Peterson, Timothy E.

Rationale: The link between obesity, hyperleptinemia, and development of cardiovascular disease is not completely understood. Increases in leptin have been shown to impair leptin signaling via caveolin-1–dependent mechanisms. However, the role of hyperleptinemia versus impaired leptin signaling in adipose tissue is not known. Objective: To determine the presence and significance of leptin-dependent increases in adipose tissue caveolin-1 expression in humans. Methods and Results: We designed a longitudinal study to investigate the effects of increases in leptin on adipose tissue caveolin-1 expression during weight gain in humans. Ten volunteers underwent 8 weeks of overfeeding, during which they gained an average weight of 4.1±1.4 kg, with leptin increases from 7±3.8 to 12±5.7 ng/mL. Weight gain also resulted in changes in adipose tissue caveolin-1 expression, which correlated with increases in leptin (rho=0.79, P =0.01). In cultured human white preadipocytes, leptin increased caveolin-1 expression, which in turn impaired leptin cellular signaling. Functionally, leptin decreased lipid accumulation in differentiating human white preadipocytes, which was prevented by caveolin-1 overexpression. Further, leptin decreased perilipin and fatty acid synthase expression, which play an important role in lipid storage and biogenesis. Conclusions: In healthy humans, increases in leptin, as seen with modest weight gain, may increase caveolin-1 expression in adipose tissue. Increased caveolin-1 expression in turn impairs leptin signaling and attenuates leptin-dependent lowering of intracellular lipid accumulation. Our study suggests a leptin-dependent feedback mechanism that may be essential to facilitate adipocyte lipid storage during weight gain.

Abstract—Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 μg/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 μmol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.

Abstract —Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours ( P <0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively ( P <0.05), and these growth factors accounted for ≈50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.

Tetrahydrobiopterin is an essential cofactor required for the synthesis of NO. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrated that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial NO synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a 2-fold increase in GTPCH I activity was detected in the aortas of caveolin-1–deficient mice, suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by the disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells, and tetrahydrobiopterin production occurs in close proximity to endothelial NO synthase. In addition, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1.

Abstract —Tissue factor (TF) is a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of arterial thrombogenicity. TF pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. The aim of this study was to define the complex interplay between TF and TFPI and the regulation of vascular thrombogenicity in a model of vascular remodeling. To determine the levels and pattern of vascular expression of TF and TFPI associated with vascular remodeling, a murine model of flow cessation was studied. TF activity of the arteries increased after ligation ( P <0.05). Quantitative analysis of homogenates of remodeled carotid arteries revealed increased TF expression but unchanged TFPI expression compared with normal carotid arteries, resulting in enhanced TF activity. To determine the potential therapeutic role of TFPI in this thrombogenic state, mice were treated with intravascular adenoviral delivery of either murine TFPI (Ad-mTFPImyc) or a control adenovirus (Ad-ΔE1). Overexpression of TFPI decreased vascular TF activity compared with viral control ( P <0.01). Overexpression of TFPI inhibited neointimal formation ( P =0.038), resulting in enhanced luminal area ( P =0.001) 4 weeks after flow cessation. In this murine model of vascular remodeling, an imbalance between TF and TFPI expression is generated, resulting in increased TF activity. Overexpression of TFPI in this model inhibits vascular TF activity and results in attenuation of vascular remodeling associated with flow interruption.

The proteasome is responsible for the degradation of oxidized proteins, and proteasome inhibition has been shown to generate oxidative stress in vitro. Atherosclerosis is thought to be initiated as a consequence of increased endogenous oxidative stress. The current study was designed to assess whether chronic proteasome inhibition is associated with early coronary atherosclerosis. Female pigs, 3 months of age, were randomized to a normal (N) or high-cholesterol (HC) diet (2% cholesterol, 15% lard) without or with twice weekly subcutaneous injections of the proteasome inhibitor (PSI) MLN-273 (0.08 mg/kg, N+PSI and HC+PSI) for a period of 12 weeks (n=5 per group). Coronary vasorelaxation to bradykinin (10 −10.5 to 10− 6.5 mol/L) and sodium nitroprusside (10 −9 to 10 −5 mol/L) was assessed by in vitro organ chamber experiments, intima–media ratio by morphometric analysis of Elastica–van Gieson–stained slides, and intima superoxide production by dihydroethidium fluorescence. Vasorelaxation to 10 −6.5 mol/L bradykinin was reduced in HC compared with N (69±7 versus 90±2%, P <0.05) and further reduced in N+PSI and HC+PSI (57±6 and 48±13%, P <0.05 versus N and HC for each). Compared with N (0.03±0.01), intima–media ratio was higher in N+PSI (0.09±0.04, P <0.01) and HC+PSI (0.15±0.06, P <0.05). Compared with N (0.6±0.9% of intima area), dihydroethidium fluorescence was higher in HC, N+PSI, and HC+PSI (8.9±1.6, 6.0±3.5, and 7.2±3.9% of intima area, P <0.05 for all). Thus, chronic proteasome inhibition is associated with increased coronary artery oxidative stress and early atherosclerosis. These findings support the significance of the proteasome and related protein quality control for vascular biology and pathology.

Objective— Vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic cytokines. Ability of VEGF to stimulate formation of superoxide anion in vivo has not been studied. We hypothesized that in vivo expression of recombinant VEGF in the rabbit carotid artery increases production of superoxide anion. Methods and Results— Plaque-forming units (10 9 ) of adenovirus-encoding human VEGF 165 (AdVEGF) or β-galactosidase (AdLacZ) were delivered into the lumen of rabbit carotid arteries. Three days after gene delivery, expression of recombinant proteins was detected in endothelium and smooth muscle cells. Endothelium-dependent relaxations to acetylcholine were impaired in AdVEGF-transduced arteries ( P <0.01; n=5). Treatment with superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10 −5 mol/L), improved relaxations to acetylcholine ( P <0.01; n=5). Western blot analysis demonstrated increased expression of p47 phox in AdVEGF-transduced arteries ( P <0.05; n=8). Lucigenin chemiluminescence showed significantly higher production of superoxide anion in AdVEGF-transduced arteries ( P <0.05; n=5 to 10). Conclusions— Our results suggest that in vivo expression of recombinant VEGF in the vascular endothelium increases local production of superoxide anion. Superoxide anion appears to be an important mediator of vascular effects of VEGF in vivo.

Objective— Endothelial progenitor cells (EPCs) display a unique aptitude to promote angiogenesis and restore endothelial function of injured vessels. How progenitor cells can execute a regenerative program in the unfavorable environment of injury/inflammation-induced oxidative stress is poorly understood. We hypothesized that EPCs are resistant to oxidative stress and that this resistance is due to high expression and activity of antioxidant enzymes. Methods and Results— EPCs outgrown from human blood of healthy subjects demonstrated a marked resistance to cytotoxic effect of LY83583 (an generator), tumor necrosis factor-α, and serum depletion. LY83583 inhibited in vitro tube formation by human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (CAECs), but not by EPCs. Compared with HUVECs and CAECs, EPCs exhibited ≈3- to 4-fold higher expression and activity of manganese superoxide dismutase (MnSOD), but not copper zinc superoxide dismutase (CuZnSOD) or catalase. The antioxidant profile in EPCs was associated with preservation of the mitochondrial network when exposed to LY83583. Moreover, cytotoxic effects of LY83583 on CAECs and HUVECs were reversed by adenoviral overexpression of MnSOD. Conclusions— Human EPCs are resistant to oxidative stress. High intrinsic expression of MnSOD is a critical mechanism protecting EPCs against oxidative stress.