Anthony G. Passerini

Triglyceride-Rich Lipoproteins Prime Aortic Endothelium for an Enhanced Inflammatory Response to Tumor Necrosis Factor-α

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-α. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor–associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-&kgr;B, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.

Spatial Regulation of Inflammation by Human Aortic Endothelial Cells in a Linear Gradient of Shear Stress

Objective: Atherosclerosis is a focal disease that develops at sites of low and oscillatory shear stress in arteries. This study aimed to understand how endothelial cells sense a gradient of fluid shear stress and transduce signals that regulate membrane expression of cell adhesion molecules and monocyte recruitment. Methods: Human aortic endothelial cells were stimulated with TNF‐α and simultaneously exposed to a linear gradient of shear stress that increased from 0 to 16 dyne/cm2. Cell adhesion molecule expression and activation of NFκ B were quantified by immunofluorescence microscopy with resolution at the level of a single endothelial cell. Monocyte recruitment was imaged using custom microfluidic flow chambers. Results: VCAM‐1 and E‐selectin upregulation was greatest between 2–4 dyne/cm2 (6 and 4‐fold, respectively) and above 8 dyne/cm2 expression was suppressed below that of untreated endothelial cells. In contrast, ICAM‐1 expression and NFκ B nuclear translocation increased with shear stress up to a maximum at 9 dyne/cm2. Monocyte recruitment was most efficient in regions where E‐selectin and VCAM‐1 expression was greatest. Conclusions: We found that the endothelium can sense a change in shear stress on the order of 0.25 dyne/cm2 over a length of ∼ 10 cells, regulating the level of protein transcription, cellular adhesion molecule expression, and leukocyte recruitment during inflammation.

IRF-1 and miRNA126 Modulate VCAM-1 Expression in Response to a High-Fat Meal

Rationale: A high-fat diet accompanied by hypertriglyceridemia increases an individual’s risk for development of atherosclerosis. An early event in this process is monocyte recruitment through binding to vascular cell adhesion molecule 1 (VCAM-1) upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect. Objective: We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects after consumption of a high-fat meal. Methods and Results: Postprandial TGRL isolated from 38 subjects were categorized as proatherogenic or antiatherogenic according to their capacity to alter the inflammatory response of HAEC. Proatherogenic TGRL increased expression of VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and E-selectin by ≈20% compared with stimulation with tumor necrosis factor-&agr; alone, whereas antiatherogenic TGRL decreased VCAM-1 expression by ≈20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, apolipoprotein (Apo)CIII, ApoE, and cholesterol. &OHgr;3-PUFA mimicked the effect of antiatherogenic TGRL by downregulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor interferon regulatory factor 1 (IRF-1) and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the proatherogenic or antiatherogenic regulation of VCAM-1. Conclusions: In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and posttranscriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a meal produced TGRL that was enriched in nonesterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.

Endothelial Heterogeneity Associated with Regional Athero-Susceptibility and Adaptation to Disturbed Blood Flow in Vivo

Endothelial phenotypic heterogeneity plays an important role in the susceptibility of the cardiovascular system to disease. Arteries and heart valves are susceptible to chronic inflammatory disease in regions of blood flow disturbance that implicates hemodynamic forces and transport characteristics as prominent influences on endothelial phenotype. By combining in vivo high-throughput genomics (discovery science) and in vitro mechanistic approaches (reductionist science), we present endothelial patho-susceptibility as an imbalance of multiple interrelated pathways that sensitize the cells to pathological change. The recently identified association of endoplasmic reticulum stress with endothelium in regions of flow disturbance is outlined as an important example of susceptible phenotype linked to proinflammatory and oxidative stress pathways.

Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal

A rise in postprandial serum triglycerides (PP-sTG) can potentiate inflammatory responses in vascular endothelial cells (ECs) and thus serves as an independent risk factor for predicting increased cardiovascular morbidity. We examined postprandial triglyceride-rich lipoproteins (PP-TGRLs) in subjects ranging from normal to hypertriglyceridemic for their capacity to alter EC acute inflammatory responses. Cultured human aortic ECs (HAECs) were conditioned with PP-TGRLs isolated from human serum at the peak after a moderately high-fat meal. VLDL particle size increased postprandially and varied directly with the subjects PP-sTG level and waist circumference. PP-TGRL particles bound to HAECs and were internalized via LDL receptor-mediated endocytosis. PP-TGRL alone did not induce an inflammatory response over the range of individuals studied. However, combined with low-dose TNF-α stimulation (0.3 ng/ml), it elicited a net 10-15% increase above cytokine alone in the membrane expression of VCAM-1, ICAM-1, and E-selectin, which was not observed with fasting TGRLs. In contrast to upregulation of ICAM-1 and E-selectin, VCAM-1 transcription and expression varied in direct proportion with individual PP-sTG and waist circumference. The extent of monocyte arrest on inflamed HAECs under shear stress also correlated closely with VCAM-1 expression induced by conditioning with PP-TGRL and TNF-α stimulation. This ex vivo approach provides a quantitative means to assess an individuals inflammatory potential, revealing a greater propensity for endothelial inflammation in hypertriglyceridemic individuals with abdominal obesity.

Shear stress modulates RAGE-mediated inflammation in a model of diabetes-induced metabolic stress

Atherosclerosis occurs preferentially at sites of disturbed blood flow despite the influence of risk factors contributing to systemic inflammation. The receptor for advanced glycation endproducts (RAGE) is a prominent mediator of inflammation in diabetes that is upregulated in atherosclerotic plaques. Our goal was to elucidate a role for arterial hemodynamics in the regulation of RAGE expression and activity. Endothelial RAGE expression was elevated at sites of flow disturbance in the aortas of healthy swine. To demonstrate a direct role for physiological shear stress (SS) in modulating RAGE expression, human aortic endothelial cells (HAEC) were exposed to high SS (HSS; 15 dyn/cm(2)), which downregulated RAGE by fourfold, or oscillatory SS (OSS; 0 ± 5 dyn/cm(2)), which upregulated RAGE by threefold, compared with static culture at 4 h. In a model of diabetes-induced metabolic stress, HAEC were chronically conditioned under high glucose (25 mM) and then simultaneously stimulated with TNF-α (0.5 ng/ml) and the RAGE ligand high mobility group box 1 (HMGB1). A 50% increase in VCAM-1 expression over TNF-α was associated with increased cytoplasmic and mitochondrial reactive oxygen species and NF-κB activity. This increase was RAGE-specific and NADPH oxidase dependent. In activated HAEC, OSS amplified HMGB1-induced VCAM-1 (3-fold) and RAGE (1.6-fold) expression and proportionally enhanced monocyte adhesion to HAEC in a RAGE-dependent manner, while HSS mitigated these increases to the level of TNF-α alone. We demonstrate that SS plays a fundamental role in regulating RAGE expression and inflammatory responses in the endothelium. These findings may provide mechanistic insight into how diabetes accelerates the nonrandom distribution of atherosclerosis in arteries.

Triglyceride-Rich Lipoprotein Modulates Endothelial Vascular Cell Adhesion Molecule (VCAM)-1 Expression via Differential Regulation of Endoplasmic Reticulum Stress

Circulating triglyceride-rich lipoproteins (TGRL) from hypertriglyceridemic subjects exacerbate endothelial inflammation and promote monocyte infiltration into the arterial wall. We have recently reported that TGRL isolated from human blood after a high-fat meal can elicit a pro- or anti-atherogenic state in human aortic endothelial cells (HAEC), defined as up- or down-regulation of VCAM-1 expression in response to tumor necrosis factor alpha (TNFα) stimulation, respectively. A direct correlation was found between subjects categorized at higher risk for cardiovascular disease based upon serum triglycerides and postprandial production of TGRL particles that increased VCAM-1-dependent monocyte adhesion to inflamed endothelium. To establish how TGRL metabolism is linked to VCAM-1 regulation, we examined endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) pathways. Regardless of its atherogenicity, the rate and extent of TGRL internalization and lipid droplet formation by HAEC were uniform. However, pro-atherogenic TGRL exacerbated ER membrane expansion and stress following TNFα stimulation, whereas anti-atherogenic TGRL ameliorated such effects. Inhibition of ER stress with a chemical chaperone 4-phenylbutyric acid decreased TNFα-induced VCAM-1 expression and abrogated TGRL’s atherogenic effect. Activation of ER stress sensors PKR-like ER-regulated kinase (PERK) and inositol requiring protein 1α (IRE1α), and downstream effectors including eukaryotic initiation factor-2α (eIF2α), spliced X-box-binding protein 1 (sXBP1) and C/EBP homologous protein (CHOP), directly correlated with the atherogenic activity of an individual’s TGRL. Modulation of ER stress sensors also correlated with changes in expression of interferon regulatory factor 1 (IRF-1), a transcription factor of Vcam-1 responsible for regulation of its expression. Moreover, knockdown studies using siRNA defined a causal relationship between the PERK/eIF2α/CHOP pathway and IRF-1-mediated VCAM-1 expression. We conclude that ER stress and the UPR contribute to the regulation of Vcam-1 transcription as a function of the atherogenic nature of TGRL.

Clinical impact of sample interference on intensive insulin therapy in severely burned patients: a pilot study.

Severely burned patients benefit from intensive insulin therapy (IIT) for tight glycemic control (TGC). The authors evaluated the clinical impact of automatic correction of hematocrit and ascorbic acid interference for bedside glucose monitoring performance in critically ill burn patients. The performance of two point-of-care glucose monitoring systems (GMSs): 1) GMS1, an autocorrecting device, and 2) GMS2, a noncorrecting device were compared. Sixty remnant arterial blood samples were collected in a prospective observational study to evaluate hematocrit and ascorbic acid effects on GMS1 vs GMS2 accuracy paired against a plasma glucose reference. Next, we enrolled 12 patients in a pilot randomized controlled trial. Patients were randomized 1:1 to receive IIT targeting a TGC interval of 111 to 151 mg/dl and guided by either GMS1 or GMS2. GMS bias, mean insulin rate, and glycemic variability were calculated. In the prospective study, GMS1 results were similar to plasma glucose results (mean bias, −0.75 [4.0] mg/dl; n = 60; P = .214). GMS2 results significantly differed from paired plasma glucose results (mean bias, −5.66 [18.7] mg/dl; n = 60; P = .048). Ascorbic acid therapy elicited significant GMS2 performance bias (29.2 [27.2]; P < .001). Randomized controlled trial results reported lower mean bias (P < .001), glycemic variability (P < .05), mean insulin rate (P < .001), and frequency of hypoglycemia (P < .001) in the GMS1 group than in the GMS2 group. Anemia and high-dose ascorbic acid therapy negatively impact GMS accuracy and TGC in burn patients. Automatic correction of confounding factors improves glycemic control. Further studies are warranted to determine outcomes associated with accurate glucose monitoring during IIT.

Shear stress modulates VCAM-1 expression in response to TNF-α and dietary lipids via interferon regulatory factor-1 in cultured endothelium

Dyslipidemia is a primary risk factor for cardiovascular disease, but the specific mechanisms that determine the localization of atherosclerotic plaques in arteries are not well defined. Triglyceride-rich lipoproteins (TGRL) isolated from human plasma after a high-fat meal modulate TNF-α-induced VCAM-1 expression in cultured human aortic endothelial cells (HAECs) via an interferon regulatory factor (IRF)-1-dependent transcriptional mechanism. We examined whether fluid shear stress acts as a mediator of IRF-1-dependent VCAM-1 expression in response to cytokine and dietary lipids. IRF-1 and VCAM-1 were examined by immunofluorescence in TNF-α-stimulated HAEC monolayers exposed to TGRL and a linear gradient of shear stress ranging from 0 to 16 dyn/cm(2) in a microfluidic device. Shear stress alone modulated TNF-α-induced VCAM-1 expression, eliciting a 150% increase at low shear stress (2 dyn/cm(2)) and a 70% decrease at high shear stress (12 dyn/cm(2)) relative to static. These differences correlated with a 60% increase in IRF-1 expression under low shear stress and a 40% decrease under high shear stress. The addition of TGRL along with cytokine activated a fourfold increase in VCAM-1 expression and a twofold increase in IRF-1 expression. The combined effect of shear stress and TGRL on the upregulation of membrane VCAM-1 was abolished by transfection of HAECs with IRF-1-specific small interfering RNA. In a healthy swine model, elevated levels of endothelial IRF-1 were also observed within atherosusceptible regions of the aorta by Western blot analysis and immunohistochemistry, implicating arterial hemodynamics in the regulation of IRF-1 expression. These data demonstrate direct roles for fluid shear stress and postprandial TGRL from human serum in the regulation of IRF-1 expression and downstream inflammatory responses in HAECs.

Alagebrium inhibits neointimal hyperplasia and restores distributions of wall shear stress by reducing downstream vascular resistance in obese and diabetic rats.

Mechanisms of restenosis in type 2 diabetes mellitus (T2DM) are incompletely elucidated, but advanced glycation end-product (AGE)-induced vascular remodeling likely contributes. We tested the hypothesis that AGE-related collagen cross-linking (ARCC) leads to increased downstream vascular resistance and altered in-stent hemodynamics, thereby promoting neointimal hyperplasia (NH) in T2DM. We proposed that decreasing ARCC with ALT-711 (Alagebrium) would mitigate this response. Abdominal aortic stents were implanted in Zucker lean (ZL), obese (ZO), and diabetic (ZD) rats. Blood flow, vessel diameter, and wall shear stress (WSS) were calculated after 21 days, and NH was quantified. Arterial segments (aorta, carotid, iliac, femoral, and arterioles) were harvested to detect ARCC and protein expression, including transforming growth factor-β (TGF-β) and receptor for AGEs (RAGE). Downstream resistance was elevated (60%), whereas flow and WSS were significantly decreased (44% and 56%) in ZD vs. ZL rats. NH was increased in ZO but not ZD rats. ALT-711 reduced ARCC and resistance (46%) in ZD rats while decreasing NH and producing similar in-stent WSS across groups. No consistent differences in RAGE or TGF-β expression were observed in arterial segments. ALT-711 modified lectin-type oxidized LDL receptor 1 but not RAGE expression by cells on decellularized matrices. In conclusion, ALT-711 decreased ARCC, increased in-stent flow rate, and reduced NH in ZO and ZD rats through RAGE-independent pathways. The study supports an important role for AGE-induced remodeling within and downstream of stent implantation to promote enhanced NH in T2DM.