bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 30: ApoE and ApoCIII Interact to Modulate the Metabolism of HDL ApoA-I in Humans
Authors: Morton Allyson , Mendivil Carlos O , Wang Liyun , Furtado Jeremy D , Sacks Frank M
Published: June 2021

ApoE has potential roles in HDL metabolism by promoting enlargement and clearance, and apoCIII could delay apoE-mediated clearance by the liver as it does for VLDL metabolism. To determine whether apoE and apoCIII modulate the kinetics of apoA-I HDL, we compared the metabolism of apoA-I in HDL subspecies that have apoE, apoCIII, both, or neither. We recruited 10 participants (4M, 6F) with low HDL-C (range 24-54 mg/dl) and BMI between 25-35 kg/m
2
. They were given an IV bolus of d3-leucine and blood collected up to 46hr. HDL was isolated from plasma by anti-apoA-I immunoaffinity chromatography, separated by sequential anti-apoE and anti-apoCIII chromatography, and size-separated using NDPAGE into alpha-1, alpha-2, alpha-3, and prebeta-1 HDL. ApoA-I was purified from HDL subspecies on SDS-PAGE, and pool size of apoA-I was determined from the protein bands, adjusted to plasma total apoA-I. D3-leucine enrichment was measured by GC-MS. We used SAAM-II modeling software to compute apoA-I fractional catabolic rates (FCR) and fluxes for each HDL subspecies using a published multicompartmental model. The main findings from our preliminary model investigation are:

- The liver secretes a range of HDL sizes for each of these HDL subspecies. About 2-6% of plasma HDL apoA-I is associated with apoE and/or apoCIII. Regardless of size, apoE- and apoCIII-containing HDL are detectable in the circulation slightly earlier after tracer administration than HDL containing neither apoE nor apoCIII.

- HDL that contains apoE but not apoCIII is especially active in size conversions, such as generating prebeta-1 HDL. Prebeta-1 HDL types are not a universal precursor of larger size HDL.

- HDL that contains apoE but not apoCIII has about a 4-fold increased FCR (range 1.3-8.8) across all sizes of HDL compared to other HDL subspecies, consistent with the role of apoE as a liver receptor ligand. When coexisting with apoE, apoCIII abolished the apoE-accelerated clearance, making the FCR similar to that of HDL that does not have apoE. But, when apoCIII is present on HDL that does not have apoE, there is no reduction in clearance compared to HDL containing neither apolipoprotein.

In conclusion, these results suggest that apoE accelerates the metabolism of HDL apoA-I, whereas apoCIII impedes this process.
Abstract 305: Exogenous Vasohibin-2 Exacerbates Angiotensin II-induced Ascending Aortic Aneurysms
Authors: Okuyama Michihiro , Uchida Haruhito A , Umebayashi Ryoko , Kakio Yuki , Tanabe Katsuyuki , Sato Yasufumi , Wada Jun
Published: June 2021

Objective:
Chronic angiotensin II (AngII) infusion promotes both ascending (TAAs) and abdominal aortic aneurysms (AAAs) in mice. Previous studies demonstrated that vascular endothelial growth factor (VEGF) enhanced AngII-induced AAA formation, and VEGF significantly increased AngII-induced AAA diameter in hypercholesterolemic mice. Vasohibin-1 (VASH-1) is induced by VEGF and inhibits angiogenesis at the termination zone under various pathologic conditions such as those of tumors, arterial intimal thickening and retinal neovasucularization. In contrast, a VASH-1 homolog, Vasohibin-2 (VASH-2), promotes angiogenesis at the sprouting front. Therefore, we hypothesized that exogenous VASH-2 influences AngII-induced TAAs in normocholesterolemic mice. The purpose of this study was to examine whether VASH-2 influenced AngII-induced TAAs.

Methods and Results:
Male C57BL/6J mice (10 weeks old) were fed a normal laboratory diet. Mice were injected 10^9 plaque-forming unit VASH-2 or Lac Z expressing adenovirus (Ad) via tail vein every other week. They were also infused subcutaneously with either AngII (1,000 ng/kg/min) or saline by osmotic mini-pumps for 3 weeks. The study mice had 4 groups: AngII + Ad VASH-2 (n=23), AngII + Ad Lac Z (n=23), Saline + Ad VASH-2 (n=13), Saline + Ad Lac Z (n=9). There were no significant differences between 4 groups in body weight. Plasma total cholesterol and VEGF concentrations were significantly increased in AngII infused groups (P < 0.05). Mortality ratio was 8.7% in AngII + Ad VASH-2 group and 4.3% in AngII + Ad Lac Z group. Intima area of aortic arch was significantly larger in AngII + Ad VASH-2 group than in AngII + Ad Lac Z group (19.78 ± 0.40 mm^3 vs 17.70 ± 0.41 mm^3, P < 0.01). AngII infusion significantly increased ex vivo maximal diameters of abdominal aorta, but there was no significant difference between VASH-2- and Lac Z-injected mice.

Conclusion:
Exogenous VASH-2 exacerbated AngII-induced ascending aortic aneurysms in male C57BL/6 mice.
Abstract 306: Murine Abdominal Aortic Aneurysms Demonstrate Heterogeneous Growth and Remodelling by High-frequency Ultrasound
Authors: Phillips Evan H , Di Achille Paolo , Bersi Matthew R , Humphrey Jay D , Goergen Craig J
Published: June 2021

In vivo
imaging of vascular disease models has been largely underutilized, but it can greatly benefit cardiovascular research. An improved understanding of the development of the angiotensin II (AngII) apolipoprotein E knockout model of abdominal aortic aneurysms (AAAs) could help patients with this life-threatening disease. The objective of this study was to investigate the early hemodynamic, biomechanical, and volumetric changes in AngII AAAs using high-frequency ultrasound. Five male apolipoprotein E-deficient C57BL/6J mice were subcutaneously implanted with AngII-loaded miniosmotic pumps (1000 ng/kg/min) and screened for appearance of AAAs. We acquired imaging data of the morphology, pulsatility, and blood flow profiles in newly formed AAAs over 7 days. We found that biomechanical and hemodynamic changes occurred during initial AAA formation alongside an increase in AAA volume. Average AAA volume increased by 140±24% between baseline and AAA diagnosis, while true lumen volume decreased by 46±12% due to formation of a focal dissection. The resulting intramural thrombus evolved in shape and volume but with variability between animals. Regional differences in blood flow velocity were apparent down the length of the largest AAAs and mean blood flow velocity significantly increased by 150±42% upon initial aortic expansion and true lumen narrowing. Mean velocity decreased over 7 days as the total AAA volume increased. Circumferential cyclic strain also significantly decreased upon initial aortic expansion and remained reduced, indicating the AAAs had stiffened vessel walls with initial aortic expansion. We are also exploring the heterogeneity of this AAA development using computational pulsatile flow models built from these ultrasound datasets. These models can provide information on site-specific changes in wall shear stress and oscillatory shear index, which are potentially predictive metrics for intramural thrombus formation and AAA growth.
Abstract 312: Macrophage Metabolic Adaptation to Heme
Authors: Bories Gaël F
Published: June 2021

Free heme is a very deleterious compound that promotes oxidation, inflammation and cell death, which contributes to numerous vascular pathologies including stroke, thrombosis and atherosclerosis. Rupture of red blood cells (RBC), also known as hemolysis, leads to the release of hemoglobin, which upon oxidation, readily releases free heme into the circulation. Due to their ability to clear heme and fight against heme-induced oxidative damage, macrophages limit tissue damage induced by hemolysis. Inside macrophages, heme is degraded by the inducible heme oxygenase 1 (Hmox-1), producing carbon monoxide (CO), iron and biliverdin. Macrophages respond to changes in their environment; they require a constant metabolic adaptation to sustain the bio-energetic demand. The pentose phosphate pathway (PPP) is the major source of NADPH, which is generated by glucose-6-phosphate dehydrogenase (G6PD) and phospho-gluconate dehydrogenase (PGD). NADPH provides reducing equivalents for numerous, Nrf2 (nuclear factor erythroid 2)-dependent anti-oxidant systems such as glutathione reductase and Hmox-1 itself. Here we show that intracellular free heme induces a specific metabolism in macrophages, characterized by increased glucose uptake, with no change in glycolysis, but a polarization towards the PPP. Pharmacologic inhibition of the PPP demonstrated that this metabolic shift is essential for macrophages to degrade heme efficiently. We also show that the metabolic adaptation to heme is dependent on Hmox activity, involving the upregulation of the glucose transporter (GLUT) 1, G6PD, and PGD. This suggests that CO is involved in the macrophage metabolic adaptation to heme. Finally we show that the PPP is increased in the liver of sickle-cell disease (SCD) mice, an accepted model of chronic intra-vascular hemolysis.

This study provides a new understanding of macrophage metabolic adaptation to efficient heme clearance, opening the field for novel therapies for hemolysis-associated diseases.
Abstract 322:
In Vivo
Vibrational Photoacoustic Tomography of Perivascular Fat in Apolipoprotein E-Deficient Mice
Authors: Sangha Gurneet S , Phillips Evan H , Goergen Craig J
Published: June 2021

Vibrational Photoacoustic Tomography (VPAT) is an emerging imaging modality that utilizes pulsed laser light to induce acoustic waves and obtain tissue specific compositional information. When used in combination with ultrasound, VPAT has the potential to identify the location, size, and distribution of lipids
in vivo
. In our study, we hypothesized that VPAT can be used to distinguish perivascular aortic fat accumulation between apolipoprotein E-deficient (apoE
-/-
) and wild-type (WT) mice. A 40 MHz central frequency transducer (Vevo2100, VisualSonics) and a ND:YAG pulsed laser (NT352C, Ekspla) were used to simultaneously obtain both long axis
in vivo
B-mode and VPAT images of the infrarenal aorta in three apoE
-/-
(8.7±0 months old) and WT controls (2.9±0 months old) mice. Pulsed laser light was specifically tuned to 1100nm and 1210nm to image blood and lipid, respectively. We then measure perivascular lipid thickness in three distinct locations along the anterior wall of the aorta. These measurements were then averaged to obtain a final perivascular fat thickness score for both the apoE
-\-
and WT groups. We observed greater 1210 nm signal in apoE
-/-
(0.47±0.14 mm) mice versus WT mice (0.32±0.11 mm; p<0.05), suggesting that apoE
-/-
mice have greater perivascular lipid accumulation than WT controls. These findings are significant as they show that VPAT can be used to image and track lipid accumulation
in vivo
. Future work will focus on monitoring luminal plaque progression in atherosclerotic murine models, including hypercholesterolemic mice on a high fat diet or rodent models of severe carotid stenosis. If successful, VPAT has the potential to be used in the clinic to 1) study atherosclerosis plaque development, 2) evaluate the effectiveness of prospective therapeutics, and 3) improve the diagnosis of atherosclerosis or related vascular diseases.
Abstract 327: Diabetes Influences Carotid Artery Endarterectomy Plaque Lipidomics
Authors: Zayed Mohamed A , Hsu Fong-Fu , Patterson Bruce W , Yan Yan , Naim Uzma , Vemuri Change , Sanchez Luis A , Semenkovich Clay F
Published: June 2021

Nearly 20% of all ischemic strokes result from an atherosclerotic embolic source at the carotid arterial bifurcation. Patients with diabetes (DM) are at significantly increased risk of developing carotid artery stenosis, which predisposes to disabling strokes. Initial evidence suggests that carotid artery phospholipids are differentially expressed in patients with advanced atherosclerotic disease. However, it is unknown whether patients with DM and/or symptomatic carotid artery stenosis have unique carotid artery lipidomic profiles. To test this hypothesis, we performed a comprehensive analysis of phospholipid expression in carotid endarterectomy tissue harvested from a cohort of 18 patients (11 DM; 7 non-DM). Diabetic patients had a higher BMI (P=0.02), and increased use of b-blockers (P<0.01), but there was no difference in age, hyperlipidemia status, statin use, smoking incidence, severity of carotid stenosis, or carotid symptoms. Maximally (MAX) diseased carotid endarterectomy plaque segments at the carotid bifurcation were analyzed relative to correspondingly minimally (MIN) diseased distal internal carotid artery segments. Mass spectrometry-derived phospholipid absolute quantity profiles between MAX and MIN diseased carotid endarterectomy segments demonstrated significantly lower phosphatidylserine (PS; P<0.01), phosphatidylinositol (PI; P=0.03), and plasminogen phosphatidylethanolamine (pPE; P<0.01) phospholipids in MAX diseased segments. Paired analyses of MAX and MIN diseased segments revealed higher pPE in diabetic patients (P<0.01), and higher PI in non-diabetic and asymptomatic patients (P=0.03 and 0.05, respectively). The arachidonic acid-containing 1-(1Z-octadecenyl)-2-arachidonoyl-
sn
-glycero-3-phosphoethanolamine (pPE38:4) and 1-(1Z,9Z-octadecedienyl)-2-arachidonoyl-
sn
-glycero-3-phosphoethanolamine (pPE38:5) were the most highly (>109% increase) and differentially expressed phospholipids in the plaque segments of DM patients (P<0.01). These findings suggest that these pre-arachidonic acid phospholipids that affect plaque inflammation are more abundant in diseased carotid arteries of DM patients, which may influence disease progression and plaque vulnerability.
Abstract 328: Effects of Unilateral Hindlimb Venous Occlusion in a Murine Model
Authors: Clark Ross M , Coffman Brittany B , Howdieshell Thomas R
Published: June 2021

Introduction:
Acute and chronic venous occlusion from thrombosis or other causes leads to long-term morbidity in a significant number of individuals. Understanding mechanisms of venous flow restoration may lead to future treatments and prevention of morbidity. Authors have previously hypothesized that this occurs by a combination of thrombus recanalization and neovascularization with collateral formation. A model of permanent hindlimb venous occlusion may allow the study of flow restoration independent of vein recanalization.

Methods:
Adult C57Bl/6 mice (n=9 total, n=6 operated, n=3 unoperated controls) were subjected to identical unilateral hindlimb venous occlusive surgeries (n=6 limbs total) involving both vein ligation and permanent ablation using an external thermal device. Contralateral limbs underwent no surgery and served as
in-vivo
controls (n=6 limbs total). Laser speckle perfusion imaging of hindlimbs was conducted for 10 post-operative days. Real-time qPCR was used to measure tissue mRNA expression of venous angiogenesis-related signaling molecules.

Results:
Mean treated limb perfusion was 165.0 perfusion units (PU), 179.9 PU, and 154.2 PU on post-operative days 3, 5 and 7, respectively. This compares to 121.5 PU, 125.0 PU, and 117.2 PU in the untreated limb (p=0.01, 0.03 and 0.04 respectively). Relative VEGFa, EphB4 and COUP-TFII expression in treated limbs immediately following surgery and at postoperative day 10 was not significantly different compared to unoperated controls.

Conclusions:
Surgical occlusion of unilateral hindlimb venous drainage leads to tissue hyperemia by laser speckle imaging 3, 5 and 7 days after surgery which resolves by post-operative day 10. Gene expression reveals no evidence of induced venous angiogenesis to account for restoration of limb drainage. Re-establishment of venous outflow may be due to recruitment of existing venous collateral tributaries rather than true neovascularization in this model.
Abstract 339: Comparison of the Antiplatelet Effect Between Ticagrelor and Clopidogrel in Chinese Patients with Acute Myocardial Infarction After Primary Percutaneous Coronary Intervention
Authors: Xu Jingjing , Yao Yi , Zhang Jiahui , Tang Xiaofang , Ma Yuanliang , Yuan Jinqing
Published: June 2021

Background:
Ticagrelor can provide effective platelet inhibition 2 hours after a loading dose, but it will take 6 hours for clopidogrel. It is not very clear whether the anthplatelet agents treated patients with AMI undergoing primary PCI can achieve ideal antiplatelet effect after 24 to 48 hours.

Purpose:
The aim is to compare the antiplatelet action between ticagrelor and clopidogrel in Chinese patients with AMI after primary PCI.

Methods:
189 patients with AMI after primary PCI were enrolled in this single center registry. All patients received loading and maintenance dose of dual antiplatelet therapy (aspirin+clopidogrel/ticagrelor). 58 cases were included in ticagrelor group. 131 patients were included in clopidogrel group. Residual platelet reactivity was assessed by VerifyNow 24-48 hours after PCI. All patients were followed-up for 30 days and 6 months after discharge.

Results:
The baseline data were well matched between the two groups. After 24-48 hours, 51 cases existed of high residual platelet response(HRPR)(platelet response unit ,PRU≥230), the ratio was 26.98%. In clopidogrel group, the average PRU was 195.8 (26~329), and 43 patients existed HRPR, ratio was 32.82%. For ticagrelor group, the average PRU was 101.8 (6~322), and 8 patients existed HRPR, ratio was 14.04%. The incidence of HRPR was significantly lower in ticagrelor group (
p
< 0.0001). Within 30 days, the incidence of MI in clopidogrel group and ticagrelor group were respectively 0.8% and 0,
p
=0.693; target lesion revascularization were 1.7% and 1.5%,
p
=0.669; death were 0.8% and 0,
p
=0.693; stroke were 0 and 0,
p
=1.000; bleeding were 2.3% and 0,
p
=0.331. In 6 months, the incidence of MI were 0.8% and 0,
p
=0.693; target lesion revascularization were 0.8% and 0,
p
=0.693; death were 0 and 0,
p
=1.000. The incidence of MACE, bleeding and stroke had no obvious difference between the two groups.

Conclusion:
24 hours after primary PCI, there are still a large proportion of Chinese patients with AMI existing insufficient platelet inhibition, no matter they take clopidogrel or ticagrelor, but ticagrelor has a significant stronger antiplatelet effect than clopidogrel. There is no obvious difference between the incidences of clinical events.
Abstract 356: IFN-γ-Dependent TET2 Repression in Coronary Allograft Vasculopathy
Authors: Ostriker Allison , Leslie Kristen , Martin Kathleen
Published: June 2021

Coronary allograft vasculopathy (CAV) occurs in 50% of heart transplant recipients at 10 years after surgery. Allograft failure secondary to CAV accounts for 30% of deaths in transplant recipients. CAV is characterized by concentric neointimal hyperplasia (NIH) in the graft vasculature results in ischemic injury. T cell-derived INF-γ is a well-established driver of NIH in CAV. The molecular mechanisms engaged by IFN-γ in vascular smooth muscle cells (VSMCs) in CAV are not fully elucidated.

Our group recently showed that TET methylcytosine dioxygenase 2 is a regulator of VSMC phenotype. We hypothesized, therefore, that IFN-γ modulates TET2 expression and/or function in VSMCs. We found, using a single minor histocompatibility mismatch aorta interposition graft model of CAV, that TET2 expression was decreased in vivo in the graft neointima as compared to in control tissue. Further, we found that IFN-y was sufficient to repress TET2 gene transcription in human VSMC in vitro, and this repression was not reversed by mTOR inhibition. We then sought to determine if repression of TET2 is sufficient to exacerbate neointimal hyperplasia in the aorta graft model. We found that NIH was exacerbated in aorta grafts from VSMC-specific, inducible, TET2 knockout (iKO) donors versus controls. Neither T cell recruitment, nor STAT1 activation, was increased in the iKO grafts versus controls; while time course analysis showed that neointimal cells were primarily recipient-derived, suggesting a non-cell autonomous mechanism. Western blot analysis of VSMCs stimulated with IFN-γ for 0-120 minutes suggested that, in addition to STAT1 activation, IFN-γ decreases β-catenin signaling. ChIP-PCR studies showed that STAT1, but not LEF1 (a β-catenin coactivator), occupancy increased at the TET2 promotor following INF-γ stimulation.

Studies are ongoing to further elucidate the requirement for STAT1 and/or β-catenin in IFN-γ-dependent repression of TET2 in VSMCs and the non-autonomous role of TET2 on mediating NIH progression in CAV. Since IFN-y-dependent repression of TET2 is not reversed by rapamycin, elucidating this pathway represents an opportunity for developing complementary therapies to mTOR inhibition that may result in improved outcomes and fewer side effects.
Abstract 357: Antioxidant and Anti-inflammatory Strategies Prevent Endothelial Dysfunction in Chronic Kidney Disease
Authors: Palomo Marta , Martin-Rodriguez Susana , Vera Manel , Cruzado Josep Maria , Rivera Jose , Cases Aleix , Escolar Ginés , Diaz-Ricart Maribel
Published: June 2021

Accelerated atherosclerosis in chronic kidney disease (CKD) is preceded by the development of endothelial dysfunction (ED), with development of a proinflammatory and prothrombotic phenotype and enhanced oxidative stress. The effect of anti-inflammatory and antioxidant strategies on the endothelium has been evaluated in an
in vitro
model of ED in uremia.

Endothelial cells (ECs) were pretreated with the antioxidant enzyme mimetics ebselen, EUK-134 and EUK-118; the flavonoids apigenin, genistein and quercetin, with both antioxidant and anti-inflammatory potential; and two commercially available compounds: N-acetylcysteine (NAC) and defibrotide (DF). There is increasing evidence demonstrating that both NAC and DF exhibit both properties. ECs were exposed to medium containing serum from patients on dialysis (n=10) or from healthy donors (n=15). Changes in the expression of the adhesion receptor ICAM-1 and the production of intracellular reactive oxygen species (ROS) were assessed. Activation of inflammation-related proteins p38 MAPK and NFkappaB (NFκB) were also evaluated.

Exposure of ECs to uremic media resulted in a significantly increased expression of ICAM-1, overproduction of ROS and activation of p38MAPK and NFκB compared to control ECs (p<0.05). Ebselen, EUK 134, and EUK118 inhibited ICAM-1 expression and ROS generation in the uremic condition (p<0.01). Regarding flavonoids, only quercetin showed a moderated but significant inhibitory effect on both parameters (p<0.05). NAC and DF exhibited a protective effect on ECs exposed to the uremic insult (p<0.05 for ICAM-1 expression and ROS generation). All the compounds reduced p38MAPK activation (p<0.05). The antioxidant-enzyme mimetics and NAC were able to inhibit the activation of NFκB induced by the uremic media (p<0.05).

Endothelial dysfunction associated with CKD is considered to be the first step in the progression of atherosclerosis. Our results indicate that the antioxidant enzyme mimetics, NAC and DF exhibit not only antioxidant but also anti-inflammatory effects on the endothelium. Therefore, further research on the protective effects of these compounds may provide new strategies for the prevention of the cardiovascular complications in uremia.
Abstract 360: Loss of Endothelial CXCR7 Exacerbates Wire-injury Induced Neointimal Formation
Authors: Hao Huifeng , Hu Sheng , Bu Dawei , Sun Xiaogang , Wang Miao
Published: June 2021

CXCR7 is a non-classical chemokine receptor for CXCL12, whose gene represents a genome-wide association locus for coronary artery disease. Global deletion of CXCR7 increased experimentally induced neointimal formation and atherosclerosis in hyperlipidemic mice, with evidence that CXCR7 modified cholesterol uptake to adipose tissue. We found that CXCR7 was expressed in endothelial cells of mouse neointima and human aortic lesions. To examine a role of endothelial CXCR7 in vascular remodeling, endothelial CXCR7 inducible knockout mice were studied for their vascular response to wire injury in femoral arteries. Tamoxifen treatment of mice harboring floxed
CXCR7
and
Cdh5
-promoter driven
CreERT2
, essentially abolished endothelial CXCR7 expression in vitro and in vivo. Postnatal deletion of endothelial CXCR7 exacerbated neointimal formation on normalipidemic background, four weeks after injury. Mechanistically, this was attributable to attenuated endothelial repair following endothelial injury. Collectively, endothelial CXCR7 is a key regulator of vascular remodeling, independent of lipid traits.
Abstract 364: TRPV4 Channel Acts as a Negative Regulator of Angiogenesis by Modulation of Rho/Rho Kinase Pathway
Authors: Cappelli Holly C , Thoppil Roslin J , Adapala Ravi K , Paruchuri Sailaja , Thodeti Charles K
Published: June 2021

Angiogenesis, the formation new blood vessels from pre-existing ones, is critical for maintenance of normal cardiovascular physiology. However, excessive or insufficient angiogenesis can contribute to various diseases including cancer, atherosclerosis, and retinopathy. While the mechanism by which angiogenesis occurs is well established, little is known about the mechanisms that negatively regulate this process. Therefore, we investigated the role of mechanosensitive ion channel, TRPV4, in the regulation of angiogenesis by employing
in vitro, ex vivo,
and
in vivo
techniques. In the present study, we first cultured aortic ring explants isolated from wild-type (WT) and TRPV4KO mice and found a significant increase in the sprouting from TRPV4KO aortic rings after 5 days. Next, we found that endothelial cells (EC) isolated from TRPV4KO mice (TRPV4KO EC) exhibited increased proliferation, migration, as well as abnormal angiogenesis
in vitro,
compared to their WT counterparts. Further,
in vivo
Matrigel plug assays revealed abnormal vascular growth in TRPV4KO mice. Mechanistically, we found that absence of TRPV4 results in a significant increase in basal Rho activity and that pharmacological inhibition of the Rho/Rho kinase pathway was able to normalize the abnormal tube formation exhibited by TRPV4KO EC
in vitro
. To confirm these findings, we examined tumor growth in TRPV4KO mice treated with Rho kinase inhibitor, Y-27632, and anti-cancer drug Cisplatin, alone and in combination. We found that Y-27632 treatment, in conjunction with Cisplatin but not alone, was able to significantly reduce the abnormal tumor growth seen in TRPV4KO mice, suggesting that Rho kinase inhibition may have normalized the tumor vasculature and improved the delivery of Cisplatin. Taken together, these data suggest that TRPV4 is a negative regulator of angiogenesis and potentially a novel target for pathological and/or therapeutic angiogenesis.
Abstract 371: Targeted Bone Marrow Cell Delivery Mediated by Nanocarriers Endowed with Molecular Recognition
Authors: Liu Zhao-Jun , Daftarian Pirouz , Kovalski Letícia , Wang Bo , Tian Runxia , Castilla Diego M , Dikici Emre , Perez Victor L , Deo Sapna , Daunert Sylvia , Velazquez Omaida C
Published: June 2021

Introduction:
Stem cell therapy has emerged as a promising approach for treatment of a number of diseases, including delayed and non-healing wounds. However, targeted systemic delivery of therapeutic cells to the dysfunctional tissues remains one formidable challenge.

Methods:
We have developed a targeted nanocarrier-mediated cell delivery method by coating the surface of the cell to be delivered with dendrimer nanocarriers modified with adhesion molecules. Infused nanocarrier-coated cells reach to destination via recognition and association with the counterpart adhesion molecules highly or selectively expressed on the activated endothelium in diseased tissues. Once anchored on the activated endothelium, nanocarriers-coated transporting cells undergo transendothelial migration,
extravasation and
homing to the targeted tissues to execute their therapeutic role. Wound healing was measured by digital photograph and ImageJ calculation. Targeted tissue homing of LacZ
+
bone marrow cells (BMC) was detected and quantified by X-gal staining, and BMC-enhanced neovascularization was examined by Dil perfusion and scanning confocal microscopy.

Results:
Soluble E-selectin (sE-sel) was successful installed on BMC surface by dendrimer nanocarriers. sE-sel-nanocarriers can associate with E-selectin ligands highly expressed on the wound endothelium, by which coated BMC are targeted delivered to skin wound tissues and grafted corneas to promote wound healing and neovascularization.

Conclusions:
We demonstrate feasibility, efficacy and safety of our targeted nanocarrier for delivery of BMC to cutaneous wound tissues and grafted corneas and its advantages over conventional BMC transplantation in mouse models for wound healing and neovascularization. This versatile platform is suited for targeted systemic delivery of virtually any type of therapeutic cell.
Abstract 373: Manganese Superoxide Dismutase: A Novel Mediator of Heart Failure Development and Progression
Authors: Miriyala Sumitra , Chandra Mini , Fox Jonathan , Kevil Christopher , Orr Wayne , Panchatcharam Manikandan
Published: June 2021

Constitutive activation mitochondrial reactive oxygen species (ROS) has been implicated in both the pathogenesis and the progression of cardiovascular disease. Absence of
SOD2
(gene that encodes MnSOD) is found to be embryonic lethal in animal models due to impairment of mitochondrial function, most noticeably in the heart. In our earlier investigation, we have shown that the MnSOD mimetic, distributes 3-fold more in mitochondria than in cytosol. The exceptional ability of MnSOD mimetic to dismute O
2
•- parallels its ability to reduce ONOO– and CO3–. Based on our earlier reports, we have generated mice that specifically lack MnSOD in cardiomyocytes (Mhy6-SOD2
Δ
). These mice showed early mortality ~4 months due to cardiac mitochondrial dysfunction. Oxidative phosphorylation (OXPHOS) in mitochondria is the predominant mode for O
2
consumption in cells, and the mitochondria are the primary source of ROS in cells due to leaked electrons. FACS analyses using Mito-Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Mhy6-SOD2
Δ
to the wild type. The rate of oxygen consumption per cells was significantly lower in Mhy6-SOD2
Δ
cardiomyocytes than that in wild type. The most noticeable difference in the O
2
consumption was found in the presence of FCCP (H+ ionophore / uncoupler). Remarkably, while the FCCP treatment increased O
2
consumption in wild type, the treatment showed no effect on the O
2
consumption in the Mhy6-SOD2
Δ
cardiomyocytes. The result indicated that the low basal OXPHOS activity in Mhy6-SOD2
Δ
was due to unusually low OXPHOS potential.
Abstract 403: Identification of CHROME as a Competing Endogenous RNA that Regulates Cholesterol Homeostasis
Authors: van Solingen Coen , Hennessy Elizabeth J , Ouimet Mireille , Rinehold Kaitlyn , Hussein Maryem , Garbedian Michael J , Teupser Daniel , Holdt Lesca M , Moore Kathryn J
Published: June 2021

The discovery of microRNAs (miRNA) targeting gene pathways involved in HDL and LDL metabolism illuminated the potent role of non-coding RNAs in the regulation of cholesterol homeostasis. Long non-coding RNAs (lncRNA) have also been identified as crucial regulators of gene expression; however, few have been fully characterized. Here we report a novel human lncRNA, CHROME (Cholesterol Homeostasis Regulator Of MicroRNA Expression), that functions as a competing endogenous RNA to regulate cellular cholesterol homeostasis. We show that CHROME has 7 broadly expressed variants that are transcriptionally regulated by the cholesterol-sensing liver X receptors. Computational analyses revealed that CHROME harbors binding sites for multiple (11) miRNAs involved in cholesterol homeostasis, including miR-27b and miR-33a/b, which function as hubs controlling the expression of genes involved in cholesterol efflux and HDL metabolism. Using CHROME knock-down and overexpression, we demonstrate that CHROME acts as a ‘miRNA sponge’ that sequesters these miRNAs, limiting their ability to repress target genes, including ABCA1, OSBPL6 and ANGPTL3. Consistent with this, we show that overexpression of CHROME increases cholesterol efflux, whereas its silencing reduces cholesterol efflux from primary human hepatocytes and macrophages. As hepatic cholesterol efflux via ABCA1 plays a central role in HDL biogenesis, we investigated the relationship of CHROME to its miRNA targets and plasma levels of HDL cholesterol in liver samples from a cohort of 200 healthy individuals. This analysis showed that CHROME is inversely correlated with miR-27b and miR-33a/b levels, and positively correlated with levels of their target genes and plasma HDL cholesterol. Collectively, these findings identify CHROME as a key regulatory component of the non-coding RNA circuitry that controls cellular cholesterol efflux and plasma HDL levels in humans.
Abstract 617: Wnt-Containing Microvesicles Are Upregulated in Vascular Smooth Muscle Cell Cultures and Plasma From SM22-Cre;LRP6(fl/fl);LDLR-/- Mice
Authors: Behrmann Abraham , McDonald Jeffrey , Mead Megan , Ramachandran Bindu , Cheng Su-Li , Stabley John , Williams Bart , Towler Dwight A
Published: June 2021

When fed a high fat diet (HFD) typical of westernized societies, male LDL receptor -deficient (LDLR-/-) mice develop obesity and insulin-resistant diabetes, with concomitant arteriosclerotic calcification and fibrosis. We recently demonstrated that noncanonical Wnt signals and ligands (including Wnt4, Wnt7b, and Wnt10a) were upregulated with LRP6 deficiency in aortic vascular smooth muscle (VSM) of LDLR-/- mice, with concomitant worsening of vascular disease. Because Wnt ligands are hydrophobic due to fatty acid acylation, we examined whether lipidaceous microvesicles (MVs) containing Wnt ligands were elaborated by VSM, and whether LRP6 impacted plasma MV numbers and contents. Microvesicles were isolated using both polymer based- and differential ultracentrifugation - based purification methods, and Malvern NS300 NanoSight nanoparticle tracking analysis implemented to quantify vesicle concentrations, focusing upon the 50-200 nm size range of exosomes and mineralizing vesicles. As compared to Cre-negative control cultures, SM22-Cre;LRP6(fl/fl);LDLR-/- VSM elaborated 2.9-fold fold higher numbers of MVs over a 2 day period (143 +/-27 million/ml +/- vs. 49+/- 5 million/ml; p = 0.01). ML141 -- a cdc42/rac1 antagonist that reduces VSM calcification and noncanonical Wnt signals activated with LRP6 deficiency — reduces MV numbers in VSM cultures (42+/- 10 million / ml with 10 uM ML141 vs.120 +/- 19 million/ ml vehicle; p< 0.001). Consistent with primary VSM results, a 2-fold increase in the circulating plasma MV concentration was observed in aging SM22-Cre;LRP6(fl/fl);LDLR-/- mice vs. LRP6(fl/fl);LDLR-/- sibling controls (4.7+/- 1.1 billion/ml vs. 2.3+/-0.3 billion/ml, p = 0.07, n = 7-8 per genotype). Western blot analysis of extracts prepared from plasma MVs confirmed the presence of Wnt5a/b, Wnt10a, Wnt4 in this circulating nanoparticle population, and that Wnt4 was significantly increased in the MV fraction with LRP6 deficiency (1.4-fold, p = 0.01). Thus, Wnt-containing microvesicles are elaborated by VSM in vitro and in vivo. Quantitative characterization of plasma MV protein and lipid composition may yield novel biomarkers of pro-sclerotic Wnt signaling in diabetic vascular disease.
Abstract 636: Accelerated Atherosclerosis in the Context of Rheumatoid Arthritis
Authors: Bäcklund Alexandra Bäcklund , Johansson Martina , Ria Massimiliano , Ketelhulth Daniel F , Tsikrika Panagiota , Chernogubova Ekaterina , Jin Hong , Bäcklund Johan , Eriksson Per , Maegdefessel Lars , Holmdahl Rikard , Hansson Göran K , Hamsten Anders
Published: June 2021

Atherosclerotic lesion development and acceleration of lesion size, leading to a cardiovascular event can be affected by many factors, where both the immune system and lipid levels have been implicated. It is well recognized that patients with chronic inflammatory diseases, such as rheumatoid arthritis (RA) have an increased risk for cardiovascular disease (CVD) compared with the general population. It has also been suggested that CVD presented in RA patients is of an altered, more aggressive, phenotype compared to subjects without RA. Therefore, the investigation of atherosclerosis lesion development during chronic inflammation may lead to novel pathways that are not only relevant to the general population but also able to target this high CVD risk patient group. Thus, there is a need to gain a deeper understanding of how the exacerbated inflammatory state of arthritis affects the atherosclerosis process when both syndromes are presented in the same individual. Such studies are hampered in humans by the influence of different factors, such as the environment and large genetic heterogeneity of the population. We have developed a novel murine model where the human relevant genes of CVD (LDLr, thus increased LDL levels) and RA (MHCII, thus susceptible to collagen-induced arthritis) have been crossed into the common C57Bl6/J strain, enabling both arthritis and atherosclerosis being presented simultaneously in a clinically relevant fashion. This model mirrors the clinical state where the systemic inflammation of arthritis enhances atherosclerosis progression, where mice presenting arthritis have a significant increase in atherosclerotic lesion progression compared with their non-arthritic littermates. Interestingly, there was an inverse correlation with cholesterol levels, but a positive correlation with macrophages, and macrophage associated cytokines but not T cells. This model thus demonstrates that the lipid levels are vital in initiation of lesion development but it is the enhanced innate immune system that is driving the lesion acceleration in a chronic inflammatory state. This novel combined model is now able to be used to investigate altered clinical treatment strategies or novel treatments of atherosclerosis in the context of arthritis
Monotherapy Anticoagulation to Expedite Home Treatment of Patients Diagnosed With Venous Thromboembolism in the Emergency Department: A Pragmatic Effectiveness Trial
Authors: Kline Jeffrey A. , Adler David H. , Alanis Naomi , Bledsoe Joseph R. , Courtney Daniel M. , d’Etienne James P. , Diercks Deborah B. , Garrett John S. , Jones Alan E. , Mackenzie David C. , Madsen Troy , Matuskowitz Andrew J. , Mumma Bryn E. , Nordenholz Kristen E. , Pagenhardt Justine , Runyon Michael S. , Stubblefield William B. , Willoughby Christopher B.
Published: June 2021

Background:

The objective was to test if low-risk emergency department patients with vitamin K antagonist (venous thromboembolism [VTE]; including venous thrombosis and pulmonary embolism [PE]) can be safely and effectively treated at home with direct acting oral (monotherapy) anticoagulation in a large-scale, real-world pragmatic effectiveness trial.

Methods:

This was a single-arm trial, conducted from 2016 to 2019 in accordance with the Standards for Reporting Implementation Studies guideline in 33 emergency departments in the United States. Participants had newly diagnosed VTE with low risk of death based upon either the modified Hestia criteria, or physician judgment plus the simplified PE severity index score of zero, together with nonhigh bleeding risk were eligible. Patients had to be discharged within 24 hours of triage and treated with either apixaban or rivaroxaban. Effectiveness was defined by the primary efficacy and safety outcomes, image-proven recurrent VTE and bleeding requiring hospitalization >24 hours, respectively, with an upper limit of the 95% CI for the 30-day frequency of VTE recurrence below 2.0% for both outcomes.

Results:

We enrolled 1421 patients with complete outcomes data, including 903 with venous thrombosis and 518 with PE. The recurrent VTE requiring hospitalization occurred in 14/1421 (1.0% [95% CI, 0.5%–1.7%]), and bleeding requiring hospitalization occurred in 12/1421 (0.8% [0.4%–1.5%). The rate of severe bleeding using International Society for Thrombosis and Haemostasis criteria was 2/1421 (0.1% [0%–0.5%]). No patient died, and serious adverse events occurred in 2.5% of venous thrombosis patients and 2.3% of patients with PE. Medication nonadherence was reported by patients in 8.0% (6.6%–9.5%) and was associated with a risk ratio of 6.0 (2.3–15.2) for VTE recurrence. Among all patients diagnosed with VTE in the emergency department during the period of study, 18% of venous thrombosis patients and 10% of patients with PE were enrolled.

Conclusions:

Monotherapy treatment of low-risk patients with venous thrombosis or PE in the emergency department setting produced a low rate of bleeding and VTE recurrence, but may be underused. Patients with venous thrombosis and PE should undergo risk-stratification before home treatment. Improved patient adherence may reduce rate of recurrent VTE.

Registration:

URL:
https://www.clinicaltrials.gov
; Unique identifier: NCT03404635
Abstract 296: Loss of Trpc6 Function in Cardiomyocytes Improves Survival and Attenuates Progression of Cardiac Dysfunction After Mi
Authors: Gross Polina , Zhang Xiaoxiao , Wang Tao , Toib Amir , Wallner Markus , Starosta Timothy , Yu Daohai , Chen Xiongwen , Houser Steven
Published: June 2021

Ischemic heart disease (IHD) commonly culminates in myocardial infarction (MI), which causes cardiac myocyte death and depressed cardiac pump function. Surviving myocytes can usually maintain pump function by increasing Ca
2+
influx and contractility. However, elevated intracellular Ca
2+
also can activate pathological hypertrophic signaling that promotes cardiac dysfunction and progression into heart failure. Ca
2+
influx through Canonical Transient Receptor Potential (TRPC) channels has been classified as a potential source of hypertrophic signaling. Specifically, TRPC6 gene expression and biological activity is significantly upregulated in cardiac myocytes after MI. Our aim was to determine if TRPC6 loss of function is cardioprotective in MI mouse model. We performed an MI study on cardiac specific dominant negative (dn) TRPC6 transgenic mice that express 3 mutated amino acids (L678A-W680A) in the pore region, which disables TRPC6 channel function. In the course of 6 weeks post MI, dnTRPC6 mice had significantly greater survival (69.2%) than wild-type (WT) mice (47.5%). Cardiac function at 2 weeks post MI was decreased to the same extent in WT and dnTRPC6 mice. Ejection fraction (EF) in WT group was 30.6% vs. 57.1% and in dnTRPC6 EF was 31% vs. 61% (MI vs. sham, respectively). The EF in dnTRPC6 mice 6 weeks post MI suggested attenuation of heart failure progression compared to WT mice (32.5% vs. 26.5%, *P=0.05). Mice in the WT group demonstrated significant elevation in end diastolic and end systolic volumes (EDV and ESV) 6 weeks post MI, while dnTRPC6 had reduced end diastolic and end systolic volumes (116μL vs. 88μL and 106μL vs. 76μL, WT vs. dnTRPC6, EDV and ESV, respectively. ***P<0.0001). Hypertrophy measures of heart weight to body weight ratio and heart weight to tibia length ratio were significantly reduced in dnTRPC6 mice 6 weeks post MI as opposed to WT mice (8.4 vs. 7.3 and 13.4 vs. 11.7, respectively *P<0.05). Hypertrophic markers of ANP, BNP and βMHC followed similar downregulation trend in the dnTRPC6 mice. In conclusion, loss of TRPC6 function slows the progression of cardiac dysfunction and cardiac remodeling in the post MI heart.
Effect of miRNA-223-3p Targeting Stromal Interaction Molecule 1 on Proliferation and Apoptosis of Hypoxia/Reoxygenation-Applied H9c2 Cardiomyocytes
Authors: Guang Bin , Liu Xiaoqin , Liang Tingchen
Published: June 2021

This study was established to determine the effect of miRNA-223-3p on the proliferation and apoptosis of hypoxia/reoxygenation-applied H9c2 cardiomyocytes and the associated mechanisms. A hypoxia/reoxygenation (H/R) model was established, with normal cells also used as a control. miRNA-NC,
miRNA-223-3p, anti-miRNA-NC, and anti-miRNA-223-3p plasmids were transfected into normally cultured cardiomyocytes, defined as the miRNA-NC, miRNA-223-3p, anti-miRNA-NC, and anti-miRNA-223-3p groups. In addition, miRNA-223-3p was co-transfected into normally cultured cardiomyocytes with pcDNA3.1
and pcDNA3.1-STIM1 plasmids, followed by treatment with H/R for cells in the miR-NC and miR-223-3p groups, defined as the H/R+miRNA-NC, H/R+miRNA-223-3p, H/R+miRNA-223-3p+pcDNA3.1, and H/R+miRNA-223-3p+pcDNA3.1-STIM1 groups. A liposome method was adopted for assessing transfection. qRT-PCR
was used to detect miRNA-223-3p expression, while western blotting was used to detect protein expression. MTT assay was used to detect cell viability, flow cytometry to detect apoptosis, and dual luciferase reporter gene assay to detect fluorescence activity. After H/R treatment, miR-223-3p,
cyclin D1, and Bcl-2 expression of cardiomyocytes decreased, p21 and Bax expression significantly increased, cell activity decreased, and the apoptosis rate increased. miRNA-223-3p achieved the targeted regulation of STIM1 expression. miRNA-223-3p overexpression promoted the H/R-induced cardiomyocyte
proliferation and inhibited cardiomyocyte apoptosis. STIM1 overexpression reversed the proliferation-promoting and apoptosis-inhibiting effects of miRNA-223-3p on cardiomyocytes treated with H/R. The findings show that miRNA-223-3p overexpression promotes H/R-induced cell proliferation, inhibits
apoptosis, and protects H/R-induced cardiomyocytes from injury, via a mechanism probably associated with STIM1 expression. miRNA-223-3p thus provides a new target for treating cardiomyocyte injury.
miR-216a-5p Suppresses the Proliferation, Invasion and Migration of Fibroblast-Like Synoviocyte in Rheumatoid Arthritis by Targeting Zinc Finger and BTB Domain-Containing Protein 2 (ZBTB2)
Authors: Zhao Jinwei , Li Ling
Published: June 2021

MicroRNAs have been reported to be associated with the initiation and progression of rheumatoid arthritis (RA). miR-216a-5p, one of the miRNAs, is involved in cancer cell proliferation, invasion and migration. However, the role of miR-216a-5p in RA remains to be explored. The expressions
of miR-216a-5p and zinc finger and BTB domain-containing protein 2 (ZBTB2) in fibroblast-like synoviocytes (FLS) of RA or healthy controls were detected by qRT-PCR and western blot analysis. Transfection of overexpressed and silenced miR-216a-5p were performed to explore the functional role
of miR-216a-5p in RA-FLS. Cell Counting Kit-8 (CCK-8) assay and transwell assay were employed to assess cell proliferation and cell invasion, respectively. Moreover, luciferase reporter assay was executed to verify the combination of miR-216a-5p and ZBTB2. The results showed that miR-216a-5p
expression in RA-FLS was downregulated than healthy controls. Overexpres-sion of miR-216a-5p inhibited RA-FLS cell proliferation, invasion and migration, while miR-216a-5p silencing revealed the opposite results. In addition, ZBTB2 was identified to be a direct target of miR-216a-5p in RA-FLS
and its expression was higher than that in healthy controls. Rescue experiments revealed that ZBTB2 overexpression reversed the effects of miR-216a-5p on the proliferation, invasion and migration of RA-FLS. These data indicated the suppressive role of miR-216a-5p in RA-FLS via the regulation
of ZBTB2, suggesting that miR-216a-5p and ZBTB2 may be the new targets for the treatment of RA.