bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 417: Endosome-Based Activation of Non-Canonical NF-kB Signaling by Complement Membrane Attack Complexes
Authors: Jane-wit Daniel , Surovtseva Yulia , Qin Lingfeng , Liu Rebecca , Clark Pamela , Manes Thomas , Wang Chen , Kashgarian Michael , Kirkiles-Smith Nancy , Tellides George , Pober Jordan
Published: March 2022

Using sera from transplant patients with high-titer panel reactive antibody (PRA), we previously described a model for studying the effects of alloantibody binding and complement activation on human endothelial cells (EC). With this model we found that complement membrane attack complexes (MAC) induced rapid post-translational stabilization of NF-kB-inducing kinase (NIK), the activator of non-canonical NF-kB signaling, which was required for inflammatory changes in cultured human EC and for vasculopathic changes in human coronary artery segments implanted into immunodeficient mouse hosts. To elucidate the mechanism of NIK stabilization, we performed a genome-wide siRNA screen and discovered a novel, TRAF3-independent signaling mechanism in which MAC induces NIK stabilization on endosomes. MAC is internalized by EC in a clathrin-, AP2-, and dynamin-dependent mechanism into Rab5+ endosomes. Activated Akt is recruited in a Rab5-dependent manner to the same endosomes which then bind and stabilize NIK and subsequently recruit and activate IKK-alpha, the catalytic activator of p52/RelB complexes, the non-canonical form of NF-kB. Pharmacological inhibition of MAC endocytosis blocked this pathway both in cultured EC and in human coronary artery xenografts. These data reveal a novel endosome-based signaling cascade for activating non-canonical NF-kB and identify new targets for inhibiting complement-mediated tissue injury such as chronic antibody-mediated rejection and cardiac allograft vasculopathy.
Abstract 422: Metabolic Regulation by miR-33 in Macrophages Controls Immune Effector Responses
Authors: Ouimet Mireille , Ediriweera Hasini , Gundra Uma Mahesh , Rayner Katey , Ramkhelawon Bhama , Rinehold Kaitlyn , van Solingen Coen , Hutchison Susan , Esau Christine , Fullerton Morgan , Steinberg Gregory , Fisher Edward , Loke P’ng , Moore Kathryn
Published: March 2022

Cellular metabolism is increasingly recognized to control immune cell fate and functions. MicroRNA-33 (miR-33) is a central regulator of cellular lipid metabolism that represses genes involved in cholesterol efflux and HDL biogenesis (Abca1, Abcg1) and fatty acid oxidation (Cpt1a, Crot, Ampk). Here we show that by altering the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation, miR-33 inhibition instructs macrophage polarization to an M2 phenotype and shapes innate and adaptive immune responses. Targeted deletion of miR-33 in macrophages increases oxidative phosphorylation, enhances spare respiratory capacity, and induces the expression of genes that define M2 macrophage polarization (Arg1, Fizz1, Cd206, Ym1). Furthermore, inhibition of miR-33 in Abca1-/- macrophages showed that these changes are independent of effects on cholesterol efflux, but instead require miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK). Notably, inhibition of miR-33 markedly increased macrophage expression of the retinoic acid-producing enzyme Aldh1a2 and retinal dehydrogenase activity both in vitro and in vivo. Consistent with the ability of retinoic acid to foster inducible regulatory T cells, these macrophages had an enhanced capacity to induce FoxP3 expression in naïve CD4+ T cells. Finally, treatment of western diet-fed Ldlr-/- mice with miR-33 inhibitors for 8 weeks (conditions that do not alter HDL cholesterol levels) reduced atherosclerosis progression by 40%, and promoted the accumulation of M2 macrophages and FoxP3+ T regulatory cells in plaques. Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and regulatory T cell induction.
Abstract 424: Hyper-Inflammatory Macrophages in Coronary Artery Disease and Rheumatoid Arthritis; A Signature of CCL18, Krüppel-like Factor 2 and 4 and Oxidative Stress Response Genes
Authors: Shirai Tsuyoshi , Schaefgen Benedikt , Nazarewicz Rafal R , Wallis Barbara B , Assimes Themistocles L , Harrison David G , Goronzy Jörg J , Weyand Cornelia M
Published: March 2022

Background:
Autoimmune disease is an independent risk factor for accelerated coronary artery disease (CAD). Patients with rheumatoid arthritis (RA) have a 2-fold increased risk for CAD, comparable to the impact of diabetes mellitus. Abnormalities in the immune system contribute to both RA and CAD pathogenesis and defining these might elucidate disease mechanisms. We studied inflammatory macrophages in RA and CAD to identify shared and disease-specific molecular pathways.

Methods:
Patients with CAD and a history of myocardial infarction, patients with RA who satisfied the 2010 RA Classification Criteria and age-matched controls were enrolled. Monocytes isolated from peripheral blood were differentiated into macrophages and further polarized into M1 or M2 with IFNγ and lipopolysaccharide or IL-4 and IL-13, respectively. The expression of 55 genes was measured by quantitative PCR. Intracellular reactive oxygen species (ROS) were quantified using CellROX and the oxidant superoxide was scavenged using the SOD mimetic Tempol.

Results:
Both CAD and RA macrophages exhibited a gene expression signature of excess inflammatory activity when compared to controls. Besides the upregulation of cytokine genes, the hyper-inflammatory signature included the loss of the inflammatory suppressors Krüppel-like factor (KLF)-2 and KLF-4, and the gain of the chemokine CCL18 and the oxidative stress response gene, NAD(P)H:quinone oxidoreductase 1 (NQO1). Quantification of intracellular ROS confirmed increased oxidative stress in the patient-derived macrophages. To examine whether superoxide affects production of CCL18, cells were treated with Tempol. ROS depletion normalized the excess production of CCL18.

Conclusion:
Pro-inflammatory macrophages in RA and CAD share molecular abnormalities characterized by excessive cytokines and chemokine production, the loss of negative regulators and the gain of oxidative stress responses. Unbalanced ROS production is upstream of abnormal chemokine production and correcting redox balance in macrophages may be helpful in suppressing the hyper-inflammatory phenotype of these cells in CAD and in the CAD-prone autoimmune disease RA.
Abstract 438: Regulation of Ischemia-induced Neovascularization by 20-HETE Involves Bone Marrow-derived EPC.
Authors: Chen Li , Zhang Frank F , Gotlinger Katherine H , Falck John R , Schwartzman Michal L , Guo Austin M
Published: March 2022

Objective:
Compensatory neovascularization is an important adaptation for recovery from critical ischemia. The CYP4A-derived 20-HETE were recently identified as a novel regulator of angiogenesis. Here, we assessed the contribution of 20-HETE to ischemia-induced neovascularization and its underlying molecular and cellular mechanisms.

Methods:
The mouse ischemia hindlimb angiogenesis assay was established to study the role of CYP4A-20-HETE in compensatory angiogenesis using the 20-HETE synthase inhibitor (DDMS, 10mg/kg/d) and the 20-HETE antagonist (6, 15-20-HEDGE, 10mg/kg/d). Hindlimb blood flow and microvessels density in ischemic hindlimb muscle were measured by Laser Doppler Perfusion Imaging and Immunofluorescent staining, respectively. 20-HETE production in ischemic muscles was measured by LC-MS-MS. Western blot was performed to explore the underlying signaling pathways. Flow cytometry analysis was used to assess the contribution of 20-HETE to mobilization and homing of bone marrow (BM)-derived endothelial progenitor cells (EPC) to ischemic neovascularization.

Results:
20-HETE inhibition by DDMS and 6, 15-20-HEDGE reduced the blood flow perfusion and microvessel formation in response to ischemia by 46±2.3% and 34±1.2%, respectively. Importantly, ischemic hindlimb muscles showed markedly elevated 20-HETE synthesis compared to non-ischemic controls (96 ± 17 pg/mg protein
vs.
non-detectable). HIF-1α, VEGF, and VEGFR2 expression were markedly induced in ischemic hindlimb muscles, but were negated by DDMS and 6, 15-20-HEDGE. Furthermore, ischemia induced BM-derived EPC mobilization and subsequent EPC homing to the ischemic hindlimb muscle by 5.5±0.7 and 20±2.8 folds, respectively, whereas DDMS and 6, 15-20-HEDGE significantly attenuated these processes.

Conclusions:
An increase in 20-HETE production in ischemic muscles regulates ischemic compensatory neovascularization
via
the induction of the HIF-1α/VEGF pathways that support the mobilization and homing of BM-derived EPC. These results strengthen the notion that 20-HETE is a key regulator of ischemic compensatory angiogenesis via regulation of EPC function and may represent a novel therapeutic target.
Abstract 460: Three Dimensional Quantification of Angiotensin II-Induced Abdominal Aortic Aneurysms Using High Frequency Ultrasound
Authors: Adelsperger Amelia R , Yrineo Alexa , Phillips Evan , Damen Fredrick , Blaize A N , Babbey Clifford , Murphey Michael , Goergen Craig
Published: March 2022

Abdominal aortic aneurysms (AAAs) are localized pathological vessel dilations and are the 13th leading cause of death in the U.S. While AAA expansion in patients is traditionally assessed with 2D ultrasound by measuring transverse diameters, recent advancements in ultrasound techniques now make it feasible to quantify AAA volumes noninvasively. High frequency ultrasound (typically up to 40 MHz) is ideal for rodent imaging when investigating experimental aneurysms. The purpose of this study was to use 3D ultrasound to measure aneurysm progression in the apolipoprotein-E-deficient (apoE-/-)/angiotensin II (AngII) mouse model that create suprarenal dissecting aneurysms. High frequency ultrasound (40 MHz, Vevo2100, VisualSonics) was used to collect images from C57BL/6 apoE-/- mice infused with AngII from subcutaneously implanted osmotic mini pumps (1000 ng/kg/min) for 28 days (n=6). We could clearly visualize, segment, and quantify 3D volumes of AAAs and visualize the dissection between medial and adventitial layers. Total AAA volume increased steadily at an average rate of 0.51±0.32 mm3/day (mean±SE) between days 3 and 14 after aneurysm formation (Figure 1). This is similar to the increase in false lumen expansion (0.42±0.30 mm3/day), but higher than the true lumen expansion that remained relatively constant over 14 days (0.09±0.06 mm3/day). These data suggest that 3D ultrasound is a technically feasible measure of aortic volume and has utility in evaluating the efficacy of therapeutic agents in animal models.
Abstract 465: Inducible Depletion of Calpain-2 Attenuates Angiotensin II-induced Abdominal Aortic Aneurysms in Male LDL Receptor Deficient Mice
Authors: Balakrishnan Anju , Howatt Deborah A. , Moorleghen Jessica J , Uchida Haruhito A , Takano Jiro , Saido Takaomi C , Subramanian Venkateswaran
Published: March 2022

Background and Objective:
Chronic infusion of angiotensin II (AngII) induces abdominal aortic aneurysms (AAAs) in normal and hypercholesterolemic mice. Recently, we demonstrated that pharmacological inhibition of calpain-1 and -2 (a class of calcium-activated, neutral cysteine proteases) attenuated AngII-induced AAAs in mice. In addition, calpain-2 compensates for the loss of calpain-1 and promoted AngII-induced AAA formation in calpain-1 deficient mice. The purpose of this study was to determine the relative contribution of calpain-2 to AngII-induced AAA development. To perform this study, calpain-2 floxed mice were bred to express an inducible form of Cre under the control of the ubiquitous promoter, chicken α-actin (Cre-ERT2).

Methods and Results:
Calpain-2 floxed mice that were hemizygous for Cre-ERT2 in an LDLr -/- background were produced by breeding male LDLr -/- x Cre-ERT2 to female LDLr -/- x calpain-2 floxed mice. At 8 weeks of age, male non-Cre littermates (Cre-) and Calp-2 x Cre-ERT2 (Cre+) mice were injected with tamoxifen (25 mg/kg, i.p.) daily for 5 consecutive days. After 2 weeks, Western blot analyses showed a complete depletion of calpain-2 protein in the aorta from Cre+ mice compared to non-Cre littermates. Male Cre- (N=23) and Cre+ (N=21) mice were fed a saturated fat-enriched diet (21% wt/wt fat; 0.15% wt/wt cholesterol) for 5 weeks. After 1 week of diet feeding, mice were infused with AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. AngII increases in systolic blood pressures were not influenced by calpain-2 depletion (post-infusion Cre-: 187 ± 5; Cre+-: 190 ± 3 mm Hg). Depletion of calpain-2 had no effect on plasma cholesterol concentrations (Cre-: 1597 ± 43; Cre+: 1541 ± 63 mg/dL) during AngII infusion. Interestingly, depletion of calpain-2 significantly attenuated AngII-induced aortic luminal dilation (Cre-: 1.70 ± 0.08; Cre+: 1.0 ± 0.03 mm; P<0.001), and expansion of ex-vivo maximal diameters of abdominal aortas in mice (Cre-: 1.90 ± 0.13; Cre+: 1.1 ± 0.07 mm; P<0.001). In addition, calpain-2 depletion significantly reduced the incidence of AngII-induced AAAs (Cre-: 91%, Cre+: 30%; P< 0.001).

Conclusion:
These findings suggest that calpain-2 plays a critical role in the development of AngII-induced AAA formation in mice.
Abstract 474: Effects of Race on Endothelial Function in Type 2 Diabetes
Authors: Brar Vijaywant , Berdy Andrew E , Panza Julio A , Campia Umberto
Published: March 2022

Background:
Patients with type 2 diabetes mellitus (DM2) have abnormal endothelial function. Healthy African Americans (AA) appear to have impaired nitric oxide-mediated vascular responses compared to Caucasians Americans (CA). Whether race-specific differences in endothelial function exist in patients with DM2 has not been investigated. We tested the hypothesis that in patients with DM2 endothelium-dependent dilation is lower in AA compared to CA.

Methods:
Retrospective analysis of data collected in a study that investigated vascular function in patients with DM2. Forearm blood flow (FBF) responses were measured by strain-gauge plethysmography during the intra-arterial infusion of acetylcholine (ACh), sodium nitroprusside (SNP), and the endothelin type A receptor blocker BQ-123 in AA and CA patients with DM2.

Results:
34 patients took part in the study. One patient was excluded from the analyses because of incomplete data set. Their baseline characteristics are reported in the table. Infusion of ACh, SNP, and BQ-123 induced a significant increase in FBF from baseline (p<0.01 for all) in both AA and CA. When compared between groups, the response to ACh was significantly lower in AA (p = 0.022 by 2-way ANOVA). However, no differences were noted in the responses to SNP and BQ-123 between AA and CA (p=0.699 and p=0.118, respectively, by 2-way ANOVA).

Conclusions:
African American patients with DM2 have lower endothelial reactivity to ACh compared with CA. Our findings suggest that the detrimental vascular effects of type 2 diabetes on vascular reactivity are additive to the already impaired endothelium-dependent vasodilation observed in healthy AA.
Abstract 493: Syndesomes: An Syndecan-4 Based Therapeutic for Effective Revascularization in Peripheral Ischemia in Diabetes
Authors: Das Subhamoy , Singh Gunjan , Monteforte Anthony J , Martinez Matthew E , Wright Catherine S , Martin Patricia E , Dunn Andrew K , Baker Aaron B
Published: March 2022

Previous research has attempted to use growth factor proteins and genes to revascularize ischemic tissues but none of the methods have found complete success in clinical trials. We have demonstrated the loss of co-receptors like syndecan-4 due to long-term diseased state and enhanced neovascularization of ischemic tissue due to co-delivery of syndesomes (syndecan-4 proteoliposomes) with FGF-2 in normal healthy rats. In this study, we used syndesomes to enhance revascularization in the ischemic hind limb of a growth factor resistant mouse model with diabetes, obesity and hyperlipidemia.

We used ob/ob mice fed with high fat diet for 10 weeks. We induced ischemia through femoral artery ligation and implanted alginate beads containing treatments into the surgical site. The recovery of perfusion in the mice was tracked using laser speckle imaging.

After 14 days, the ischemic limbs of the mice treated with FGF-2 with syndesomes had significantly higher perfusion compared to the FGF-2 alone group (A, B). Morphometric analysis on sections from the calf and thigh muscle of the mice demonstrated increased ischemic changes in the ischemic limbs of the FGF-2 alone group. Immunostaining for von Willebrand factor revealed increased neovascularization corresponding with the increase perfusion measured in the FGF-2 with syndesomes group (C, D). Immunostaining for pro-inflammatory M1 macrophage marker CD86 showed similar levels in both treatment groups (E, F). However, immunostaining for pro-wound healing M2 macrophage marker revealed significantly higher levels in the syndesome with FGF-2 treatment group (G, H).

Taken together, these preliminary studies support that co-delivery of syndecan-4 with FGF-2 significantly enhances revascularization in the ischemic hind limb in a clinically relevant diseased mouse model. Our treatment restores the signaling pathway components that are lost due to diseased state causing tissues to become resistant to growth factor therapies.
Abstract 538: A PCSK9 Antibody that Blocks Binding to LDLR while Allowing Normal PCSK9 Inactivation by Furin is Afforded a Reduced Clearance Rate and a Longer Duration of Effect in Mice
Authors: Beyer Thomas P , Eacho Patrick I , Schroeder Krista M , Hansen Ryan J , Wroblewski Victor J , Han Bomie , Pickard Richard T , Kowala Mark C
Published: March 2022

Introduction:
Monoclonal antibodies (Mabs) that neutralize proprotein convertase subtilisin-kexin type 9 (PCSK9) have been shown to lower LDL-C in human trials. It is known that PCSK9 is cleaved by furin at Arg218 and that the cleaved PCSK9 is inactive in modulating LDLR. Antibodies whose epitopes are near the EGFA binding domain are more efficacious in increasing LDLR but may interfere with furin cleavage and thus normal PCSK9 clearance. Here we describe a unique antibody (LY) that both lowers LDL-C and allows for normal PCSK9 cleavage by furin.

Hypothesis:
An antibody with an epitope that permits furin cleavage of PCSK9 will show more durable LDL lowering compared to one with an epitope that interferes with cleavage.

Methods:
Furin cleavage of recombinant PCSK9 was evaluated by electrophoresis in the presence of antibodies. Antibodies were studied in mice expressing wild-type or a non-cleavable variant of human PCSK9.

Results:
We determined that LY3015014 (LY) permits furin cleavage of PCSK9 while Mab A, which shares the epitope of a Mab tested in humans, does not. LY’s epitope is completely N-terminal of the cleavage site, while the conformational epitope of Mab A spans the Arg218 cleavage site. To assess the impact of PCSK9 cleavage on antibody efficacy in vivo, we generated two human PCSK9 overexpressing mouse models; one with WT PCSK9 and one with a non-cleavable variant (R215A, R218A). In mice expressing WT human PCSK9, LY showed a longer duration of LDL lowering and a slower clearance rate than Mab A. Additionally, we found that PCSK9 did not accumulate in the serum of mice given LY, likely due to cleavage and release from the antibody. In mice expressing the non-cleavable variant PCSK9, the clearance and LDL durability benefits of LY compared to Mab A were lost. Additionally, in the non-cleavable model, PCSK9 accumulated in the serum of mice given LY, presumably due to lack of furin cleavage.

Conclusions:
LY allows normal cleavage and inactivation of bound PCSK9 while maintaining LDL-C lowering. This proteolysis reduces the antibody’s clearance rate and extends the therapeutic durability of LY in mice relative to Mab A which interferes with proteolytic degradation. We have shown that cleavage is the mechanism of durability through the use of non-cleavable PCSK9.
Abstract 544: A Novel Peroxisome Proliferator Response Element Modulates LDL-receptor Transcription in Response to PPAR Agonists.
Authors: Shende Vikram R , Liu Jingwen
Published: March 2022

Hepatic LDL receptor (LDLR) binds to and internalizes circulating LDL-cholesterol thereby facilitating its clearance from the serum. LDLR is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein (SREBP1/2). Additionally, SRE-dependent modulation of LDLR by peroxisome proliferator-activated receptor gamma (PPARγ) has also been previously described. However whether additional promoter elements directly regulate LDLR transcription is not very well understood. We observed that treatment with the PPARδ agonist L165041 elevated luciferase-reported activity of human LDLR promoter as well as the mRNA and protein expression of LDLR in both, human and mouse cell lines. The induction of LDLR mRNA in HepG2 cells was dose-dependent with maximal expression observed at 20uM. We also observed that the half-life of LDLR mRNA was similar in control and L165041 treated samples, suggesting that the induction was a result of transactivation of mRNA synthesis. Combinatorial treatment of HepG2 cells with L165041 and rosuvastain (RSV) demonstrated additive effects of the two compounds on LDLR mRNA as well as protein expression. Transcription factor prediction programs indicated presence of a single PPAR-response element (PPRE)-like sequence motif in the human LDLR promoter, located at -768 to -752 bases upstream of the translation start site. Luciferase reporter analyses indicated that mutation or deletion of this PPRE-like element resulted in attenuation of L165041 mediated upregulation of LDLR promoter luciferase activity. Electrophoretic mobility shift assays further confirmed the binding of PPARδ/RXR heterodimers to the LDLR-PPRE site. Taken together, our studies identify a novel PPRE in the proximal promoter of LDLR and suggest that combinatorial treatment of statin with PPAR agonist may have advantageous effects on LDLR expression.
Abstract 548: Association Between Serum Omentin-1 Level and Coronary Artery Disease: A Meta-Analysis
Authors: Agasthi Pradyumna , Aloor Sivakanth , Axiyan Maihemuti , Onwuanyi Anekwe
Published: March 2022

Background:
In recent years, visceral adipose tissues and adipokines have been implicated to play a crucial role in the pathogenesis of atherosclerosis and coronary artery disease (CAD). Omentin-1(OM-1) is a novel adipokine shown to be associated with chronic inflammatory diseases, insulin resistance, obesity and carotid atherosclerosis. Recent studies have shown OM-1 as a modulator of vascular function via endothelium-dependent vasodilation. OM-1 plays a role in vascular inflammation as it suppresses cytokine-stimulated expression of adhesion molecules in endothelial cells. The association of CAD and OM-1 remains unclear. The aim of the present study is to conduct a meta-analysis to determine the relationship between serum OM-1 levels and CAD.

Methods:
We searched MEDLINE, CIINHAL and COCHRANE databases for studies reporting serum OM-1 levels in the CAD and non-CAD study population. We included case controls, cohort and cross-sectional studies. We calculated the weighted standardized mean difference (SMD) in serum OM-1 levels between the CAD and control groups.

Results:
Our search strategy yielded 31 articles and we included 5 case control and 1 cross-sectional study enrolling 844 participants. The median age of the CAD group was 64yrs (IQR 62.3 - 65.76) compared to 61.93yrs (IQR 60.18 - 62.97) in the control group. The median percentage of female population in the CAD group was 39.25(IQR 29.13 - 53.7) compared to 43.5(IQR 26.92 - 43.5) in the control group. The unweighted median serum OM-1 levels in the CAD group were 103.6ng/ml (IQR 39.2 - 110.91) compared to 130.9ng/ml (IQR 62.6 - 370.65) in the control group. The SMD of OM-1 level was -2.27 (95% CI -3.44, -1.09) p<0.001 comparing those in the CAD group and control group.

Conclusion:
Serum OM-1 level is independently and negatively associated with CAD. Further studies are needed to define the role of OM-1 in the pathogenesis of CAD and its potential to serve as a novel biomarker.
Abstract 550: Regulation of Macrophage Functions by a Synthetic ROR Ligand
Authors: Billon Cyrielle , Burris Thomas
Published: March 2022

In the United States, approximately 16 million people suffer from coronary heart disease (CHD) and its various complications. Despite significant research effort and advances in the field of cardiovascular diseases, atherosclerosis remains the leading cause of mortality. Atherosclerosis is a multi-step inflammatory disease characterized at the early steps by accumulation of cholesterol in the arterial wall followed by recruitment of immune cells (macrophages and lymphocytes).

Previous studies suggest that a member of the nuclear receptor family, retinoic-acid-receptor-related orphan receptors alpha (RORα), may play an important role in the development of atherosclerosis but mechanisms are still unclear. Recent work from our lab shows that ROR is involved in inflammatory process. We hypothesize that synthetic ROR ligands can be used as a tool to investigate the role of ROR in the regulation of inflammation and cholesterol handling and could have a therapeutic potential in the treatment of atherosclerosis.

We developed a selective RORα/γ inverse agonist, SR1001, that blocks ROR activity both in vivo and in vitro. To identify the role of ROR in macrophage function, we used bone marrow derived macrophages from WT mice treated with SR1001 or DMSO control. Using an in vitro model of macrophages polarization and bio-molecular techniques, we monitor the effect of ROR in M1/M2 polarization of macrophages and NLRP3-inflammasome activity.

Our results show that the ROR ligand blocks the macrophages M2 polarization and increases M1 function such as cytokines production and secretion. The ROR antagonist induces inflammation by stimulating the caspase-1-activating NLRP3 inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Decreasing M2 polarization and induction of IL-1β is associated with increased atherosclerosis. Thus, Our data suggests that ROR could play a critical role in atherosclerosis development.

In conclusion, ROR could be a good target to fight inflammatory diseases such as atherosclerosis by targeting macrophages.
Abstract 562: Evidence of Protozoan Biofilm Communities in Atheromatous Debris
Authors: Heuser Richard , Fry Stephen E , Ellis Jeremy , Schwartz Renata , Martinez Delyn L , Shabilla Matthew A , Henonburg Avory M , Schwartz Boris
Published: March 2022

Background:
Evidence that vascular inflammation is an important mechanism involved in all stages of atherogenesis continues to accumulate. We hypothesize that common complex microbial involvement may be present in atheromatous debris removed from treated lesions. We evaluated this debris with metagenomic analysis.

Methods:
After informed consent, fifteen cases of vascular aspirate or explanted embolic filters were examined. Thirteen cases were in patients undergoing carotid stenting (3 MoMa, 10 embolic filters). One case was in a saphenous vein graft intervention (aspirate and filter) and one case was after SFA orbital atherectomy (aspirate and filter). Fluorescence microscopy, bacterial and protozoan metagenomic analysis using the RIDI™ Next Generation Sequencing (NGS) analysis system, and specific protozoan multiplex PCR probes were used to assess the presence and composition of biofilm populations.

Results:
Bacteria were not detected in peripheral blood; however, 4 of 12 filters and 2 of 5 atheroma debris samples had identified bacterial populations (2 patients had atheroma debris and filter evaluated). Evidence of protozoan populations was obtained in 4 of 15 peripheral blood samples, 11 of 12 filters and 4 of 5 atheroma debris samples. Microscopy illustrated a complex composition of biofilm communities in blood, devices, and atheroma debris samples. The identified bacterial taxa in atheroma debris suggest a diverse and novel population composition. Biofilm dwelling bacteria, while present in several atheroma or filter samples, were not detectable in peripheral blood and were not universally present in atheroma or filter. Taxonomic comparisons of sequenced protozoa are consistent with a diverse array of organisms similar to poorly characterized environmental protozoa.

Conclusion:
Of 15 patients, 6 patients had evidence of bacteria and 13 had evidence of protozoa in debris and 14 exhibited evidence of complex biofilm communities. This data suggests that biofilm forming protozoa may play a key role in arterial vascular disease.
Abstract 578: Spotty Calcification as a Marker of Vulnerable Plaque: Novel Findings From
in vivo
Study in Survivors of Cardiac Arrest and
in vitro
Study in Autopsied Patients of Sudden Cardiac Death
Authors: Pu Jun , Zhang Pei , Mintz Gary S , He Ben
Published: March 2022

Backgrounds:
We previously reported that spotty calcification detected by intravascular ultrasound (IVUS) was often associated with histological fibroatheromas (JACC, 2014, 63:2220-2233). However, the exact mechanisms underlying spotty calcification and the associated risk for ischemic events remain poorly understood. In the present study, we performed a multimodality imaging study on spotty calcification using IVUS, near-infrared spectroscopy (NIRS) and optical coherent tomography (OCT) in survivors of cardiac arrest and autopsied patients of sudden cardiac death (SCD).

Methods and Results:
IVUS, NIRS and OCT were performed in 62 vessels from patients who had a documented sudden cardiac arrest but successfully resuscitated and 52 vessels from autopsied patients of SCD. Spotty calcification was detected in 88.5% survivors of cardiac arrest; 78.3% of them had spotty calcium in superficial location. About 83.3% superficial spotty calcium co-existed with echo-attenuated plaques on IVUS, 73.9% co-existed with thin-cap fibroatheroma (TCFA) or plaque rupture on OCT, and 88.8% of contained lipid core plaque on NIRS (Figure 1). The arc of spotty calcium was negatively correlated with lipid core burden index on NIRS (Spearman ρ=-0.40,
P
=0.03), and positively correlated with cap thickness on OCT (Spearman ρ=0.41,
P
=0.01). In
in vitro
study, IVUS and OCT detected spotty calcification in 80.6% SCD patients. On pathological analyses, the arc of spotty calcium was negatively correlated with 1) inflammation of fibrous cap, 2) levels of oxidative/nitrative stress, and 3) apoptosis and necrotic core size. Compared with spotty calcium in deep location, that in superficial location was associated with larger size of necrotic core (
P
=0.02) and more TCFA (
P
<0.001) and plaque rupture (
P
=0.006).

Conclusions:
Spotty calcification, especially when superficial in location, is a marker of vulnerable plaque in survivors of cardiac arrest and autopsied patients of SCD.
Abstract 579: Lack of Interleukin-19 Exacerbates Experimental Atherosclerosis
Authors: Ray Mitali , Richards James , Gabunia Khatuna , Kelemen Sheri , Autieri Michael
Published: March 2022

Interleukin-19 (IL-19) is a Th2 interleukin and a member of an IL-10 subfamily. Previous studies from our laboratory suggest an anti-inflammatory role for IL-19 outside the immune system, and for the vasculature in particular. Significantly more IL-19 is expressed in human atherosclerotic plaque from symptomatic rather than asymptomatic patients. However, addition of rIL-19 to LDLR-/- mice could significantly reduce atherosclerosis, suggesting that IL-19 expression is a compensatory, counter-regulatory mechanism. The purpose of this study was to test the hypothesis that absence of IL-19 results in increased atherosclerosis, and to characterize potential mechanisms for these effects. LDLR-/- mice were crossed with IL-19-/- mice, and homozygous double knock outs (DKO) were fed a high fat atherogenic diet for 14 weeks and compared with LDLR-/- mice. En face oil red O staining demonstrated that loss of IL-19 significantly exacerbated development of plaque in the aortic arch (13.41%+/-1.04 vs 21.13) +/-1.18, P<0.0001 for LDLR and DKO). Preliminary experiments were performed in which DKO were injected with 10ng/g/day rIL-19 or saline as control, and plaque quantitated en face staining. DKO mice rescued with rIL-19 had less plaque compared with saline controls (15.85+/-2.09% vs 22.43+/-2.80%, P=0.07). There was no significant difference in weight gain or serum cholesterol levels between these mice. As a first step to identify mechanisms, differences in cholesterol uptake in macrophage and VSMC were examined. Cholesterol uptake in BMDM from IL-19-/- mice were significantly decreased compared to wild-type controls (P<0.001). Conversely, cholesterol uptake in VSMC from IL-19-/- mice was significantly increased compared to wild-type (P<0.001). These data suggest that IL-19 plays an important part in development of atherosclerosis, with one potential mechanism being alterations in cholesterol homeostasis.
Abstract 58: Differential Signaling by CRP-Rac1-NOX1 and Thrombin-Rac1-NOX2 Axes Regulates Ros Generation and Platelet Activation
Authors: Akbar Huzoor , Duan Xin , Saleem Saima , Davis Ashley K , Zheng Yi
Published: March 2022

Agonist induced generation of reactive oxygen species (ROS) enhances platelet aggregation and hence the risk of thrombosis. Rac1 GTPase has been shown to be critical in ROS generation by both NOX1 and NOX2. However, its role in agonist induced ROS generation in platelets remains to be determined. We have shown previously that gene targeting or inhibition of Rac1 GTPase inhibits platelet secretion and aggregation. In this study we tested our hypothesis that Rac1 regulates platelet activation, at least in part, by regulating ROS generation through the NOX enzymes by using Rac1 and NOX2 gene targeted mice and/or pharmacologic inhibitors. ROS generation was determined by flow cytometry in dcf-da loaded washed platelets. Collagen-related peptide (CRP) or thrombin induced ROS generation in a time- and a concentration-dependent manner. A two minute pre-incubation of platelets with NSC23766 (3-30 μM), a Rac GTPases inhibitor, or Phox-I (1-10 μM), an inhibitor of NOX2 that blocks Rac1-p67
phox
interaction, diminished CRP or thrombin induced ROS generation, ATP secretion and aggregation. However, treatment of platelets with NSC23766 or Phox-I did not inhibit phosphorylation of p47
phox
, a critical subunit of NOX. Addition of CRP or thrombin induced significantly less ROS generation in platelets from Rac1
-/-
, as compared to Rac1
+/+
, mice. Addition of CRP generated significantly less ROS and exhibited diminished aggregation in NOX2
-/-
platelets than NOX2
+/+
platelets. A two minute incubation of platelets with ML171 (0.1 -1.0 μM), a selective inhibitor of NOX1, inhibited CRP induced but not thrombin induced ROS generation, ATP secretion and aggregation. Addition of a combination of ML171 and NSC23766, Phox-I and NSC23766 or ML171and Phox-I to platelets did not result in additive inhibitory effect on CRP induced ROS generation. Together, these results show for the first time that Rac1 GTPase: (a) is essential for agonist induced ROS generation in platelets; (b) inhibits ROS generation by inhibiting Rac1-p67
phox
interaction but not activation of p47
phox
; (c) is involved in ROS generation by both NOX1 and NOX2. The differential signaling of CRP activated Rac1-NOX1-ROS and thrombin-stimulated Rac1-NOX2-ROS play an important role in platelet secretion and aggregation.
Abstract 588: Cap Inflammation Leads to Large Plaque Cap Stress Decrease and Strain Increase: MRI-PET/CT-Based FSI Modeling
Authors: Yang Chun , Tang Dalin , Huang Sarayu , Mani Venkatesh , Fayad Zahi A
Published: March 2022

Introduction:
Recent studies indicated that plaque rupture may be associated with: a) extreme mechanical stress/strain conditions; b) large lipid-rich necrotic core; c) thin and weakening fibrous cap; d) cap inflammation; e) intraplaque hemorrhage. Fayad et al and others have been developing multi-modality imaging technology using PET/CT (Positron Emission Tomography/ Computed Tomography) and MRI (magnetic resonance imaging) to identify inflammation in arteries. The goal of this paper is to investigate possible impact of cap inflammation on plaque stress/strain and flow shear stress conditions.

Method:
An MRI-PET/CT-based modeling approach is proposed to develop fluid-structure interaction (FSI) models for human carotid plaque assessment and quantify the effect of inflammation on plaque stress/strain conditions. The combined PET/CT and MRI data was acquired from a 74 year old male patient (with informed consent) to assess arterial inflammation. The patient was imaged with dark blood multi-contrast MRI and 18F-fluorodeoxyglucose (18F-FDG) PET/CT in separate imaging sessions 12 days apart. For the 15-slice MRI/PET/CT data set, inflammation was identified for S4-S11. The 3D FSI model was built following established procedures. Material stiffness for the fibrous cap was adjusted lower to reflect the cap weakening causing by cap inflammation. Setting stiffness ratio (SR) to be 1.0 for baseline, results for SR=0.5, 0.25, and 0.1 (smaller SR value indicates softer cap stiffness) were obtained.

Results:
Mean plaque cap stress (PC-Stress), strain (PC-Strain) and flow shear stress (PC-FSS) are reported in Table 1. Roughly, 30-50% decrease in PC-Stress and 50-100% increase in PC-Strain were observed. Greater changes were observed for SR=0.1.

Conclusion:
Since cap inflammation and extreme stress/strain conditions are of great interest, further investigations are warranted to validate our findings.
Abstract 595: Proof-of-concept Studies for Sirna-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit
Authors: Chen Zhu , Cai Tian-Quan , Luo Bin , Xu Yiming , Wu Weizhen , Clark Seth , Hoos Lizbeth , Zhou Yuchen , Jochnowitz Nina , Lubbers Laura , Seiffert Dietmar , Tadin-Strapps Marija
Published: March 2022

Multiple coagulation factors in the intrinsic cascade are emerging targets for new anticoagulant therapies. Rat and rabbit are two commonly utilized preclinical species for studying thrombosis and hemostasis. The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target coagulation factors in rat and rabbit. Plasma kallikrein was selected for rat studies as plasma kallikrein knockout mouse had been described before in thrombosis and hemostasis models. Factor X (FX) was selected for rabbit studies as a rich dataset of FXa inhibitors in rabbits exists in literature. siRNAs that produced over 90% knockdown of rat plasma prekallikrein mRNA and rabbit FX mRNA in vitro were identified from cellular screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA
in vivo
delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day seven post dosing in both species. Both prekallikrein siRNA and FX siRNA resulted in dose-dependent and selective knockdown of target gene mRNA in the liver of respective species with maximum reduction of over 90% on day 7 following a single dose of the siRNA-LNP. Plasma prekallikrein siRNA at the highest dose (0.5 mg/kg) produced 92% knockdown on the circulating zymogen in the rats, associated with modest (~45%) clot weight reduction in the arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. These observations are consistent with the modest antithrombotic phenotype and absence of bleeding diathesis reported for the knockout mouse. FX siRNA at the highest dose (1.0 mg/kg) produced 98% knockdown on the circulating zymogen in the rabbits, accompanied with significant prolongation in ex vivo aPTT (103%) and PT (202%) measurements, consistent with the literature findings on pharmacodynamic effects of FXa inhibitors in rabbit. Results thus fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat and rabbit, via systemic delivery of siRNA. The titratable knockdown platform will not only strengthen target validation in thrombosis research but also facilitate studies on hemophilia diseases and hemostatic agents thereof.
Abstract 6: Continuous Plasma S1p-dependent Signaling by Apically Polarized S1Pr1 Supports Endothelial Barrier Function
Authors: Wilson Stephen J , Wilsbacher Lisa D , Kazmi Sabeen A , Baluk Peter , McDonald Donald M , Coughlin Shaun R
Published: March 2022

Sphingosine-1-phosphate, generated by sphingosine kinases (Sphk1 and 2), acts at its receptor S1Pr1 to maintain vascular homeostasis. The gradient of S1P concentration (low micromolar in plasma vs. low nanomolar in tissue) suggests two non-mutually exclusive models via which S1P maintains the endothelial barrier: 1) plasma S1P communicates continuously with the endothelium to maintain vascular integrity and regulate vascular leak, or 2) compartmentalization of S1P and S1Pr1 enables sensing of plasma extravasation and a dynamic response to leak. To distinguish between these models, we examined endothelial responses to apical vs. basolateral application of S1P in vitro and S1Pr1 subcellular localization in vivo. Addition of S1P to the upper (apical) chamber of transwells containing EA.hy926 endothelial monolayers triggered ERK activation. Surprisingly, addition of these agonists to the lower (basolateral) chamber was without effect. Similar results were obtained with the S1Pr1 agonist SEW2871, but the PAR1 agonist SFLLRN triggered ERK activation from either chamber. By immunofluorescence staining and surface biotinylation of EA.hy926 cells, S1Pr1 protein was present predominantly on the apical surface. Similarly, staining of mouse trachea microvessels revealed S1Pr1 on CD31-positive membranes apical, but not basal, to the endothelial cell nucleus. S1Pr1 staining co-localized with the apical membrane marker podocalyxin but not the basal marker β1-integrin. The level of S1Pr1 protein in endothelial cells in mice lacking plasma S1P (Sphk1 flox/-; Sphk2 -/-; Mx-1 cre) was twice that in littermate controls. Deletion of S1Pr1 from the endothelium (S1Pr1 flox/-; Cdh5 Cre) resulted in a 6-fold increase in vascular permeability compared to littermate controls. Preliminary data suggests that S1Pr1 inactivation alters the phosphorylation state of VE-cadherin. These results suggest that S1Pr1 is polarized to the luminal membrane of endothelial cells where it is continuously activated but only partially downregulated by plasma S1P and that ongoing activation of S1Pr1 by S1P in plasma is required to maintain endothelial barrier function.
Abstract 612: Regulation of Bone Marrow Stem/Progenitor Cell Mobilization by Leptin
Authors: Jarajapu Yagna P , Vasam Goutham
Published: March 2022

Background:
Leptin, an adipocyte-derived hormone, elicits a variety of physiological functions by acting on leptin receptor (LepR). LepR is expressed in bone marrow hematopoietic stem/progenitor cells (BMSPCs) as well as in mesenchymal stromal cells, which play an important role in the maintenance and retention of BMSPCs. Cytokines such as G-CSF induce mobilization of BMSPCs, in part, via acting on MSCs.

Aim:
In this study the role of leptin in the physiological, and G-CSF-induced mobilization of BMSPCs was evaluated.

Methods:
The study was carried out in control and streptozotocin (STZ)-induced diabetic mice (C57Bl/6j), and in mice with genetic ablation of leptin receptor (LepR+/- and LepR-/- (db/db) mice). Circulating white blood cells (WBCs), lineage- cells (Lin-), and Lin-Sca-1+cKit+ (LSK), cells were enumerated by flow cytometry in different treatment groups. Mobilization of different cell populations was induced by G-CSF (125μg/Kg, s.c., twice a day, 4 days). Effects of leptin (600μg/Kg, i.p., 5 days) or LepR antagonist, PESLAN-1 (5mg/Kg, s.c., once-in-two-days, 14 days, were evaluated on basal as wells as G-CSF-mobilized levels of different cell populations.

Results:
Leptin treatment increased basal levels of WBCs (P<0.005), and decreased Lin- (P<0.05) or LSK (P<0.05) cells compared to the control (n=5). Leptin treatment also decreased Lin- (P<0.005) and LSK (P<0.005) cells but WBCs mobilized by G-CSF. PESLAN-1 exacerbated G-CSF-induced mobilization of LSK cells (P<0.05 vs PESLAN-1-untreated). G-CSF-mobilized WBCs, Lin- and LSK cells were higher in LepR+/- or LepR-/- (P<0.05 to 0.001, n=6) mice compared to wild-type (WT) control (LepR+/+) mice. Since db/db mice are diabetic, we verified the effect of diabetes in WT/LepR+/+ mice. In STZ-induced diabetic mice (n=7), G-CSF- induced mobilization was reduced (WBCs (P<0.05), Lin- (P<0.005), and LSK (P<0.001) compared to nondiabetic, n=7) in contrast to the observations in diabetic LepR-/- mice.

Conclusions:
These results suggest that in physiological conditions, leptin-LepR signaling tends to retain BMSPCs, and oppose G-CSF-induced mobilization. Furthermore, these results imply that LepR blockade could overcome diabetes-induced dysfunction of BMSPC mobilization.
Abstract 623: The Contribution of Hiv to Brain Arterial Remodeling: A Case-control Study
Authors: Gutierrez Jose , Menshawy Khaled , Elkind Mitchell S , Marshall Randolph S , Morgello Susan
Published: March 2022

Background:
The incidence of cerebrovascular events is rising in the HIV population but little is known about the mechanisms of vascular injury among patients with HIV population.

Methods:
Large cerebral arteries were obtained from autopsies in 80 HIV+ cases and 80 HIV- controls, matched for sex and age ± 3 years, range 30-84. Arterial size, atherosclerosis phenotype, luminal stenosis, and media thickness were obtained in each artery. The average pixel intensities (AI) of metalloproteinase (MMP)-2, MMP-3, MMP-9, tissue inhibitor of MMP (TIMP)-1 and TIMP-2 was assessed with immunohistochemistry defining high intensity as an AI in the upper tertile. Inflammation was assessed with CD68 staining. All estimates were adjusted for age, sex, ethnicity, vascular risk factors and cocaine use.

Results:
Arterial inflammation was associated with greater staining intensity of MMP3 (Beta=0.36, P=0.003) and TIMP2 (Beta=0.29, P=0.002) in HIV+ cases, but not in HIV- controls. The association of HIV with MMP3 was independent of whether the inflammation was located in the media, intima, or adventitia, while for TIMP-2 the association was only significant with intima inflammation (Beta=-0.36, P=0.03). Cerebral arteries in HIV + cases with high intensity of MMP3 staining had thinner media (Beta=-2.3, P= 50 viral copies/mL (B=0.62, P=0.04), Nucleoside Reverse Transcriptase Inhibitor (B=1.08, P=0.009) or protease inhibitor use at the time of death (B=-0.64, P=0.05), vascular risk factors (B=0.51, P=<0.001), and diabetes (B=1.2, P=<0.001), were associated with higher expression of MMP3. There were no significant associations between inflammation and MMP2, MMP9 and TIMP1 intensity staining.

Conclusions:
Arterial inflammation in HIV+ cases is associated with greater expression of MMP3, and arteries with higher expression of MMP3 have a thinner media, despite thicker walls, and greater degrees of luminal narrowing. Through thinning of the media, MMP3 may play a role in HIV-associated dolichoectasia; through secondary effects, it may contribute to paradoxical luminal stenosis.