (0.49±0.12 pmol neopterin/mg protein; P<0.05 vs. control: 0.92±0.07 pmol neopterin/mg). Furthermore, LY83583 translocated eNOS from caveolae to cytosol in Ad-ΔE1 treated endothelial cells (P<0.05; n=3). This effect was specific because there was no change in caveolin-1 expression and no decrease in cell viability. Treatment of HUVECs with Ad-GTPCH I significantly increased GTPCH I activity in caveolae-enriched membranes (1.37±0.22 pmol neopterin/mg; P<0.05; n=4). Interestingly, LY83583-induced dislocation of eNOS from caveolar membrane fraction was significantly prevented by overexpression of GTPCH I (P<0.05; n=3). Our results suggest that overexpression of GTPCH I increases its enzymatic activity in caveolae. This effect preserves strategically key localization of eNOS in cell membrane during oxidative stress. This research has received full or partial funding support from the American Heart Association, National Center.