Dan E Berkowitz

Johns Hopkins University, Baltimore, Maryland, United States

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Publications (145)772.96 Total impact

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    ABSTRACT: Background: The loss of structural and functional integrity of red blood cells (RBCs) during storage, collectively referred to as "storage lesion," has been implicated in reduced oxygen delivery after transfusion. RBCs are highly susceptible to oxidative damage from generation of reactive oxygen species by autoxidation of hemoglobin. Therefore, we examined whether increased oxidative stress (OS) in stored RBCs is associated with impaired cell membrane deformability before or after transfusion. Study design and methods: Thirty-four patients undergoing multilevel spine fusion surgery were enrolled. OS in RBCs was assessed by the presence of fluorescent heme degradation products and methemoglobin, which were measured with fluorimetric and spectrophotometric methods, respectively. Deformability and aggregation were determined by ektacytometry in stored RBCs, autologous salvaged RBCs, and posttransfusion blood samples. Results: OS in stored RBCs was significantly increased with longer storage (R = 0.54, p = 0.032) and significantly higher than that in fresh RBCs (9.1 ± 1.3 fluorescent arbitrary units vs. 7.7 ± 0.9 fluorescent arbitrary units, p < 0.001). Deformability decreased (R = -0.60, p = 0.009) with increasing storage duration. OS was elevated (p < 0.05) and deformability was decreased (p < 0.05) in postoperative blood from patients who had undergone moderate (≥4 RBC units) but not minimal or no transfusion. Neither the decrease in deformability of RBCs nor the aggregation changes were correlated with OS. Conclusions: Although stored RBCs show signs of increased OS and loss of cell membrane deformability, these changes were not directly correlated and were only evident after moderate but not lower dose transfusion in postoperative surgical patients. These findings suggest that factors other than OS may contribute to impaired rheology with stored RBCs in the clinical setting.
    No preview · Article · Jan 2016 · Transfusion
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    ABSTRACT: Adults older than 65years undergo more than 120,000 coronary artery bypass (CAB) procedures each year in the United States. Chronological age alone, though commonly used in prediction models of outcomes after CAB, does not alone reflect variability in aging process; thus, the risk of complications in older adults. We performed a prospective study to evaluate a relationship between senescence marker p16(INK4a) expression in peripheral blood T-lymphocytes (p16 levels in PBTLs) with aging and with perioperative outcomes in older CAB patients. We included 55 patients age 55 and older, who underwent CAB in Johns Hopkins Hospital between September 1st, 2010 and March 25th, 2013. Demographic, clinical and laboratory data following outline of the Society of Thoracic Surgeons data collection form was collected, and p16 mRNA levels in PBTLs were measured using TaqMan® qRT-PCR. Associations between p16 mRNA levels in PBTLs with length of hospital stay, frailty status, p16 protein levels in the aortic and left internal mammary artery tissue, cerebral oxygen saturation, and augmentation index as a measure of vascular stiffness were measured using regression analyses. Length of hospital stay was the primary outcome of interest, and major organ morbidity, mortality, and discharge to a skilled nursing facility were secondary outcomes. In secondary analysis, we evaluated associations between p16 mRNA levels in PBTLs and interleukin-6 levels using regression analyses. Median age of enrolled patients was 63.5years (range 56-81years), they were predominantly male (74.55%), of Caucasian descent (85.45%). Median log2(p16 levels in PBTLs) were 4.71 (range 1.10-6.82). P16 levels in PBTLs were significantly associated with chronological age (mean difference 0.06 for each year increase in age, 95% CI 0.01-0.11) and interleukin 6 levels (mean difference 0.09 for each pg/ml increase in IL-6 levels, 95% CI 0.01-0.18). There were no significant associations with frailty status, augmentation index, cerebral oxygenation and p16 protein levels in blood vessels. Increasing p16 levels in PBTLs did not predict length of stay in the hospital (HR 1.10, 95% CI 0.87-1.40) or intensive care unit (HR 1.02, 95% CI 0.79-1.32). Additional evaluation of p16 levels in PBTLs as predictor of perioperative outcomes is required and should include additional markers of immune system aging as well as different outcomes after CAB in addition to length of hospital stay.
    No preview · Article · Dec 2015 · Experimental gerontology
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    ABSTRACT: Hydrogen sulfide (H2S) has emerged as an important gasotransmitter in the vasculature. In this study, we tested the hypothesis that H2S contributes to coronary vasoregulation and evaluated the physiological relevance of two sources of H2S, namely, cystathionine-gamma-lyase (CSE) and 3-mercaptypyruvate sulfertransferase (MPST). MPST was detected in human coronary artery endothelial cells as well as rat and mouse coronary artery; CSE was not detected in the coronary vasculature. Rat coronary artery homogenates produced H2S through the MPST pathway but not the CSE pathway in vitro. In vivo coronary vasorelaxation response was similar in CSE knockout mice, wild-type mice (WT), and WT mice treated with the CSE inhibitor propargylglycine, suggesting that CSE-produced H2S does not have a significant role in coronary vasoregulation in vivo. Ex vivo, MPST substrate 3-mercaptopyruvate (3-MP) and H2S donor sodium hydrosulfide (NaHS) elicited similar coronary vasoreactivity responses. Pyruvate did not have any effects on vasoreactivity. The vasoactive effect of H2S appeared to be NO-dependent: H2S induced coronary vasoconstriction in the presence of NO and vasorelaxation in its absence. Maximal endothelial-dependent relaxation was intact after 3-MP, and or NaHS induced an increase in pre-constriction tone, suggesting that eNOS activity was not significantly inhibited. In vitro, H2Sreacted with NO, which may, in part explain the vasoconstrictive effects of 3-MP and NaHS. Taken together, these data show that MPST rather than CSE generates H2S in coronary artery, mediating its effects through direct modulation of NO. This has important implications for H2S based therapy in healthy and diseased coronary arteries.
    No preview · Article · Oct 2015 · AJP Heart and Circulatory Physiology
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    ABSTRACT: Pulse wave velocity (PWV) and pulse pressure (PP) are blood pressure (BP)-dependent surrogates for vascular stiffness. Considering that there are no clinically useful markers for arterial stiffness that are BP-independent, our objective was to identify novel indices of arterial stiffness and compare them with previously described markers. PWV and PP were measured in young and old male Fisher rats and in young and old male spontaneously hypertensive rats (SHR) over a wide range of BPs. The BP dependence of these and several other indices of vascular stiffness were evaluated. An index incorporating PWV and PP was also constructed. Both PWV and PP increase in a non-linear manner with rising BP for both strains of animals (Fisher and SHRs). Age markedly changes the relationship between PWV or PP and BP. The previously described Ambulatory Arterial Stiffness Index (AASI) was able to differentiate between young and old vasculature, whereas the Cardio-Ankle Vascular Index (CAVI) did not reliably differentiate between the two. The novel Arterial Stiffness Index (ASI) differentiated stiffer from more compliant vasculature. Considering the limitations of the currently available indices of arterial stiffness, we propose a novel index of intrinsic arterial stiffness, the ASI, which is robust over a range of BPs and allows one to distinguish between compliant and stiff vasculature in both Fisher rats and SHRs. Further studies are necessary to validate this index in other settings.Hypertension Research advance online publication, 22 October 2015; doi:10.1038/hr.2015.109.
    No preview · Article · Oct 2015 · Hypertension Research
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    ABSTRACT: Recent research suggests that priapism in sickle cell disease (SCD) is due to dysregulation of penile erection homeostasis including alteration of nitric oxide synthase (NOS) and phosphodiesterase type 5 (PDE5) activities by excessive levels of reactive oxygen species (ROS) released during hemolysis. It is unknown if subacute exposure to hemolysis is sufficient or if chronic reconditioning of erectile tissues is required for perturbation of homeostatic pathways and whether PDE5 inhibitor (PDE5I) treatment can restore erectile homeostasis in the subacute setting.
    No preview · Article · Sep 2015 · Journal of Sexual Medicine
  • A. Scott · N. Enika · D. Johnson · V Barodka · D. Berkowitz · S. Frank

    No preview · Article · Aug 2015 · Transfusion
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    ABSTRACT: Hyperglycemia-induced reactive oxygen species (ROS) production plays a major role in the pathogenesis of diabetic vascular dysfunction. However, the underlying mechanisms remain unclear. Toll-like receptor 4 (TLR4), a key component of innate immunity, is known to be activated during diabetes. Therefore, we hypothesize that hyperglycemia activates TLR4 signaling in vascular smooth muscle cells (VSMCs) that triggers ROS production and causes vascular dysfunction. Rat mesenteric VSMCs exposed to high glucose (25 mmol/l) increased TLR4 expression and activated TLR4 signaling via upregulation of myeloid differentiation factor 88 (MyD88). TLR4 inhibitor CLI-095 significantly attenuated elevated levels of ROS and nuclear factor-kappa B (NF-κB) activity in VSMCs exposed to high glucose. Mesenteric arteries from streptozotocin-induced diabetic rats treated with CLI-095 (2 mg/kg/day) intraperitoneally for 2 weeks exhibited reduced ROS generation and attenuated noradrenaline-induced contraction. These results suggest that hyperglycemia-induced ROS generation and NF-κB activation in VSMCs are at least, in part, mediated by TLR4 signaling. Therefore, strategies to block TLR4 signaling pathways pose a promising avenue to alleviate diabetic-induced vascular complications. High glucose-induced TLR4 activation in vascular smooth muscle cells. Inhibition of TLR4 attenuated high glucose-induced ROS production and NF-κB activity in VSMC. Suppression of TLR4 signaling attenuated mesenteric contraction in diabetic rat.
    No preview · Article · Jul 2015 · Journal of Molecular Medicine
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    ABSTRACT: Emerging evidence strongly supports a role for HDAC2 in the transcriptional regulation of endothelial genes and vascular function. We have recently demonstrated that HDAC2 reciprocally regulates the transcription of Arginase2, which is itself a critical modulator of endothelial function via eNOS. Moreover HDAC2 levels are decreased in response to the atherogenic stimulus OxLDL via a mechanism that is apparently dependent upon proteasomal degradation. NEDDylation is a post-translational protein modification that is tightly linked to ubiquitination and thereby protein degradation. We propose that changes in NEDDylation may modulate vascular endothelial function in part through alterations in the proteasomal degradation of HDAC2. In HAEC, OxLDL exposure augmented global protein NEDDylation. Pre-incubation of mouse aortic rings with the NEDDylation activating enzyme inhibitor, MLN4924, prevented OxLDL-induced endothelial dysfunction. In HAEC, MLN enhanced HDAC2 abundance, decreased expression and activity of Arginase2, and blocked OxLDL-mediated reduction of HDAC2. Additionally, HDAC2 was shown to be a substrate for NEDD8 conjugation and this interaction was potentiated by OxLDL. Further, HDAC2 levels were reciprocally regulated by ectopic expression of NEDD8 and the de-NEDDylating enzyme SENP8. Our findings indicate that the observed improvement in endothelial dysfunction with inhibition of NEDDylation activating enzyme is likely due to an HDAC2-dependent decrease in Arginase2. NEDDylation activating enzyme may therefore be a novel target in endothelial dysfunction and atherogenesis.
    No preview · Article · Apr 2015 · Journal of Molecular and Cellular Cardiology
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    Full-text · Dataset · Jan 2015

  • No preview · Article · Jan 2015 · Journal of Sexual Medicine
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    ABSTRACT: We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol. This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.
    Full-text · Article · Dec 2014 · Journal of Visualized Experiments
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    ABSTRACT: Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO(.)), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-β subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α(C42S) mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation. © 2014 American Heart Association, Inc.
    Full-text · Article · Dec 2014 · Hypertension
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    ABSTRACT: Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.
    Preview · Article · Nov 2014 · Proceedings of the National Academy of Sciences

  • No preview · Article · Nov 2014 · Nitric Oxide

  • No preview · Article · Nov 2014 · Free Radical Biology and Medicine
  • D. Pandey · G. Sikka · Y. Bergman · J. Kim · L. Romer · D. Berkowitz

    No preview · Article · Jul 2014 · Basic & Clinical Pharmacology & Toxicology
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    ABSTRACT: Rationale: Increased arginase activity contributes to endothelial dysfunction by competition for l-arginine substrate and reciprocal regulation of nitric oxide synthase (NOS). The rapid increase in arginase activity in human aortic endothelial cells exposed to oxidized low-density lipoprotein (OxLDL) is consistent with post-translational modification or subcellular trafficking. Objective: To test the hypotheses that OxLDL triggers reverse translocation of mitochondrial arginase 2 (Arg2) to cytosol and Arg2 activation, and that this process is dependent on mitochondrial processing peptidase, lectin-like OxLDL receptor-1 receptor, and rho kinase. Methods and results: OxLDL-triggered translocation of Arg2 from mitochondria to cytosol in human aortic endothelial cells and in murine aortic intima with a concomitant rise in arginase activity. All of these changes were abolished by inhibition of mitochondrial processing peptidase or by its siRNA-mediated knockdown. Rho kinase inhibition and the absence of the lectin-like OxLDL receptor-1 in knockout mice also ablated translocation. Aminoterminal sequencing of Arg2 revealed 2 candidate mitochondrial targeting sequences, and deletion of either of these confined Arg2 to the cytoplasm. Inhibitors of mitochondrial processing peptidase or lectin-like OxLDL receptor-1 knockout attenuated OxLDL-mediated decrements in endothelial-specific NO production and increases in superoxide generation. Finally, Arg2(-/-) mice bred on an ApoE(-/-) background showed reduced plaque load, reduced reactive oxygen species production, enhanced NO, and improved endothelial function when compared with ApoE(-/-) controls. Conclusions: These data demonstrate dual distribution of Arg2, a protein with an unambiguous mitochondrial targeting sequence, in mammalian cells, and its reverse translocation to cytoplasm by alterations in the extracellular milieu. This novel molecular mechanism drives OxLDL-mediated arginase activation, endothelial NOS uncoupling, endothelial dysfunction, and atherogenesis.
    No preview · Article · Jun 2014 · Circulation Research
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    ABSTRACT: Arginase 2 (Arg2) is a critical target in atherosclerosis because it controls endothelial nitric oxide, proliferation, fibrosis, and inflammation. Regulators of Arg2 transcription in the endothelium have not been characterized. The goal of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial Arg2 transcription and endothelial function. The HDAC inhibitor trichostatin A increased levels of Arg2 mRNA, protein, and activity in both human aortic endothelial cells and mouse aortic rings. These changes occurred in both time- and dose-dependent patterns and resulted in Arg2-dependent endothelial dysfunction. Trichostatin A and the atherogenic stimulus oxidized low-density lipoprotein enhanced the activity of common promoter regions of Arg2. HDAC inhibition with trichostatin A also decreased endothelial nitric oxide, and these effects were blunted by arginase inhibition. Nonselective class I HDAC inhibitors enhanced Arg2 expression, whereas the only selective inhibitor that increased Arg2 expression was mocetinostat, a selective inhibitor of HDACs 1 and 2. Additionally, mouse aortic rings preincubated with mocetinostat exhibited dysfunctional relaxation. Overexpression of HDAC2 (but not HDAC 1, 3, or 8) cDNA in human aortic endothelial cells suppressed Arg2 expression in a concentration-dependent manner, and siRNA knockdown of HDAC2 enhanced Arg2 expression. Chromatin immunoprecipitation indicated direct binding of HDAC2 to the Arg2 promoter, and HDAC2 overexpression in human aortic endothelial cells blocked oxidized low-density lipoprotein-mediated activation of the Arg2 promoter. Finally, overexpression of HDAC2 blocked oxidized low-density lipoprotein-mediated vascular dysfunction. HDAC2 is a critical regulator of Arg2 expression and thereby endothelial nitric oxide and endothelial function. Overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis.
    No preview · Article · May 2014 · Arteriosclerosis Thrombosis and Vascular Biology
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    ABSTRACT: Both cardiopulmonary bypass (CPB) and red blood cell (RBC) storage are associated with detrimental changes in RBC structure and function that may adversely affect tissue oxygen delivery. We tested the hypothesis that in cardiac surgery patients, RBC deformability and aggregation are minimally affected by CPB with autologous salvaged blood alone but are negatively affected by the addition of stored allogeneic blood. In this prospective cohort study, 32 patients undergoing cardiac surgery with CPB were divided into 3 groups by transfusion status: autologous salvaged RBCs alone (Auto; n = 12), autologous salvaged RBCs + minimal (<5 units) stored allogeneic RBCs (Auto+Allo min; n = 10), and autologous salvaged RBCs + moderate (≥5 units) stored allogeneic RBCs (Auto+Allo mod; n = 10). Ektacytometry was used to measure RBC elongation index (deformability) and critical shear stress (aggregation) before, during, and for 3 days after surgery. In the Auto group, RBC elongation index did not change significantly from the preoperative baseline. In the Auto+Allo min group, mean elongation index decreased from 32.31 ± 0.02 (baseline) to 30.47 ± 0.02 (nadir on postoperative day 1) (P = 0.003, representing a 6% change). In the Auto+Allo mod group, mean elongation index decreased from 32.7 ± 0.02 (baseline) to 28.14 ± 0.01 (nadir on postoperative day 1) (P = 0.0001, representing a 14% change). Deformability then dose-dependently recovered toward baseline over the first 3 postoperative days. Changes in aggregation were unrelated to transfusion (no difference among groups). For the 3 groups combined, mean critical shear stress decreased from 359 ± 174 mPa to 170 ± 141 mPa (P = 0.01, representing a 54% change), with the nadir at the end of surgery and returned to baseline by postoperative day 1. In cardiac surgery patients, transfusion with stored allogeneic RBCs, but not autologous salvaged RBCs, is associated with a decrease in RBC cell membrane deformability that is dose-dependent and may persist beyond 3 postoperative days. These findings suggest that autologous salvaged RBCs may be of higher quality than stored RBCs, since the latter are subject to the so-called storage lesions.
    No preview · Article · May 2014 · Anesthesia and analgesia
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    ABSTRACT: Glycosphingolipids are integral components of the cell membrane and have been shown to serve as messengers, transducing growth factor initiated phenotypes. Here we have examined whether inhibition of glycosphingolipid synthesis could ameliorate atherosclerosis and arterial stiffness in transgenic mice and rabbits. Apolipoprotein E-/- mice (12 weeks of age, n = 6) were fed regular chow or a western diet (1.25% cholesterol, 2% fat). Mice were fed 5mg/kg (mpk) or 10mpk of an inhibitor of glycosphingolipid synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), solubilized in vehicle (5% Tween-80 in PBS) and the placebo group received vehicle only. At 20 and 36 weeks of age, serial echocardiography was performed to measure aortic intima medial thickening (IMT). Aortic pulse wave velocity (PWV) measured vascular stiffness. Feeding mice a western diet markedly increased aortic PWV, IMT, oxidized LDL, Ca(2+) deposits, and glucosyl- and lactosylceramide synthase activity. These were dose-dependently decreased by feeding D-PDMP. In liver, D-PDMP decreased cholesterol and triglyceride levels by raising the expression of SREBP2, LDL-r, HMGCo-A reductase, and cholesterol efflux genes (e.g., ABCG5, ABCG8). D-PDMP affected VLDL catabolism by increasing the gene expression for LPL and VLDLr. Rabbits fed a western diet for 90 days had extensive atherosclerosis accompanied by a 17.5-fold increase in total cholesterol levels and a 3-fold increase in lactosylceramide levels. This was completely prevented by feeding D-PDMP. Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in ApoE-/- mice and rabbits. Thus, inhibition of glycosphingolipid synthesis may be a novel approach to ameliorate atherosclerosis and arterial stiffness.
    Full-text · Article · Apr 2014 · Circulation

Publication Stats

5k Citations
772.96 Total Impact Points

Institutions

  • 1998-2015
    • Johns Hopkins University
      • • Department of Biomedical Engineering
      • • Department of Surgery
      • • Department of Anesthesiology and Critical Care Medicine
      • • Department of Medicine
      Baltimore, Maryland, United States
  • 1997-2015
    • Johns Hopkins Medicine
      • • Department of Anesthesiology and Critical Care Medicine
      • • Department of Biomedical Engineering
      • • Department of Medicine
      Baltimore, Maryland, United States
  • 2011
    • Pennsylvania State University
      • Department of Kinesiology
      University Park, Maryland, United States
  • 1994-1996
    • Duke University Medical Center
      • Department of Anesthesiology
      Durham, North Carolina, United States
    • Duke University
      • Department of Surgery
      Durham, North Carolina, United States