bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 179: Rho Kinase Isoform Contribution to Endothelial Contractility
Authors: Valent Erik , Krishnan Ramaswamy , van Hinsbergh Victor , van Nieuw Amerongen Geerten P
Published: July 2021

Background:
Rho kinase (ROCK) is the major effector protein of RhoA and is known to mediate F-actin stress fibers. ROCK activates myosin motors. Actomyosin-generated contractile forces can be transmitted to cell-cell junctions, resulting in increased junctional tension, junctional disassembly and endothelial hyperpermeability. In apparent contrast, basal ROCK activity is required for maintenance of barrier integrity
.
We hypothesize that these dual roles of ROCK could be contributed to the two highly homologous expressed isoforms.

Methods:
Traction force microscopy was utilized to study the endothelium-generated contractile forces after depletion of ROCK1 and/or ROCK2 by SiRNA approach in the absence and presence of the vaso-active agent thrombin.

Results:
The traction force landscape showed that ROCK is a major contributor to endothelial contraction and permeability. Depletion of ROCK1 and ROCK2 resulted in a blockade of contractile response to thrombin and a marked reduction in endothelial hyperpermeability. Detailed force distribution maps revealed that the absence of ROCK1, but not of ROCK2, resulted in an increase in baseline contractile forces (siROCK1: 88.1 ± 4.1 Pa, siROCK2: 53.6 ± 3.4 Pa, Control: 56.2 ± 6.1 Pa), which was associated with the formation of numerous inter-endothelial gaps upon stimulation with thrombin. Knockdown of ROCK2 stabilized the endothelial barrier and largely prevented traction force enhancements. Exposure to thrombin resulted in all conditions in an increase of traction forces and hyperpermeability.

Conclusion:
Both ROCK isoforms have a distinct function in endothelial contractility and permeability. ROCK1 is predominantly contributing to barrier maintenance under baseline conditions, whereas ROCK2 mediates the thrombin-induced contractile force enhancements and subsequent barrier dysfunction.

Funding:
Dutch Heart Foundation 2011T072
Abstract 183: Carbon Monoxide Induces Macrophage VEGF Production and Promotes Angiogenic Behavior in Endothelial Cells
Authors: Leake Andrew E , Hong Guiying , Washington Christopher B , Shukla Ankur J , Sachdev Ulka , Tzeng Edith
Published: July 2021

Introduction:
Carbon monoxide (CO) has potent anti-inflammatory and pro-healing properties. We have previously shown that CO enhances endothelial cell (EC) angiogenic behavior in vitro. More striking, however, is that conditioned medium (CM) from macrophages isolated from CO treated rats can promote angiogenesis indirectly. We sought to examine the mechanism of this indirect angiogenic effect of CO.

Methods:
Peritoneal macrophages were collected from rats after inhaled CO treatment (250 PPM for 1 hr) or air treatment. Macrophages were cultured overnight and CM was collected to treat human umbilical vein ECs (HUVECs). VEGF and eNOS expression was assessed by Western blot.

Results:
CM from macrophages isolated from CO treated rats (CO) showed a 3-fold increase in VEGF vs Air CM (Ratio CO/Air=2.96, Range 1.6-4.2) (Figure). VEGF levels were similar between the macrophages from CO or air treated rats (CO/Air=0.89). HUVECs treated with CO or Air CM also showed no difference in VEGF levels (CO/Air=1.07). However, eNOS expression was significantly increased in HUVECs treated with CO CM vs. Air CM (CO/Air=26.4, Range 20-32; p=0.004). HUVECs cultured in a CO chamber or a standard incubator exhibited no change in eNOS expression (CO/Air, 1.38 p=0.11).

Conclusion:
In vivo CO promotes macrophage secretion of VEGF. These cells can promote angiogenic behavior in ECs through this VEGF and other secreted products. These macrophage products upregulate eNOS but not VEGF expression in HUVECs. The effect of the CO CM on eNOS expression in HUVECs could not be reproduced by direct CO treatment of the cells. Future studies will further characterize the phenotypic change of the macrophages induced by inhaled CO that favors angiogenesis and healing.
Abstract 188: Differentiation of Preexisting Cardiosphere-Derived Stem Cells Was Modulated by Telocyte Niche
Authors: Yu Xi-Yong , Huang Yu-Kai , Pan Yu , Li Xiao-Hong , Lin Qiu-Xiong , Shan Zhi-Xin , Deng Chun-Yu , Yang Min , Geng Qing-Shan , Lin Shu-Guang
Published: July 2021

Background:
Cardiosphere-derived stem cells (CSCs) were found to be unusual pre-existing cells, comparing with other kinds of stem cells, showing the greatest cardiomyocytes differentiation potency. This study is to explore whether the microenvironment provided by telocytes can improve differentiation efficiency of CSCs.

Methods and Results:
The CSCs and telocytes were isolated from myocardium of SD rats. The experiment was divided into control group, T-group (CSCs co-culture with adult rat telocytes), N-group (CSCs co-culture with neonatal rat caridomyocytes). Q-PCR was used to check the change of stem cell markers and cardiomyocyte markers. Immunofluorescence was used to detect the expression of cTnI protein in each group. The expression of Isl-1 in T-group was higher than other groups at 3 days, Nanong was higher at 5d, Gata4 and cTnI were higher at 9d. There are almost no expressions of cTnI protein in control group and N-group, but higher expressions of cTnI protein in T-group. The data of PCR-array showed that the most biomarkers of stem cells increased when CSCs co-culture with telocytes for 3 days, but the most biomarkers of cardiac progenitor cells increased when CSCs co-culture with telocytes for over 9 days. Finally, the CSCs induced by telocytes (iCSCs) were injected into borders of cardiac scar tissue 1 wk after experimental infarction. The results showed a nearly normal ejection fraction (EF) in iCSCs-treated rats but a lower EF in all PBS-treated animals.

Conclusion:
There are bilateral regulations of the microenvironment provided by telocytes which not only maintain the stemness of CSCs at the early stage, but also improve the differentiation efficiency of CSCs at the late stage. Telocyte is an important chaperone of CSCs, and helps for repairing damaged heart muscle. This work was supported by NSFC (81330007) and 973 program of China (2012CB526600).
Abstract 191: Endothelial Dysfunction Potentiates Deep Venous Thrombosis in a Mouse Model of Sepsis
Authors: Obi Andrea T , Kessinger Chase W , Luke Catherine E , Elfline Megan , Siahaan Teruna J , Jaffer Farouc A , Raghavendran Krishnan , Wakefield Thomas W , Henke Peter K
Published: July 2021

Objective:
Sepsis is a strong risk factor for the development of deep venous thrombosis (DVT). However, little is understood about the underlying pathobiology responsible for this phenomenon. This study was undertaken to evaluate the endothelial response of cellular adhesion molecules (CAM) involved in early thrombogenesis in a mouse model of sepsis.

Methods:
Wild-type mice underwent treatment with vehicle or LPS (10mg/kg), or followed by inferior vena cava (IVC) ligation to generate a venous thrombus (VT). Tissue and plasma were harvested at 2 hours post-intervention and were assessed for CAMs, thrombus size, and markers of extracellular DNA (NETs). Pre-treatment (12 hours prior and at time of thrombosis) with P/E selectin (sel) and ICAM exogenous inhibition was undertaken. Antibody depletion was accomplished in selected mice with anti-Ly6G antibody. To understand leukocyte trafficking to the vein wall under sepsis conditions, a separate group of animals underwent LPS or vehicle injection followed by intravital fluorescence microscopy of rhodamine-6G-labeled leukocytes.

Results:
Pre-thrombosis, IVC CAMs (P-sel p=0.15, E-sel, p<0.01 and ICAM-1 p=0.11, all n=6) were elevated in mice treated with LPS. Consistently, intravital microscopy of leukocyte behavior revealed increased leukocyte rolling (n=6, p< .0001) and decreased rolling velocity (n=75/group, p<0.0001). Post-thrombosis, LPS treated mice formed larger thrombi (6.4±0.8mg v. 9.9±1.1mg, p=0.03) and exhibited elevations in circulating P-sel, E-sel, and ICAM-1 (all p<0.05, n=10). NETs markers H3 and elastase were not elevated. Pre-treatment with an ICAM-1 inhibitor restored thrombus size (6.6±1.5 mg, n=5) and circulating ICAM-1 levels to that of vehicle treated mice, while P/E sel inhibition resulted in moderately reduced thrombus size (7.3±1.2mg, n=10). Exogenous PMN depletion did not decrease thrombus weight.

Conclusions:
In a mouse model of endotoxemia, CAM elevation and leukocyte trafficking occurs in the serum and local vein wall; and with stasis insult, larger VT form. Inhibitor experiments suggest that ICAM may be central to the sepsis related early VT, while PMNs and P/E sel may be less important. CAM inhibition may be a unique strategy to prevent sepsis-associated DVT.
Abstract 193: Antithrombin Perfluorocarbon Nanoparticles Ameliorate Renal Injury Following Transient Warm Ischemia
Authors: Vemuri Chandu , Chen Junjie , Palekar Rohun U , Allen John S , Yang Xiaoxia , Zhang Hiuying , Wickline Samuel A
Published: July 2021

Objective:
Thrombin mediated microvascular thrombosis plays a crucial role in the pathogenesis of acute renal reperfusion injury following transient ischemia. We hypothesize that anti-thrombin nanoparticles will ameliorate acute renal injury by inhibiting microvascular thrombosis.

Methods:
Adult, male Sprague Dawley rats were randomized into two groups of 5 to receive tail vein injections of saline or nanoparticles loaded with Phe[D]-Pro-Arg-Chloromethylketone (NP-PPACK). Immediately following injection, all animals underwent operative bilateral renal artery occlusion to create 45 minutes of warm ischemia, followed by restoration of renal blood flow. Blood samples were drawn daily and animals were euthanized on day 1 or 7 for histologic analysis of kidney injury (H&E, TUNEL and thrombin staining).

Results:
Histologic analysis of renal tissue revealed significant apoptosis, necrosis and thrombin accumulation 1 day after ischemia-reperfusion, confirming acute kidney injury. The peak creatinine (mg/dl) on day 1 was significantly lower in NP-PPACK treated animals (0.57 +/- 0.07 (SEM)) than in saline treated controls (1.40 +/- 0.20 (SEM); p-value <0.01). Furthermore, animals treated with NP-PPACK continued to exhibit less renal dysfunction for 7 days after injury (Figure 1).

Conclusion:
Histologically confirmed intrarenal thrombosis was detected one day after ischemia-reperfusion injury. Targeted inhibition of thrombin with NP-PPACK prevented a decline in renal function following transient occlusion. Future work will focus on defining the underlying mechanisms of this effect.
Abstract 196: NADPH Oxidase Isoforms NOX1 and NOX2 Differentially Regulate GPVI- and Non-GPVI-Dependent ROS Generation and Platelet Activation
Authors: Akbar Huzoor , Duan Xin , Saleem Saima , Davis Ashley K , Zheng Yi
Published: July 2021

Reactive oxygen species (ROS) play an important role in platelet activation. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Platelets express two NOX isoforms, NOX1 and NOX2. In this study we investigated the effects of ML171 (0.1-1 μM), a selective NOX1 inhibitor (ACS Chem Biol. 5, 981-993, 2010), and Phox-I (1-10 μM), a rationally designed small molecule inhibitor of NOX2 (Chem. Biol. 19, 699-710, 2012), to define the roles of NOX1 and NOX2 in ROS-mediated platelet activation by diverse agonists. A two-minute pre-incubation of washed human platelets with ML171 or Phox-I inhibited CRP, a GPVI agonist, induced ROS generation, ATP secretion and platelet aggregation. In contrast, only platelets treated with Phox-I, but not ML171, inhibited CRP induced binding of PAC-1 to platelets and expression of P-selectin. Treatment of platelets with Phox-I, but not ML-171, inhibited thrombin or U46619, a stable analog of TXA
2
, induced ROS generation, P-selectin expression, ATP secretion and aggregation. Both Phox-I and ML171 inhibited CRP induced phosphorylation of myosin light chain (MLC) and Akt. However, only Phox-I inhibited thrombin induced phosphorylation of MLC or Akt. Treatment of platelets with Phox-I or ML171 only minimally or partially inhibited CRP induced phosphorylation of p38-MAPK or ERK. In other experiments, incubation of platelets with varying concentrations of both ML171 and Phox-I did not exhibit additive effect on CRP induced ROS generation. These results imply that ROS generation by these NOX isoforms may be sequential. These data suggest that NOX1 is specifically involved in GPVI mediated, whereas NOX2 is involved in GPVI- and non-GPVI-dependent, signals in ROS generation and platelet activation.
Abstract 234: Changes in Plasma Nitration of High-Density and Low-Density Lipoproteins in Patients With Chronic Kidney Disease Receiving Kidney Transplants
Authors: Bakillah Ahmed , Tedla Fasika , Ayoub Isabelle , John Devon , Norin Allen , Hussain Mahmood M , Brown Clinton
Published: July 2021

Background:
Functional abnormalities of high-density lipoprotein (HDL) and elevated concentration of low-density lipoprotein (LDL) could contribute to cardiovascular disease (CVD) in chronic kidney disease (CKD) patients. Both qualitative and quantitative changes in HDL have been described in patients with CKD. Specifically, HDL abundance is reduced and HDL acquires a pro-inflammatory properties. In this study, we hypothesized that a functioning kidney transplant reduces serum nitrated apoA-I concentration.

Methods:
Concentrations of nitrated apoA-I, nitrated apoB, total apoA-I and total apoB were measured using indirect sandwich ELISA on sera collected from each transplant subject pre-transplant and at 1, 3, and 12 months post-transplant. Patients were excluded if they had a history of diabetes, prednisone dose > 15 mg/day, nephrotic range proteinuria, serum creatinine (Cr) > 1.5mg/dL or active inflammatory disease, or were treated with lipid-lowering medication or HIV protease inhibitors. Paired values of percent nitrated Apo A-I or nitrated apoB before and after kidney transplantation were compared using Wilcoxon signed rank sum test.

Results:
We screened 75 transplant patients, and 14 were found to meet the selection criteria. Amongst these patients, twelve and eight patients had Cr < 1.5 mg/dL at 3 and 12 months post-transplant, respectively. There was a significant reduction in % nitrated apoA-I at 12 months post-transplant compared to pre-transplant values in patients with Cr<1.5 mg/dL (p=0.04) but neither at 3 months post-transplant nor in patients with Cr >1.5. Reduction of nitrated apoA-I was associated with slight increase in HDL levels 12 months post-transplantation. In contrast to apoA-I, there was no significant change in % nitrated apoB at 3 months and 12 months post-transplant. No significant corelation was observed between nitrated lipoproteins and CRP levels.

Conclusion:
Patients with well functioning grafts had significant reduction in percent nitrated apoA-I without any effect on apoB nitration 12 months after kidney transplantation. Further studies are needed in a large cohort to determine if nitrated apoA-I can be used as a valuable marker for cardiovascular risk stratification in CKD.
Abstract 24: Integrin αIIb Tail Participates in Inside-Out αIIbß3 Activation by Providing a Steric Lever Arm for Talin
Authors: Ye Feng , Li Ang , Guo Qiang , Hu Weiming
Published: July 2021

Increases in ligand binding to integrins (“activation”) play critical roles in platelet and leukocyte function. Integrin activation requires talin and kindlin binding to integrin β cytoplasmic tails. Much research has focused on the conserved GFFKR motif in integrin αIIb tails for its importance in keeping integrins inactive and integrin β cytoplasmic tails and their interacting partners. However, the roles of αIIb tail distal of GFFKR motif are unexplored. Here, we examine the role of αIIb tail distal of GFFKR in talin-mediated inside-out integrin activation, αIIbβ3 outside-in signaling and αIIbβ3-talin interactions. Deletion of amino acid residues after the GFFKR motif in αIIb tail abolished talin-induced inside-out αIIbβ3 activation without affecting αIIbβ3-talin interaction or outside-in αIIbβ3 signaling in modeled cell systems, measured by cell spreading and Src phosphorylation. Addition of non-homologous or non-specific amino acids to the GFFKR motif restored the capacity of talin to activate αIIbβ3 in modeled cells. Moreover, thrombin-stimulated αIIbβ3 activation in megakaryocytic leukemia cells (CMK cells) are similarly abolished by truncation after GFFKR and restored by adding non-specific sequences. Furthermore, Molecular modeling indicates that β3-bound talin sterically clashes with the αIIb tail in the αIIbβ3 complexes, potentially disfavoring the α-β interactions that keep αIIbβ3 inactive. Thus, the αIIb tail sequences distal of GFFKR participate in talin-mediated inside-out αIIbβ3 activation through its steric clashes with β3-bound talin.
Abstract 244: The Flavonoids Naringenin and Nobiletin Stimulate the AMPKinase Pathway in Primary Mouse Hepatocytes
Authors: Samsoondar Joshua P , Bojic Lazar A , Sutherland Brian G , Steinberg Gregory R , Edwards Jane Y , Telford Dawn E , Huff Murray W
Published: July 2021

Dyslipidemia associated with insulin resistance and obesity are core features of the metabolic syndrome and type 2 diabetes, which contribute significantly to atherosclerosis. In mouse models of diet-induced metabolic dysregulation, the citrus flavonoids naringenin and nobiletin prevent obesity, hepatic steatosis, apoB100 overproduction, dyslipidemia, insulin resistance and atherosclerosis. To elucidate the mechanism of action in liver we assessed flavonoid-induced activation of AMP-activated protein kinase (AMPK), the major regulator of cellular energy homeostasis, in primary mouse hepatocytes. Stimulated AMPK activity promotes catabolic, ATP-generating processes such as fatty acid (FA) oxidation while inhibiting anabolic processes such as FA synthesis. In primary C57BL/6 (WT) hepatocytes, naringenin and nobiletin increased phosphorylation (P) of AMPK and its downstream target acetyl-CoA carboxylase (ACC) in a time- and dose-dependent manner. This was associated with decreased apoB100 secretion. Phosphorylation of ACC by AMPK inhibits the formation of malonyl-CoA reducing substrate for FA synthesis in the cytosol while relieving inhibition of mitochondrial FA oxidation by malonyl-CoA. Under insulin resistant conditions stimulated by high glucose media, reduced pAMPK and pACC were reversed by flavonoid treatment in WT hepatocytes, whereas these effects were lost in Ampkβ1-/- hepatocytes. Sterol receptor element binding protein-1c, which stimulates lipogenesis, was also phosphorylated (inhibited) by flavonoid-induced AMPK activation. BAPTA, a calcium chelator or STO609, an inhibitor of Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKβ), did not block flavonoid-induced pACC, suggesting that CaMKKβ is not required for AMPK activation by flavonoids. In chow-fed Ldlr-/- mice, acute i.p. injection of nobiletin following a fasting-refeeding protocol, depressed the respiratory exchange ratio indicative of a switch to FA oxidation. Freeze-clamped liver samples from these mice sacrificed 90 min. post injection showed marked induction of pAMPK and pACC. These results suggest that naringenin and nobiletin attenuate hepatic steatosis and metabolic dysregulation, in part, through activation of hepatic AMPK.
Abstract 248: Hepatocyte Nuclear Factor 4α Regulates Lipoprotein Metabolism and Atherosclerosis
Authors: Xu Yang , Zalzala Munaf , Li Yuanyuan , Zhang Yanqiao
Published: July 2021

Hepatocyte nuclear factor 4alpha (HNF4a) is a nuclear hormone receptor and highly expressed in the liver. Mutations in human HNF4a causes maturity-onset diabetes of the young type 1 (MODY1) and lipid disorder.

We investigated the role of loss of hepatic HNF4a in lipid and lipoprotein metabolism using both adenovirus-mediated gene knockdown (Ad-shHNF4a) and liver-specific HNF4a knockout (L-HNF4a-/-) mice. Our data show that loss of hepatic HNF4a caused hypolipidemia and fatty liver disease. Plasma total cholesterol and triglyceride levels were significantly reduced whereas hepatic triglyceride levels were markedly increased. There effects were caused by impaired VLDL secretion. We further determined the effect of loss of hepatic HNF4a in atherosclerosis using ApoE-/- or Ldlr-/- mice. Our data show that loss of hepatic HNF4a caused a marked reduction in the atherosclerotic lesions when mice were challenged with a Western diet. Our data indicate that hepatic HNF4a plays a critical role in modulating lipid and lipoprotein metabolism and the development of atherosclerosis. Since human HNF4a mutations are found in MODY1 patients, this study may be of clinical importance.
Abstract 251: Investigating the Role of the Hexosamine Biosynthesis Pathway in Diabetic Atherosclerosis
Authors: Beriault Daniel , Werstuck Geoff
Published: July 2021

Background:
Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. We have established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted, cell-specific, response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesize that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways.

Results:
STZ-induced hyperglycemic and glucosamine supplemented apoE-/- mice had significantly larger and more advanced atherosclerotic lesions compared to the control mice. Indications of ER stress were increased in atherosclerotic lesions of hyperglycemic and glucosamine-supplemented mice but not in the controls. In glucosamine-supplemented mice the accelerated atherosclerosis was independent of detectable changes in blood glucose concentration, glucose tolerance, plasma insulin, or plasma lipid levels. Treatment with either valproate or 4-phenylbutyrate, two small molecules that have been shown to block ER stress signaling, attenuated hyperglycemic and glucosamine-induced atherogenesis. Our data suggest that elevated concentrations of glucose/glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation.

Conclusions:
Similar to hyperglycemia, glucosamine-supplementation promotes ER stress and accelerated atherosclerosis in apoE-/- mice. These findings support a model by which conditions of hyperglycemia promote vascular complications via a glucosamine-intermediate.
Abstract 252: Is Vascular Disease a Result of Too Much Vascular Damage? Evidence for an Active Endogenous Plasma Membrane Repair System in Endothelial Cells
Authors: Bernatchez Pascal-Nicolas , Sellers Stephanie
Published: July 2021

Many studies have correlated vascular disease with excessive vascular damage. For instance, vascular endothelial cells (EC) at sites of arterial branching are exposed to turbulent flow, a highly atherogenic factor, whereas chronic hypertension accelerates endothelial dysfunction. At the molecular level, EC are routinely exposed to damaging oxidants, cholesterol and cigarette smoke chemicals. However, how the vascular endothelium ‘repairs’ after ‘damage’ was until recently unknown.

Recently, we have shown that EC express FERLINS ‘repair’ proteins, documented to allow the fusion of lipid ‘patches’ to a damaged plasma membrane. Indeed, Dysferlin and Myoferlin mRNA and proteins were found to be highly expressed in every EC line and human/rodent blood vessel investigated. Proteomics analyses revealed that both Dysferlin and Myoferlin are caveolae/lipid raft resident proteins, well known to be rich in signaling proteins at the heart of laminar flow sensing/ mechanotransduction. In vitro, loss of Dysferlin and Myoferlin resulted in attenuated Platelet Endothelial Cellular Adhesion Molecule-1 (PECAM-1) and Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) expression, respectively, as well as blunted membrane repair following physical injury. In vivo, Dysferlin-null and Myoferlin-null mice show blunted angiogenic response in response to various stimuli. Together, these data clearly show that the vasculature has complex, endogenous physical repair machinery at the plasmalemma, that the lipid ‘patches’ likely contain transmembrane protein cargo, and that Ferlins clearly play a role in vascular homeostasis.

Finally, recent preliminary data from our laboratory suggests that Dysferlin is highly overexpressed in human atherosclerotic sections, and that Dysferlin-null mice are more susceptible to cardiovascular disease that their WT counterparts, indicating a protective role for Ferlin-associated repair in the cardiovascular system.
Abstract 262: Anti-miR33 Therapy Promotes PGC1α Expression, Mitochondrial Function and Immunometabolic Pathways in Macrophages in Atherosclerosis
Authors: Karunakaran Denuja , Thrush A Brianne , Ramphos Eleni , Wu Zongbin , Geoffrion Michele , Esau Christine , Moore Kathryn J , Harper Mary-Ellen , Rayner Katey J
Published: July 2021

Macrophage foam cells are central to atherosclerotic plaque formation and therapies that increase macrophage cholesterol efflux remain an attractive yet elusive target. Therapeutically inhibiting microRNA-33, a critical regulator of lipid metabolism, increases circulating HDL and reduces atherosclerosis. However, the precise molecular mechanisms that regulate these beneficial effects are unknown. Thus, we sought to uncover novel gene networks regulated by miR33 that may contribute to atherosclerosis.

Results:
Microarray analysis of macrophages from anti-miR33 treated Ldlr-/- mice revealed several novel miR33 target genes that were de-repressed. Among these genes was PPARγ coactivator 1α (PGC1α), a crucial gene that controls cellular energy homeostasis and mitochondrial metabolism. 3’UTR luciferase assays verified that PGC1α is a specific and direct target of miR33. Macrophages transfected with miR33 mimics or anti-miR33 had decreased or increased PGC1α mRNA and protein respectively. Bioinformatic pathway analysis predicted that in addition to PGC1α, miR33 also regulates multiple mitochondrial proteins, such as pyruvate dehydrogenase kinase 4 (PDK4), solute carrier 25 A25 (SLC25A25) and Acyl-CoA Dehydrogenase (ACADSB). Anti-miR33 treatment of macrophages increases PDK4, SLC25A25 and ACADSB mRNA expression, validating these genes as miR33 targets. Seahorse XF Flux Analyzer studies show that macrophages treated with anti-miR33 have increased basal and maximal oxygen consumption rates, confirming that anti-miR33 therapy impacts positively on mitochondrial respiration and function. miR33 inhibition increased macrophage cholesterol efflux, which was abolished by oligomycin- a mitochondrial respiration inhibitor. This suggests that miR33 regulates cholesterol efflux at least in part by regulating mitochondrial function. Lastly, western-diet-fed apoE-/- mice treated with anti-miR33 injections (10mg/kg) for 8 weeks had a marked reduction in aortic sinus lesion area relative to control (p≤0.05), despite no change in HDL levels.

Conclusion:
Anti-miR33 therapy can positively impact on mitochondrial function, promoting macrophage cholesterol efflux and may contribute to HDL-independent anti-atherosclerotic effects.
Abstract 271: Thrombospondin-1 Plays a Direct Role in Leptin-Induced Atherosclerosis
Authors: Sahu Soumyadip , Ganguly Rituparna , Ohanyan Vahagn , Chilian William M , Raman Priya
Published: July 2021

Hyperleptinemia, characteristic of diabetes and a hallmark feature of obesity, contributes to the increased risk of atherosclerotic complications. We recently reported that leptin has a direct regulatory effect on the expression of a potent proatherogenic and antiangiogenic matricellular protein thrombospondin-1 (TSP-1)
in vitro
and
in vivo
. While leptin and TSP-1 are independently linked to atherosclerosis, a potential cross-talk
in vivo
remains unknown. The present study is the first evidence that TSP-1 plays a direct role in leptin-induced atherosclerosis. Briefly ApoE
-/-
and TSP-1
-/-
/ApoE
-/-
double knockout (dKO) mice at 17 wks of age, on regular chow diet, were injected i.p. with murine recombinant leptin (5μg/g body weight) or PBS (vehicle control) once daily for 3 wks and mice were sacrificed at 20 wks of age. Using
en face
atherosclerotic lesion assay shown by Oil red O staining, we demonstrated that while leptin
in vivo
significantly increased (2.5-fold) lesion area in the aortic walls of ApoE
-/-
mice, TSP-1
-/-
/ApoE
-/-
dKO mice developed relatively fewer lesions in response to exogenous leptin. Using high-frequency ultrasound imaging we found that in ApoE
-/-
mice leptin significantly decreased diameter of transaortic arch, ascending and descending aorta (p < 0.01), while TSP-1
-/-
/ApoE
-/-
dKO mice were completely protected against this leptin-induced reduction in aortic vessel diameter. Ultrasound imaging of common carotid artery further revealed that leptin-treated TSP-1
-/-
/ApoE
-/-
dKO mice were protected against development of vascular lesions while leptin-treated ApoE
-/-
mice showed more pronounced lesions and a significant reduction in carotid artery internal diameter (p ≤ 0.02). Finally, while leptin
in vivo
significantly increased plasma cholesterol (~40%) and triglyceride (2.5 fold) levels in ApoE
-/-
mice, this leptin-induced increase in plasma lipid levels was attenuated in TSP-1
-/-
/ApoE
-/-
dKO mice. Together, our results suggest that knockout of TSP-1 protects ApoE
-/-
mice against leptin-induced atherosclerosis
in vivo
, underscoring a novel role of TSP-1 in hyperleptinemia-induced macrovascular complications.
Abstract 285: Bisphenol A Increases Atherosclerosis Mediated by the Human Pregnane X Receptor
Authors: Sui Yipeng , Park Se-Hyung , Helsley Robert N , Zhou Changcheng
Published: July 2021

Objective:
Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA has recently been associated with increased risk of cardiovascular disease (CVD) in multiple large-scale human population studies but the underlying mechanisms remain elusive. We previously reported that BPA activates the pregnane X receptor (PXR) which acts as a xenobiotic sensor to regulate xenobiotic metabolism and has pro-atherogenic effects in animal models upon activation. BPA is a potent agonist of human PXR but has no effects on mouse or rat PXR activity, which confounds the use of rodent models to evaluate mechanisms of BPA-mediated CVD risk. This study aims to investigate the atherogenic mechanism of BPA using a novel PXR-humanized mouse model.

Approach and Results:
We generated PXR-humanized ApoE deficient (huPXR•ApoE
-/-
) mice that respond to human PXR ligands and therefore constitute a model to study the atherogenic effects of BPA. Feeding studies were performed to determine the effects of BPA exposure on atherosclerosis development. We found that exposure to BPA increased atherosclerosis in huPXR•ApoE
-/-
mice but not their control littermates. BPA exposure did not affect plasma lipid levels but increased CD36 expression and lipid accumulation in macrophages of huPXR•ApoE
-/-
mice.

Conclusions:
Our findings provide a molecular mechanism linking BPA exposure to increased risk of CVD in exposed individuals. PXR is therefore a relevant target for future risk assessment of BPA and related environmental chemicals in humans.
Abstract 295: The Stem Cell Therapy to Prevent Expansion of Abdominal Aortic Aneurysm (STOP-AAA) Trial: Rationale and Design
Authors: Murphy Michael P ,   The Investigators of the Cardiovascular Cell Therapy Rsch Network
Published: July 2021

Background:
Abdominal aortic aneurysm (AAA) is the 13
th
leading cause of death in the United States. The only options for treatment at present for AAA are open surgical and endovascular repair, both of which are associated with significant morbidity, mortality, and expense.Thus AAA represents an unmet medical need. AAA pathogenesis is a multifactorial inflammatory process characterized by activation of T-cells,macrophages and neutrophils resulting in degradation of the aortic wall. Mesenchymal stromal cells (MSCs) suppress T-cell and macrophage activation, inhibit matrix metalloproteinases and have been shown to decrease aneurysm expansion in murine models.

Methods:
The STOP-AAA trial, from the NHLBI funded Cardiovascular Cell Therapy Research Network, is a randomized, double-blind, placebo-controlled trial evaluating the safety and efficacy of autologous bone marrow derived MSCs in suppressing expansion of small AAA (35-50mm).Forty patients will be randomized in a 1:1 fashion to receive systemic administration of placebo or 3 doses of 2x10
6
MSC/kg. at baseline, 24, and 52 weeks. The primary endpoint will be change in AAA diameter at 18 months as measured by a single blinded observer using contrast enhanced helical computed tomographic angiography (CTA).The secondary endpoints include time to elective repair, incidence of AAA related deaths and rupture,change in aortic mural inflammation at 18 months as quantified by F18-fluorodeoxyglucose (FDG) uptake with integrated positron emission tomography and computed tomography (PET/CT), changes in circulating mRNA, microRNA, and inflammatory cell profiles, changes in serum cytokine levels, and major adverse cardiac events.

Conclusion:
The STOP-AAA will be the first in man study to assess the efficacy of autologous bone marrow derived MSCs to suppress AAA expansion. Furthermore the data collected from this novel trial will provide unique mechanistic insights in the immunomodulatory properties of this stem cell population.
Abstract 309: Vasovasorum Volume Can Be Measured by Contrast-Enhanced 3D Carotid Duplex Ultrasound: Concept and Design of a Novel Method for Predicting Stroke Risk in Patients With Carotid Artery Stenosis
Authors: Arhuidese Isibor J , Nodel Alexander , Qazi Umair , Call Diana , Perler Bruce , Malas Mahmoud B
Published: July 2021

Introduction:
Stroke remains a leading cause of death and disability. The reliance on the occurrence of symptoms and degree of stenosis for selecting patients with carotid stenosis for intervention is not ideal because it is often seen that patients with severe stenosis remain asymptomatic while many patients with moderate stenosis experience stroke. Furthermore, the majority of patients are asymptomatic until they experience stroke. It is known that intimal neovascularization flourishes as atherosclerotic disease progresses; however no technique in current use adequately correlates neovascularization to stroke risk.

Objective:
With seed grant support from the Society for Vascular Surgery Foundation we are executing a study based on our hypothesis that Vasovasorum Volume (VVV) measured using CE-3DCDU as a valid tool for mapping stroke risk.

Method:
We are recruiting symptomatic and asymptomatic patients adjudged to have >50% and >70% stenosis respectively on routine duplex ultrasound. Vasovasorum volume is measured using CE-3DCDU in patients who are eligible for carotid endarterectomy. Plaque removed during surgery is marked, decalcified and immunostained with CD34. Thereafter, VVV is measured in the excised plaque using 3D reconstruction histometry. We then evaluate the reliability and accuracy of CE-3DCDU in relation to the histopathology and compare VVV in symptomatic and asymptomatic patients.

Results:
The preliminary study included six patients and the results show that VVV measurement in carotid ultrasound and histopathology is feasible and reproducible (Figures 1 and 2).

Conclusion:
Vasovasorum volume is a promising predictor of stroke risk. By identifying patients who are truly at high risk for stroke, VVV measured by CE-3DCDU will aid precise patient selection for intervention, thus prevent stroke, save lives, limit disability and expend health care resources in an informed manner. The next phase of this project involves the establishment of efficacy and a population based multi-center clinical trial to generate evidence required to incorporate VVV measured using CE-3DCDU into clinical practice.
Abstract 319: Intermedin Attenuates Vascular Calcification by Upregulating Klotho via CRLR/RAMP3 Receptor Complex and cAMP/PKA Signaling Pathway in Rats With Chronic Kidney Disease
Authors: Chang Jin-Rui , Hou Yue-Long , Lu Wei-Wei , Zhang Jin-Sheng , Yu Yan-Rong , Jia Mo-Zhi , Zhang Jing , Zhu Yi , Tang Chao-Shu , Qi Yong-Fen
Published: July 2021

Vascular calcification (VC) is highly associated with increased morbidity and mortality in patients with advanced chronic kidney disease(CKD). We previously reported that paracrine/autocrine factor intermedin (IMD) could protect against VC. In the present study we assessed the hypothesis that IMD inhibits VC by upregulating klotho protein. VC in CKD rat was induced by 5/6 nephrectomy plus vitamin D
3
administration and vascular smooth muscle cells (VSMCs) calcification was induced by calcifying media containing
β
-glycerophosphate and CaCl
2
. IMD (100 ng kg
-1
h
-1
) was systemically administered by a mini-osmotic pump. CKD rat aortas showed lower IMD content and increased expression of its receptors (calcitonin receptor-like receptor,CRLR/receptor activity-modifying protein 3, RAMP3), along with increased aortic alkaline phosphatase (ALP) activity and calcium deposition. In vivo administration of IMD significantly reduced aortic ALP activity and calcium deposition in CKD rats when compared with vehicle treatment, which was further confirmed in cultured VSMCs. Concurrently, the loss of smooth muscle lineage markers and klotho protein in aortas was rescued by administering IMD to CKD rats with VC. However, the inhibitory effects of IMD on VC were abolished upon pre-treatment with small interfering RNA to reduce klotho. Moreover, the increased effects of IMD on klotho were abolished upon pretreatment with small interfering RNA to reduce its receptors or with PKA inhibitor H89. These results demonstrated that IMD attenuates VC by upregulating klotho via CRLR/RAMP3-cAMP/PKA signaling pathway in rat with CKD. IMD is an important paracrine/autocrine protective factor for VC.
Abstract 324: RAGE Deficiency Contributes to Increased Induction of Angiogenesis in Hindlimb Ischemia in Diabetic Mice by Modulation of Hypoxia- and Inflammatory Dependent Gene Expression
Authors: Xu Yunlu , Shen Xiaoping , Rosario Rosa , Schmidt Ann Marie , Yan Shi-Fang
Published: July 2021

People with diabetes are at higher risk of developing ischemic vascular disease, including peripheral artery disease (PAD). Prior studies highlighted the importance of midkine for induction of angiogenesis associated with vascular repair process in the hindlimb ischemia model and suggested that the hypoxia inducible factor (HIF) pathway may be involved in midkine expression in human polymorphonuclear neutrophils (PMN), monocytes and endothelial cells under hypoxia. Our previous data showed that genetic deletion of receptor for advanced glycation endproducts (RAGE) improved restoration of angiogenesis and blood flow in hindlimb ischemia in diabetic mice. Here, we tested the hypothesis that RAGE deficiency would upregulate expression of certain genes responsible for the coordination of inflammatory response and angiogenesis in hindlimb ischemia in diabetic mice. Wild type (WT) and RAGE
-/-
mice were rendered diabetic (D) with streptozotocin (stz); non-diabetic (ND) cohorts were treated with buffer alone. After two months of diabetes, at age 16 weeks mice were subjected to femoral artery (FA) ligation. RNA was prepared from muscle tissues on day 7 after surgery. Using “Angiogenesis” focused PCR array, gene expression profiling identifies upregulation of inflammation and angiogenesis-related genes impacted by diabetes and RAGE in hindlimb ischemia. In addition to our earlier findings of striking increase in monocyte chemotactic protein-1 (
Mcp-1
/
Ccl2
) and CD68
+
cells in D RAGE
-/-
mice vs D WT mice, there were ≈ 6.5-, 4-, 3.8-, 4.8-, 4.7- and 4.3-fold increases in midkine (
Mdk
),
Hif-1
α
,
interleukin (
IL
)-
1
β
,
Itg
β
3
, chemokine (C-X-C motif) ligand 2 (
Cxcl2
) and matrix metalloproteinases (
Mmp
)
19
transcripts in response to ischemia in D RAGE
-/-
vs. D WT mice. These data link RAGE deficiency to increased expression of the above inflammatory and/or angiogenic genes in ischemia, especially in diabetes. This may provide a basis for further elucidating the molecular mechanisms by which RAGE modulates hypoxia and inflammation-modulating gene expression for induction of angiogenesis in the setting of limb ischemia in diabetes, and may have the potential to provide a novel therapeutic target for diabetic complications.
Abstract 351: Development of an Engineered Base Cerebrovasculature Model to Study Alzheimer’s Disease in vitro
Authors: Robert Jerome , Stukas Sophie , Wellington Cheryl L
Published: July 2021

Alzheimer disease (AD) is the most common cause of cognitive decline in the elderly, and affects more than 40% of people over 85 years of age. In addition to the defining neuropathological hallmarks of neurofibrillary tangles and parenchymal amyloid deposits within brain tissue, over 80% of AD patients also develop cerebrovascular amyloid deposits known as cerebral amyloid angiopathy (CAA). As the presence of cardiovascular and metabolic risk factors including hypertension, type II diabetes and dyslipidemia also increase AD risk, cerebrovascular function is believed to play a critical role in the etiology of AD, likely through regulating the clearance of amyloid-beta (Aβ) peptides from the brain. The cerebrovasculature has two major known mechanisms to facilitate Aβ clearance. First, Aβ interacts with the LDL receptor related protein (LRP) to directly cross the blood brain barrier (BBB). Second, Aβ can be cleared via interstitial fluid drainage pathways that form the equivalent of the lymphatic system in the central nervous system (CNS). However, a critical roadblock to progress in delineating how cerebrovascular function contributes to AD pathogenesis is the lack of model systems that recapitulate the complexity of cerebral blood vessels.

We recently engineered a novel model system using primary human endothelial cells (EC) and smooth muscle cells (SMC) cocultured under pulsatile, native-like flow condition that produces a three dimensional (3D) artery equivalent. Histological analyses showed the formation of a dense tissue composed of a tight monolayer of endothelial cells supported by a basement membrane and multiple smooth muscle cell layers. We will in a close future investigate if the engineered model developed CAA when expose to Aβ.
Abstract 376: Berry Anthocyanins and Their Metabolite Ameliorate High Glucose-Induced Adhesion of Monocytes to Human Aortic Endothelial Cells by Modulating the Cross-Talk Between eNOS and NFκB Signaling
Authors: Pon Velayutham Anandh Babu , Kim Eui Min , Panneerseelan-Bharath Leena , Muthuswamy Vasanthi , Jalili Thunder , Namakkal Soorappan Rajasekaran , Symons J David , Jia Zhenquan
Published: July 2021

Introduction:
In diabetes, hyperglycemia-induced vascular inflammation, resulting in the adhesion of monocytes to vascular endothelial cells and their subsequent migration into the subendothelial space, plays a major role in the development of atherosclerosis. ROS induce the inflammatory events involved in diabetic vascular disease by suppressing endothelial nitric oxide synthase (eNOS) through activation of Inhibitor κB kinase (IKKβ), an activator of nuclear factor-κB (NFκB).

Aim:
To determine the effects of cyanidin-3-glucoside (a major anthocyanin abundant in berry fruits; cyn-3-glu), protocatechuic acid (a major metabolite of anthocyanins; PCA), and bilberry anthocyanins (a mixture of 15 anthocyanins present in bilberry; BBA) on endothelial inflammation induced by hyperglycemia.

Methods:
Human aortic endothelial cells (HAEC) were cultured in normal (5 mM) or high-glucose (25 mM) ± 1 nM - 10 μM cyn-3-glu, PCA or BBA for 72 h. Cell adhesion (assessed via fluorescent probe labeled THP-1, a human monocytic cell line), intracellular ROS (assessed via DCFH-DH), mRNA expression of eNOS, NFκB-p65, and IKKβ (assessed via qPCR), and cell viability (assessed via CellTiter-Blue assay) were quantified in response to the described treatments.

Results:
Relative to results obtained from HAEC exposed to 5 mM glucose, ROS generation (42±4%), monocyte adhesion (35±2%), NFκB-p65 expression (80±13%), and IKKβ expression (50±11%) were elevated (p<0.05), whereas eNOS expression was suppressed (50±2%; p<0.05), in cells challenged with high-glucose (n=3-4). Cell viability was maintained regardless of the treatment. The effects of high-glucose on ROS generation, monocyte adhesion, and NFκB-p65, IKKβ and eNOS expression were ameliorated by concurrent treatment with cyn-3-glu or PCA (100 nM). Further, BBA treatment improved the effects of high glucose on ROS and monocyte adhesion.

Conclusion:
Berry anthocyanins and their metabolite, at concentrations achievable in human plasma by consumption of berry fruits, attenuate glucotoxic vascular inflammation in HAEC by ameliorating NFκB mediated suppression of eNOS signaling. Anthocyanin consumption might be a novel ancillary treatment to prevent vascular complications associated with diabetes.
Abstract 389: Gestational Inflammation and Perinatal Arterial Ischemic Stroke: A Causal Connection?
Authors: Guiraut Clemence , Cauchon Nicole , Lepage Martin , Sebire Guillaume
Published: July 2021

Introduction:
Perinatal arterial ischemic strokes affect about 1/3,000 newborn and are the main cause of hemiplegic cerebral palsy. The large cerebral arteries from the anterior system, namely the intra-cranial carotid bifurcation, are the most affected, ischemic stroke being located in its territory in 85% of cases. The classic, but unproven, pathophysiological hypothesis postulated that arterial occlusion was caused by emboli from placental origin. This remains controversial due to the major unbalance of brain infarcts between anterior and posterior distribution, and to the absence of associated extra-cerebral infarcts. A new pathophysiological perspective emerged from the epidemiological association between gestational inflammation and perinatal stroke. Our hypothesis is that materno-foetal inflammation, induced by gestational exposure to pathogens, leads to a site-specific vasculitis affecting the carotid bifurcation and then triggering a focal thrombosis.

Material and methods:
Dams were injected with saline or lipopolysaccharide (LPS) from Escherichia coli (200 μg/kg/12h) between gestational day (G) 21 and 22. Brains were harvested at G21, G22 and postnatal day 1 (P1). At P1, a prothrombotic stress (transcutaneous photothrombosis) was applied on middle cerebral arteries to compare its susceptibility to thrombosis between LPS-exposed or unexposed pups. Immunohistochemistry and ELISA detected maternal, placental and fetal/neonatal inflammatory markers.

Results:
Our results showed a maternal, placental and fetal inflammation mediated by IL-1β, TNF-α and MCP-1 as well as an arterial inflammation in relation with the clinical pattern of perinatal arterial ischemic strokes. LPS+photothrombosis pups presented ischemic strokes and motor impairments, which were not detected when photothrombosis was applied without prior treatment with LPS.

Conclusion:
Preliminary results from our new pre-clinical model support our hypothesis of increased susceptibility of anterior cerebral arteries to gestational inflammation, and open a new vasculitic pathophysiological avenue to understand perinatal stroke.