bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 164: Action of Nicotinic Acid on Adiponectin, Leptin and Plasminogen Activator Inhibitor 1 Production and Expression In 3T3-L1 Adipocytes Under Oxygen Deprivation.
Authors: Nakamichi Renata , Quinto Beata M , Zanella Maria T , Batista Marcelo C
Published: March 2022

Obesity has been considered one of the major contributors to the current global mortality. Recent studies suggest the dysregulation production of adipokines is partly responsible for obesity linked metabolic disorders.

The aim of this study was to investigate the effect of nicotinic acid (NA) on adipokines production and expression in 3T3-L1 adipocytes before and after hypoxia.

The mature adipocytes were pretreated with NA for 24h and induced by hypoxia for 4, 8 and 12h. Levels of adipokines in medium and total hypoxia inducible factor-1 alpha (HIF-1α) were quantified using immunoassay. Adipokines expression was analyzed by real time PCR.

Adipocytes treated with NA had lower leptin production in comparison to spontaneous production and an increase on leptin and Plasminogen Activator Inhibitor 1 (PAI-1) production associated with different periods of oxygen deprivation. It was observed a reduction of leptin levels in cells pretreated with NA compared to cells submitted to hypoxia. The same effect could be observed on PAI-1 production.

NA increased the production of adiponectin comparing to spontaneous production and cells pretreated witch NA increased adiponectin production during hypoxia.

Adipocytes were submitted to periods of oxygen deprivation had raised HIF-1α production comparing to spontaneous production and NA treatment. It also observed a reduction of HIF-1α in cells pretreated with NA and submitted to hypoxia.

Real time PCR showed a raise of adiponectin mRNA and a decreased PAI-1 in adipocytes treated with NA in comparison to spontaneous expression, but didn’t exert effect on spontaneous leptin.

Hypoxia, in all periods increased the leptin and PAI-1 expression and decreased adiponectin expression. In adipocytes pre-treated with NA blunted the production of leptin in spontaneous conditions and under hypoxia. It also increased PAI-1 production and expression. Adiponectin production and expression raised under NA treatment. The translation of such“in vitro” nicotinic acid benefits to the clinical setting should be object of future studies.
Abstract 167: Dietary Medium-chain Triglyceride Supplementation Has No Effect on Apolipoprotein B-48 and Apolipoprotein B-100 Kinetics in Insulin-resistant Males
Authors: Tremblay André J , Lamarche Benoit , Lepine Marie-Claude , Lemelin Valery , Couture Patrick
Published: March 2022

Medium-chain triglyceride (MCT) supplements are used by clinicians to treat patients with severe hypertriglyceridemia at risk for pancreatitis. However, the potential mechanisms underlying the triglyceride-lowering effects of MCT have not been thoroughly examined yet in human. This double-blind crossover study compared the impact of a 4-week supplementation with MCT oil 20 g/d vs corn oil 20 g/d on the intravascular kinetics of apolipoprotein (apo) B-48-containing triglyceride-rich lipoproteins (TRL) and apoB-100-containing VLDL as well as on the expression of key intestinal genes involved in lipid metabolism in 28 obese insulin-resistant males. In vivo kinetics of TRL apoB-48 and VLDL apoB-100 were assessed using a primed-constant infusion of L-[5,5,5-D3]leucine for 12h in the fed state. Expression of key intestinal genes involved in lipid metabolism was assessed in a subgroup of participants (n=16) by real-time PCR quantification in duodenal biopsy performed at the end of each phase of the supplementation in the fasted state. Compared with corn oil, MCT supplement had no significant effect on plasma lipoprotein profile and the TRL apoB-48 and VLDL apoB-100 kinetics. There were significant positive correlations at the end of the MCT supplementation between expression of SREBP-2 and expression of HNF-4α (r=0.51; P<0.05), HMGCoAR (r=0.75; P<0.001), ACAT-2 (r=0.51; P<0.05), NPC1L1 (r=0.66; P<0.05), ACS-1 (r=0.74; P<0.001), LDL receptor (r=0.67; P<0.05) and PCSK-9 (r=0.60; P<0.05) in intestinal tissue. However, MCT supplement had no significant impact on the expression of those key lipid and fatty acid regulation genes compared with corn oil. These data indicate that 4-week supplementation with MCT in obese insulin resistant men has no effect on TRL apoB-48 and VLDL apoB-100 kinetics and on the intestinal expression of genes involved in lipid and fatty acid metabolism.
Abstract 185: Infliximab Suppresses TNF-a Induced Inflammatory Phenotype in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Formation
Authors: Ali Muhammad S , Starke Robert M , Jabbour Pascal M , Tjoumakaris Stavropoula I , Gonzalez L. Fernando , Rosenwasser Robert H , Koch Walter J , Dumont Aaron S
Published: March 2022

Background

Cerebral Aneurysm rupture results in significant morbidity and mortality. TNFα has been associated with cerebral aneurysms, but potential mechanisms have not been established, and its role in phenotypic modulation is unknown. Therapy targeting TNFα may represent a novel treatment strategy for cerebral aneurysms.

Hypothesis

Infliximab inhibits TNFα induced phenotypic modulation and upregulation of pro-inflammatory mediators.

Methods

Cultured cerebral VSMC were incubated with increasing concentrations of TNFα and gene expression measured with qPCR. Cerebral aneurysm induction surgery was performed in rats. Animal were euthanized at 4 wks for qPCR and immunohistochemical analysis of the circle of Willis. Animals undergoing aneurysm induction and Infliximab (5mg/kg) treatment were compared to animals undergoing aneurysm induction alone and untreated controls.

Results

In vitro experiments demonstrated suppression of differentiation markers and induction of pro-inflammatory genes. In vivo, TNFα mRNA and protein expression was significantly elevated following aneurysm induction compared to untreated controls. Expression of inflammatory markers including chemokines (MCP1 and VCAM1), transcription factors (NF-κB, KLF4), matrix remodeling proteins (MMP-2 & 3), IL-1β and iNOS was also increased. Furthermore, aneurysm induction resulted in phenotypic modulation by decreasing expression of SMC differentiation marker gene SM-α−Actin. Treatment with Infliximab significantly suppressed TNFα expression, inhibited expression of inflammatory markers, and decreased the number of CD68+ cells during aneurysm formation. Infliximab also reversed phenotypic modulation in vascular SMC following aneurysm induction.

Conclusions

TNFα plays a pivotal role in SMC phenotypic modulation with induction of pro-inflammatory genes and inhibition of SMC differentiation marker genes both in cultured cerebral vascular SMC and in vivo following cerebral aneurysm induction. Infliximab promotes anti-inflammatory effects in cerebral vessels by inhibiting TNFα and suppressing a pro-inflammatory cascade, which has important implications for the mechanisms behind intracranial aneurysm formation and potential therapeutic options.
Abstract 196: Effects of an Engineered Herpes Simplex Virus (HSV-1) on Vascular Cell Adhesion
Authors: McCormick Susan , He Qi , Weichselbaum Ralph , Skelly Christopher L
Published: March 2022

Introduction

The genetically engineered HSV R7020 is known to block vascular smooth muscle cell (SMC) proliferation while allowing for re-endothelialization after balloon angioplasty. This HSV-1 anti-proliferative effect requires MEK activation. However, it is unknown what effect R7020 has on vascular inflammation and monocyte adhesion. We hypothesized that exposure of SMCs to HSV R7020 would not affect vascular adhesion molecules.

Methods

Human aortic SMCs were exposed to R7020 for 10 minutes in the absence and presence of TNF-α (10 ng/ml). After washing cells were cultured for 24 hours in the absence and presence of TNF-α. Vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecue-1 (ICAM-1) levels were determined using immunocytochemistry and image J. The adhesion of calcein AM labeled PBMCs to these cells were also analyzed at 24 hours. VCAM-1 and ICAM-1 levels were measured in the carotids of New Zealand White Rabbits that underwent balloon injury and exposed to R7020 (n=4) or saline (n=4) for 10 minutes using microarray analysis.

Results

There was no significant difference in ICAM-1 levels in the R7020 treated and control SMCs (219±11 vs 223±1; p=0.758); there was an 8 fold increase in ICAM-1 in the TNF-α treated SMCs (219±11 vs 1587±68; p=1.2x10
-13
). Results were similar for VCAM-1. R7020 caused an increase in the adhesion strength of SMCs to collagen coated glass slides at 24 hours. Microscopic experiments (n=3) showed PBMCs adhered similarly to SMCs treated with TNF-α in the absence and presence of the virus (4 times that of control cells as measured by PBMC adhesion assay). Interestingly, after adhesion, Calcein AM was frequently observed in virus infected SMCs in the presence and absence of TNF-α. In cells not exposed to R7020, this rarely occurred. Microarray analysis demonstrated a 2.3 fold reduction in VCAM-1 expression in the R7020 treated rabbits (p=2.8x10
-14
) and no significant difference in expression in ICAM gene expression (p=0.3).

Conclusions

In contrast to TNF-α, R7020 does not increase ICAM-1 or VCAM-1. Both treatments domonstrate PBMC SMC adhesion with no additive or synergistic effect. However, R7020 results in intracellular transfer from PBMCs to SMCs suggesting the virus changes PBMC-SMC interplay.
Abstract 203: Smooth Muscle Cells from Abdominal Aortic Aneurysms are Unique and Can Independently and Synergistically Degrade Insoluble Elastin
Authors: Airhart Nathan , Brownstein Bernard , Cobb Perren , Schierding William , Arif Batool , Grapperhaus Kathy , Ennis Terri , Thompson Robert , Curci John A
Published: March 2022

Background

The purpose of this study was to further elucidate the role of the vascular smooth muscle cell (SMC) in abdominal aortic aneurysm (AAA) disease. We hypothesized that that AAA-SMC are unique and actively participate in the process of degrading the aortic matrix.

Methods and Results

Whole-genome expression profiles of SMC from AAA, normal abdominal aorta (NAA) and carotid endarterectomy (CEA) were compared. We quantified elastolytic activity by culturing SMC in [3H]elastin-coated plates and measuring solubilized tritium in the media after 7 days. MMP-2 and MMP-9 production was assessed using real-time PCR, zymography and western blotting. Each SMC type exhibited a unique gene expression pattern. AAA-SMC had greater elastolytic activity than NAA (+68%, p<0.001) and CEA-SMC (+45%, p<0.001). Zymography showed an increase of active-MMP-2 (62kD) in media from AAA-SMC. AAA-SMC demonstrated 2-fold greater expression of MMP-2 mRNA (p<0.05) and 7.3-fold greater MMP-9 expression (p<0.01) than NAA-SMC. Culture with U937 monocytes caused a synergistic increase of elastolysis by AAA-SMC (41%, p<0.001) but not NAA or CEA (p=0.99). Co-culture with U937 caused a large increase in MMP-9 mRNA in AAA and NAA-SMC (p<0.001). MMP-2 mRNA expression was not affected. Western blots of culture media showed a 4-fold increase of MMP-9 (92kD) protein in AAA-SMC/U937 compared to NAA-SMC/U937 (p<0.001) and a large increase in active-MMP2 (62kD) which was less apparent in NAA/U937 media (p<0.01).

Conclusions

AAA-SMC have a unique gene expression profile and a pro-elastolytic phenotype that is augmented by macrophages. This may occur via a failure of post-transcriptional control of MMP-9 synthesis.
Abstract 217: Coronary Endothelial Dysfunction is Associated With Increased Risk of Osteoporosis
Authors: Prasad Megha , Reriani Martin , Khosla Sundeep , Lennon Ryan , Gulati Rajiv , Prasad Abhiram , Lerman Lilach O , Lerman Amir
Published: March 2022

Background

A growing body of evidence links coronary artery atherosclerosis and calcification to osteoporosis in women. Coronary endothelial dysfunction (CED) is associated with adverse cardiovascular events. We aimed to study the association of CED and development of osteoporosis.

Methods

Coronary vascular reactivity was evaluated in 225 women, greater than age 50, with non-obstructive coronary arteries, by administration of intracoronary acetylcholine during diagnostic angiography. CED was defined as <50% increase in coronary blood flow (CBF) from baseline in response to maximal dose. After a median follow-up of 7 years, patients were assessed by a questionnaire for development of osteoporosis.

Results

The average age of the cohort was 60 years. Of 225 women, 60% (135) had CED. 18.2% (41 of 225 women) developed osteoporosis during follow-up. Women with CED were twice more likely to develop osteoporosis vs. women without CED (23% vs. 11.1%, p=0.024). Women who developed osteoporosis compared to those who did not had a markedly lower response (% change) in CBF: median (IQR)-4.3 (-28.3, 51.7) vs. 39.9 (-8.2, 106.0), p=0.007. In univariate analysis, CED was significantly associated with development of osteoporosis (p=0.024, relative risk 2.07, 95% CI: 1.07, 4.00). Multivariate analysis indicated CED as an independent predictor of osteoporosis (p=0.04).

Discussion

Endothelial dysfunction may account for or share similar mechanisms with development of osteoporosis in cardiac patients.
Abstract 218: Orphan Nuclear Receptor Nur77 is a Novel Negative Regulator of ET-1 Expression in Vascular Endothelial Cells
Authors: Qin Qing , Chen Ming , Yi Bing , Li Pan , You Xiaohua , Sun Jianxin
Published: March 2022

Objective

Endothelin-1 (ET-1), a vasoactive peptide mainly produced by vascular endothelial cells, plays an important role in the regulation of vascular tone and the development of cardiovascular diseases. The objective of the present study is to identify the novel regulators implicated in the regulation of ET-1 expression in vascular endothelial cells (ECs), and to determine the molecular mechanism involved.

Method and Results

by using quantitative real-time PCR (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA), we showed that either ectopic expression of Nur77 or pharmacological activation of Nur77 by 6-mercaptopurine (6-MP) substantially inhibited the ET-1 expression, under both basal and thrombin stimulated conditions, in human umbilical vein endothelial cells (HUVECs). Furthermore, knockdown of Nur77 enhances both basal and thrombin induced ET-1 expression, suggesting that Nur77 is a negative regulator of the ET-1 expression in vascular ECs. Inhibition of ET-1 expression by Nur77 mainly occurs at the gene transcriptional levels, since Nur77 potently inhibited the ET-1 promoter activity, without affecting the ET-1 mRNA stability. As shown in electrophoretic mobility shift assay (EMSA), Nur77 over-expression substantially inhibited both basal and thrombin stimulated transcriptional activity of AP-1, which is a critical transcriptional factor implicated in the ET-1 expression in ECs. Mechanistically, we found that Nur77 specially interacts with c-Jun in the nuclear fraction of endothelial cells by immunoprecipitation and inhibits the AP-1 dependent c-Jun promoter activity, which eventually leads to the decreased expression of c-Jun, a critical component for both AP-1 transcriptional activity and ET-1 expression in ECs.

Conclusions

These results demonstrate for the first time that Nur77 is a novel negative regulator of the ET-1 expression in vascular ECs through an inhibitory interaction with c-Jun/AP-1 pathway. Targeting Nur77 may represent a potentially novel therapeutic strategy for preventing certain cardiovascular diseases, such as atherosclerosis, vascular complications of diabetes, and pulmonary artery hypertension.
Abstract 228: Distinct Roles of PDGF-C and VEGF in Impaired Angiogenesis of Diabetes and Its Implication in Therapeutic Angiogenesis
Authors: Moriya Junji , Ferrara Napoleone
Published: March 2022

Background

Platelet-derived growth factor-C (PDGF-C) is known to promote angiogenesis independently of vascular endothelial growth factor (VEGF), although its significance in postnatal angiogenesis
in vivo
remains poorly understood.

Methods and Results

We employed murine model of hind limb ischemia and found that expression of PDGF-C and its receptor PDGFR-alpha were markedly up-regulated in ischemic limbs. Treatment with a neutralizing antibody against PDGF-C significantly impaired blood flow recovery and neovascularization after ischemia almost to the same extent as treatment with a VEGF neutralizing antibody. Moreover, PDGF-C- deficient mice showed a reduced blood flow recovery after ischemia compared to wild-type mice, confirming a strong pro-angiogenic activity of PDGF-C.

Next we injected an expression vector encoding the PDGF-C gene into ischemic limbs. Blood flow recovery and neovascularization after ischemia were significantly improved in the PDGF-C-treated group compared with the control group.

It has been reported that angiogenic response to ischemia is attenuated in diabetes even after VEGF treatment, although a precise mechanism remains unknown. We hypothesized that PDGF-C might relate to the impaired angiogenesis of diabetes. To test this hypothesis, we created murine model of diabetes by intraperitoneal injection of streptozotocin (STZ). We found that expression levels of PDGF-C at baseline and after ischemia were both significantly lower in limb tissues of diabetic mice than in those of control mice, while expression levels of VEGF and other members of the PDGF family were not changed or even higher in diabetic mice. Unlike in non-diabetic mice, introduction of VEGF gene into ischemic limbs did not improve blood flow recovery in diabetic mice. On the other hand, these changes were effectively reversed by introduction of the PDGF-C gene.

Conclusion

These results indicate that down-regulation of PDGF-C expression in limb tissues of diabetic mice contribute to impaired angiogenesis and suggest that introduction of PDGF-C would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective, such as in the diabetic state.
Abstract 23: miR-455-3p Overrides Transcription and Inhibits Cholesterol Biosynthesis
Authors: Toth Cynthia L , Levin Michael G , Wassif Christopher A , Porter Forbes D , Remaley Alan T , Vickers Kasey C
Published: March 2022

Cholesterol biosynthesis is under tight control by sterol sensing proteins and transcriptional regulation; however, recent evidence suggests that this pathway may also be post-transcriptionally regulated by microRNAs (miRNA). miR-455-3p was found to be significantly up-regulated in mouse livers with high-fat diets, which coincided with significant repression of miR-455-3p target genes, as determined by cumulative distribution fraction analysis of gene expression microarray data. Ten of fourteen cholesterol biosynthetic enzymes were also found to be significantly down-regulated in the livers of these mice; however, sterol regulatory element binding transcription factor 2 (SREBF2) activity was found to be significantly increased, not decreased. Multiple enzymes in the cholesterol biosynthetic pathway were predicted to be miR-455-3p targets; therefore, we hypothesized that miR-455-3p is a master regulator of cholesterol production through direct and indirect targeting of enzymes in the biosynthetic pathway. Furthermore, inhibition of miR-455-3p with locked-nucleic acids (LNA) significantly increased mRNA levels of all cholesterol enzymes analyzed in vitro, including HMGCR, CYP51A1, DHCR7, SC4MOL, and SC5DL. Conversely, miR-455-3p over-expression resulted in decreased mRNA levels of specific cholesterol biosynthesis enzymes. Most importantly, miR-455-3p inhibition significantly increased cholesterol biosynthesis in multiple cell lines from multiple species, as determined by radiolabeled acetate incorporation assays; whereas miR-455-3p transfection decreased cholesterol biosynthesis. Using gene reporter (luciferase) assays, miR-455-3p was found to directly target the 3’ untranslated region of 7-dehydrocholesterol reductase (DHCR7). Inhibition of miR-455-3p in DHCR7 mutated cell lines representing Smith-Lemli Opitz Syndrome, which results from non-complete dysfunction of DHCR7, significantly increased DHCR7 mRNA levels and cholesterol biosynthesis. In summary, miR-455-3p is a powerful inhibitor of cholesterol biosynthesis through post-transcriptional regulation that likely overrides SREBF2 transcriptional regulation within cholesterol metabolism.
Abstract 238: Aortic Intima-Media Thickness by M-mode or 2-Dimensional Semi-Automated Imaging: Do These Methods Provide Equivalent Results?
Authors: Macias Leonardo , Schevchuck Alex , Roldan Paola C , Qualls Clifford R , Sibbitt Wilmer L , Middendorf Julia , Rodan Carlos A
Published: March 2022

Background

Aortic (Ao) atherosclerosis is best defined by manual quantitation of intima-media thickness (IMT) using M-mode. However, M-mode imaging is time consuming and requires experience. Thus, we aimed to determine if Ao IMT assessed by M-mode and 2-dimensional (2-D) semi-automated imaging provide similar results.

Methods

57 volunteer subjects (51 women, age 36±12 years) underwent multiplane transesophageal echocardiography (TEE) using a 7 MHz phased array transducer with a 0.1 mm axial resolution. At low depth (3-4cm) and with a narrow sector scan to improve image resolution, 2-D guided M-mode images were obtained to assess IMT of the anterior wall of the proximal, mid, and distal descending thoracic Ao. At each Ao level, 3 IMT measurements were performed from Ao long axis views during end-diastole. Studies were measured off-line using electronic calipers by an experienced observer. A second observer unaware of M-mode results and using 2-D semi-automated imaging performed Ao IMT measurements from long axis images at each Ao level and same site where M-mode measurements were obtained.

Results

IMT of proximal, mid, and distal Ao by M-mode was similar to that obtained by 2-D semi-automated imaging (p≥0.19) (
Table
). Less variability of IMT values occurred by 2-D than by M-mode imaging (coefficient of variation 17% vs 38%). Both imaging methods were highly correlated (overall r=0.69, p<0.001).

Conclusion

Assessment of Ao IMT by TEE M-mode and 2-D semi-automated imaging provide equivalent results.
Abstract 24: Thrombus Formation is Impaired in Glutamate Receptor Interacting Protein 1 Deficient Mice
Authors: Modjeski Kristina L , Ture Sara K , Field David J , Morrell Craig N
Published: March 2022

Platelets contribute to the pathogenesis of myocardial infarction and stroke. Understanding mechanisms of platelet activation and thrombus formation may provide new targets for anti-platelet therapies. GRIP1 (glutamate receptor interacting protein 1) is a 7 PDZ domain scaffolding protein which localizes receptors at the membrane in a regulated manner. Previous work has shown that glutamate receptors increase platelet activation through glutamate receptor signaling and we wanted to investigate the regulation of these receptors. In the neuron, GRIP1 regulates surface expression of GluR2, however, GRIP1 KO mice are embryonic lethal due to hemorrhage and hypovolemia indicating severe vascular defects. Endothelial cells do not appear to express GRIP1, therefore, we hypothesize that GRIP1 mediates platelet activation by PDZ domain coordination of protein-protein interactions. We have discovered that GRIP1 is present in mouse and human platelets. To explore the role of GRIP1 in platelets we have created a platelet-specific GRIP1
-/-
mouse. GRIP1
-/-
mice have normal platelet counts but significantly decreased thrombus formation (p<0.05) during ferric chloride injury and their bleeding time is significantly longer (p<0.05) in tail bleeding assays. They have no observed spontaneous bleeding or premature death. CD62P and JON/A expression after stimulation with thrombin, 2-meADP,
U46619
, or convulxin was unchanged compared to WT mice. To determine which protein-protein interactions could be mediating the GRIP1 effects in platelets, we performed IP of GRIP1 and mass spectrometry. GRIP1 showed an association with the GP1b complex. Aggregation after stimulation with thrombin and 2-meADP is also impaired. Ongoing studies will determine whether specific members of the GP1b complex associate with GRIP1 in IP assays, flow chamber adhesion, and botrocetin aggregation to complement our in vivo data. Based on our data, GRIP1 has a central role in platelet thrombus formation through non-glutamate receptor dependent interactions.
Abstract 241: MicroRNA-24 Controls Macrophage Survival in Murine Abdominal Aortic Aneurysm Via Chi3l1
Authors: Spin Joshua M , Maegdefessel Lars , Raaz Uwe , Toh Ryuji , Adam Matti , Nakagami Futoshi , Deng Alicia , Eriksson Per , Tsao Philip S
Published: March 2022

Abdominal aortic aneurysm (AAA) is a major source of vascular mortality, and effective treatments remain elusive. MicroRNAs (miRs) are key regulators of vascular homeostasis and pathology. Array profiling and qRT-PCR of aneurysmal aortic tissue in two murine AAA models (elastase-infused C57/Bl6, and angiotensin II(AngII)-treated ApoE-/- mice) showed significant down-regulation of miR-24. Levels of a recently identified inflammatory mediator and predicted target of miR-24, YKL-40/Chi3l1 (chitinase 3 like-1), were inversely correlated with miR-24 expression in both models. Previously we showed that aortic over-expression of miR-24 significantly reduced inflammatory activity and macrophage survival, leading to a dramatic decrease in AAA expansion in both mouse models.

We explored the roles of miR-24 and Chi3l1 in macrophages
in vitro
. Specifically, we examined peritoneal macrophages obtained from AngII-treated ApoE-/- mice and RAW (mouse leukemic monocyte/macrophage) cells, stimulated with interleukin-6 (IL6) to trigger inflammation and apoptosis. In both cell types, modulation of miR-24 through transfection with anti-miR-24 (to inhibit) or pre-miR-24 (to overexpress) altered apoptotic response to IL6 by caspase and annexin assays. Pre-24 significantly decreased macrophage survival, while anti-24 abrogated the pro-apoptotic effects of IL6. Again, Chi3l1 was inversely correlated with miR-24 expression, and treatment with recombinant Chi3l1 led to dose-dependent attenuation of apoptosis. As expected, IL6 treatment decreased miR-24, and increased Chi3l1. This effect was mediated by the NF-kB pathway. Prior to IL6 treatment, macrophages were transfected with siRNA against either RelA (p65) or Nfkb1 (p50) (key components of NF-kB signaling). Knock-down was >75% by qRT-PCR, and both demonstrated significant decreases in resultant NF-kB activity. Both methods attenuated the impact of IL6 treatment on miR-24 (vs. neg. control siRNA). These results suggest that the apoptotic effects of inflammatory stimuli on macrophages in AAA are opposed by a drop in miR-24 and a rise in its target Chi3l1, permitting macrophage infiltration and survival. This would explain in part the positive effects of pre-miR-24 on AAA progression
in vivo
.
Abstract 250: Surface Microscale Topograhical Features Promotes Endothelial Cell Proliferation and Migration on a Nanocomposite Vascular Graft Material
Authors: Chong Debra S , Dalby Matthew J , Gadegaard Nikolaj , Seifalian Alexander M , Hamilton George
Published: March 2022

Objectives

Micro and nanoscale surface topographical features have been shown to have an influence on cellular behaviour. Endothelisation of small diameter vascular grafts has long been thought to have beneficial and protective effects as well as ensuring patency of the graft. Our objective is to explore the potential of using surface topographical features on a biocompatible nanocomposite polymeric material to produce a ‘self-endothelising’ vascular graft.

Methods

Photolithography was used to fabricate microscale topographical grooves onto a mask. These features were then heat-casted onto a nanocomposite polymeric material made of polyhedral oligomeric silsesquioxane (POSS) and poly (carbonate urea) urethane (PCU). Fidelity of the microscale grooves were confirmed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Human umbilical vein endothelial cells (HUVECs) were seeded onto POSS-PCU sheets with different microscale grooves and cellular behaviour was monitored daily. Cell proliferation and metabolic activity was measured using a colorimetric assay. Adhesion of the cells were confirmed using fluorescence microscopy.

Results

Our results show that grooves can be reproduced with high fidelity using masks produced by photolithography onto POSS-PCU nanocomposite polymer. Ridges of width 12.5 μm with channels widths of 25μm and subsequent depths of 300 - 700 nm were measured and confirmed using SEM and AFM These ordered grooves enhance cell spreading and adhesion compared with flat polymer surfaces. Furthermore, cell proliferation is further improved on these microscale features compared to featureless surfaces.

Conclusion

Microscale grooves on the surface of a nanocomposite polymeric material promotes increased endothelial cell adhesion, migration and proliferation. These ordered topographical features could be used to produce a new generation of vascular grafts with ‘self-endothelising’ potential.
Abstract 264: Edaravone Injected at the Start of Reperfusion Suppresses Lung Damage Caused by Myonephropathic Metabolic Syndrome in Rats
Authors: Yamamura Mitsuhiro , Mitsuno Masataka , Tanaka Hiroe , Ryomoto Masaaki , Fukui Shinya , Miyamoto Yuji
Published: March 2022

Introduction & Hypothesis

Free radicals have been implicated in myonephropathic metabolic syndrome (MNMS), which damages not only muscles but also lungs. At ATVB 2012, we previously reported that radical scavenger, edaravone (Radicut®, Mitsubishi Tanabe Pharma Co., Japan) injected at the start of reperfusion suppresses muscle injury following leg ischemia in rats. In this study, we evaluated whether edaravone can suppress MNMS-induced lung damage following leg ischemia, if injected at the start of reperfusion.

Methods

The MNMS models (Male Lewis rats, 525 ± 78 g, n = 10) were induced, by clamping the bilateral common femoral arteries for 5 hours and then de-clamping. The rats were intraperitoneally injected with either 9.0 mg / kg of edaravone (edaravone group; n = 5), or saline (MNMS group; n = 5) at the same time as de-clamping, corresponding to the start of reperfusion. Five hours after de-clamping, the both lungs were harvested and stained with hematoxylin & eosin (HE). Normal lungs without reperfusion were also harvested as control (n = 3). Lung damage was expressed as the percentage area of the alveolar wall thickness, determined by computerized densitometry (NIH Image Program J, Ver. 1.37).

Results

The lungs of MNMS group had extensive swelling. But the lungs of the edaravone group were filled with air, similar to that of normal lungs (Figure attached, original 200x). The mean percentage area in the edaravone group was significantly lower than that in MNMS group (17.2 ± 5.2 vs. 63.6 ± 5.6%, p < 0.001).

Conclusions

Our results suggest that edaravone injected at the start of reperfusion can suppress MNMS-induced lung damage following leg ischemia in rats.
Abstract 271: Scavenger Receptor Bi is a Key Determinant and HDL is a Critical Modulator of Thymocyte Apoptosis in Sepsis
Authors: Li Xiang-an , Guo Ling , Zheng Zhong , Ai Junting , Howatt Deborah , Daugherty Alan
Published: March 2022

Background-

Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood.

Methods and Results-

We induced sepsis in scavenger receptor BI (SR-BI) null and wild type littermates with cecal ligation and puncture (CLP). CLP induced profound thymocyte apoptosis in SR-BI
+/+
mice, but no thymocyte apoptosis in SR-BI
-/-
mice, indicating that SR-BI is a key determinant of thymocyte apoptosis in sepsis. SR-BI controlled inducible glucocorticoid (GC) generation during sepsis, which is required for triggering thymocyte apoptosis. Unexpectedly, supplementation with GC only partly restored thymocyte apoptosis in SR-BI
-/-
mice. We demonstrated that HDL is a critical modulator for thymocyte apoptosis. SR-BI
+/+
HDL significantly enhanced GC-induced thymocyte apoptosis but SR-BI
-/-
HDL had no such activity. Further study revealed that SR-BI
+/+
HDL modulates GC-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI
-/-
HDL loses such regulatory activity. To understand why SR-BI
-/-
HDL loses its regulatory activity, we analyzed HDL cholesterol contents and found that there was 3-fold enrichment of unesterified cholesterol in SR-BI
-/-
HDL compared with SR-BI
+/+
HDL. Importantly, normalization of unesterified cholesterol in SR-BI
-/-
HDL by probucol administration restored GC-induced thymocyte apoptosis both
in vitro
and
in vivo
, and incorporating unesterified cholesterol into SR-BI
+/+
HDL rendered SR-BI
+/+
HDL dysfunctional.

Conclusions-

The findings in this study reveal a previously unrecognized regulatory mechanism of thymocyte apoptosis in sepsis, involving SR-BI as a key determinant and HDL as a critical modulator.
Abstract 274: HDL-Mediated Cellular Cholesterol Efflux in Patients with Severe Carotid Atherosclerosis
Authors: Hafiane Anouar , Doonan Robert J , Daskalopoulou Styliani , Genest Jacques
Published: March 2022

The objective of this study was to examine the function of high-density lipoproteins (HDL) in patients with severe carotid atherosclerosis undergoing carotid endarterectomy (CEA) and in patients with coronary artery disease (CAD).

We examined clinical, biochemical and lipoprotein profiles and HDL cholesterol efflux capacity (CEC) in 155 patients undergoing CEA and compared the results with patients with an acute coronary syndrome (ACS time 0 n=26) measured within 48 hours of the event, and again at 12 weeks (ACS recovery, n=26), in patients with chronic, stable CAD (sCAD) n=27 and in healthy controls. There were 110 men (71%) and 45 women (29%) with a mean age 69±10 years in the CEA group. Mean HDL-C was 0.96±0.26 mmol/L (38±10 mg/dL). There were no significant differences in HDL-C between CEA, ACS and sCAD subjects.

HDL was obtained after depletion of apo B-containing lipoproteins with PEG precipitation. Cellular efflux capacity was determined by incubating HDL in cAMP-stimulated J774 mouse peritoneal macrophages for 6 hours. Specific cholesterol efflux was obtained by subtracting total efflux from efflux in non-cAMP stimulated cells. The procedure was standardized to enable medium throughput. Coefficient of variability was approximately 5%.

HDL from CEA patients differ in its ability to promote cholesterol efflux. The range of CEC observed was: min 14.7%, max 34.0% mean 22.4±0.6%; (IQR 18.9-27.3% median 22.0). We found a weak correlation between HDL-C and CEC in patients CEA (r= 0.20, P=0.012); a significant correlation was found between apo A-I and CEC (r=0.23, P=0.005). Subjects with similar HDL-C or apoA-I differed in their ability to promote cellular cholesterol efflux, suggesting that the HDL-C mass does not reflect functionality. We found similar CEC in patients with CAD and ACS recovery.

Here, we report that patients with severe carotid atherosclerosis have low HDL-C and a CEC similar to that observed in CAD patients. The function of HDL to promote cellular cholesterol efflux, as determined by CEC, varied over a wide range and was only weakly correlated with HDL-C. The significant correlation between apo AI and CEC suggests that HDL particle size -and composition may also be an important determinant of the ability to promote cellular cholesterol efflux.
Abstract 281: Cyclophilin A is a Novel Biomarker for Oxidative Stress and Atherosclerotic Diseases
Authors: Satoh Kimio , Fukumoto Yoshihiro , Sugimura Koichiro , Miura Yutaka , Aoki Tatsuo , Nochioka Kotaro , Tatebe Shunsuke , Tatebe Shunsuke , Yamamoto Saori , Shimizu Toru , Takagi Yusuke , Tsuburaya Ryuji , Ito Yoshitaka , Matsumoto Yasuharu , Nakayama Masaharu , Takeda Morihiko , Takahashi Jun , Ito Kenta , Yasuda Satoshi , Shimokawa Hiroaki
Published: March 2022

Background

We have recently demonstrated that oxidative stress induces secretion of cyclophilin A (CyPA) from vascular smooth muscle cells, which plays a crucial role in the pathogenesis of atherosclerosis and abdominal aortic aneurysms (AAA) in mice. Thus, we tested our hypothesis that plasma CyPA levels could be a biomarker of atherosclerotic diseases such as coronary artery disease (CAD) and AAA in humans.

Methods and Results

In consecutive 320 patients undergoing coronary angiography, we examined the relationship between the severity of CAD and plasma CyPA levels measured by an immunoassay. Plasma CyPA levels were significantly higher in patients with significant coronary stenosis (>50%, n=189) compared to those without it (n=131) (P<0.001). A significant positive correlation was noted between plasma CyPA levels and significant coronary stenosis even after adjustment for age, sex, hypertension, diabetes, dyslipidemia and smoking. The average number of coronary arteries with significant stenosis and the need for coronary intervention were significantly increased in the quartiles of higher CyPA levels (both P<0.001). In addition, plasma CyPA levels were significantly correlated with the number of coronary arteries with significant stenosis (P<0.001). Indeed, plasma CyPA levels were a strong predictor of CAD (adjusted odds ratio for CAD, 6.20; 95% confidence interval, 3.14-12.27; P<0.001). Interestingly, plasma levels of CyPA increased according to the number of atherosclerotic risk factors that enhance oxidative stress. Furthermore, plasma CyPA levels significantly reduced after medical treatment of those risk factors. Finally, pathological examinations showed that CyPA was strongly expressed in atherosclerotic coronary plaque of patients with myocardial infarction. In a separate study, we examined the plasma CyPA levels in patients with aortic aneurysms. Plasma CyPA levels were significantly higher in patients with aortic aneurysms (n=58) compared to those without it (n=69) (P<0.001). Importantly, plasma CyPA levels were significantly higher in patients coexisting with CAD (n=18) compared to those only with aortic aneurysms (n=40) (P=0.012).

Conclusion

Plasma CyPA level is a novel biomarker of atherosclerotic diseases.
Abstract 288: Differences in Proliferative and Migratory Capability of Human Myoblasts Isolated From CLI and Control Patients
Authors: Yu Rebekah Sian Hwee , Abraham David , Baker Daryll , Tsui Janice
Published: March 2022

Objectives

Patients with critical limb ischemia (CLI) have attributable myopathy that impairs their functional ability as well as dampening the positive effect of successful revascularisation outcomes. Adult skeletal muscle retains the potential to repair following different injury patterns. We aim to investigate the potential differences in proliferation and migration capability of human myoblasts with CLI and asymptomatic control patients.

Methodology

Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated and cultured from each sample and stained with the myoblast marker desmin. Confirmed myoblast cultures (<5% alternative cell type contamination) were then used to investigate the proliferative capability (MTT assay) as well as migratory potential (scratch-wound assay) under both ischaemic and normoxic conditions of each group.

Results

Myoblasts isolated from CLI patients demonstrated greater proliferative ability in comparison to control samples. However, control samples revealed greater capacity to heal scratch wounds made in a monolayer of myoblasts in both normoxia and hypoxic conditions.

Conclusion

Isolation of human myoblasts, especially from disease groups provides a useful platform to perform in vitro analysis to better understand and characterise pathology. CLI myoblasts exhibited enhanced proliferative but diminished migratory potential in comparison to control myoblasts.
Abstract 291: In Situ Re-endothelialization via Multifunctional Nanoscaffolds
Authors: Su Lee-Chun , Xu Hao , Tran Richard T , Tsai Yi-Ting , Tang Liping , Yang JIan , Nguyen Kytai T
Published: March 2022

The endothelium monolayer lining in the luminal side of blood vessels provides critical anti-thrombotic activities. Damage to these cells will expose a highly thrombogenic subendothelium that stimulates thrombosis. Standing on the concept of tissue engineering and ligand-receptor targeting strategy, we developed a novel biodegradable urethane-doped polyester (UPE) multifunctional targeting nanoparticle (MTN) scaffold system with dual ligands: 1) GP1b-α to target the injured arterial endothelium and subendothelium and 2) anti-CD34 antibodies to capture endothelial progenitor cells (EPCs) for endothelium regeneration. The fabricated spherical MTNs of 400 nm were shown to be biocompatible and hemocompatible. Both the
in vitro
and
ex vivo
targeting of these nanoscaffolds not only showed improved binding efficiency of MTNs onto the von Willebrand Factor (vWF) coated surfaces presented as the injured arterial walls, but also outperformed platelets for binding onto these injured sites. Our
in vivo
study further showed 57% neointimal hyperplasia suppression and a 70% increase in endothelium regeneration within 21 days after delivering of MTNs upon vascular injury. These results indicate that our injectable MTN nanoscaffolds show a potential non-invasive way for endothelium regeneration
in situ
.
Abstract 30: Leveraging the Innate Immunity Pathway for Transdifferentiation of Fibroblasts to Endothelial Cells
Authors: Sayed Nazish , Wong Wing Tak , Cooke John P
Published: March 2022

Background

Cell-based approaches to regenerate the endothelium holds promise, with one such candidate source being induced pluripotent stem cells (iPSCs). We have investigated the potential of endothelial cells (ECs) derived from iPSCs to promote the perfusion of ischemic tissue in a murine model of peripheral artery disease (PAD). However, it may be more efficient to transdifferentiate fibroblasts to ECs directly. Recent reports have suggested that direct reprogramming to ECs is feasible, however still requires the use of viral vectors encoding transcription factors, thus making them clinically unsafe.

Hypothesis

We recently discovered that retroviral vectors encoding the reprogramming factors, by activating the Toll-like receptor 3 (TLR3) pathway, make nuclear reprogramming possible by increasing epigenetic plasticity and favoring an open chromatin state (Lee & Sayed et al. Cell). Based on this recognition that innate immunity favors an open chromatin state, we hypothesized that activation of TLR3, together with external microenvironmental cues that drive EC specification, might induce transdifferentiation of fibroblasts into ECs (“iECs”).

Results

Intriguingly, our preliminary data showed that TLR3 agonist Poly I:C, combined with exogenous endothelial growth factors, was sufficient to transdifferentiate fibroblasts into iECs (in the absence of viral vectors or transcription factors). These iECs exhibited all the characteristics of EC phenotype comparable to HMVECs including ability to form capillary-like structures and incorporating acetylated-LDL. Moreover, loss-of function studies showed that activation of TLR3 plays a role in the efficient transdifferentiation of human cells when using Poly I:C. Furthermore, iECs significantly improved limb perfusion and neovascularization in the ischemic hindlimb compared to control groups.

Conclusion

This study is a first step toward development of a regenerative strategy for PAD on the use of ECs derived from small molecules and growth factors without use of viral vectors encoding transcription factors. Moreover, it has allowed us to generate a small molecule strategy for therapeutic transdifferentiation that might be applied for direct reprogramming in vivo.
Abstract 305: Role of Endothelin-1 Signaling in Osteogenic Differentiation and Matrix Calcification of Vascular Smooth Muscle Cells
Authors: Miller Stephanie L , Nguyen Ngoc B , Speer Mei Y
Published: March 2022

Vascular calcification (VC), the “hardening” of blood vessels and valves, is prevalent in patients with type II diabetes mellitus (T2DM). Although often asymptomatic at early stages, patients with VC have increased risk for cardiovascular morbidity and mortality. It is presently unknown whether diabetes is causative and what regulatory molecules and pathways are critical for initiation and progression of this complication. In a diabetic VC mouse model recently examined in our laboratory, vascular smooth muscle cells (SMCs) were found to be the major contributing cell source, with increased susceptibility to osteochondrogenic differentiation and calcification under hyperglycemia. Endothelin-1 (ET1) was high in diabetic mouse sera and cartilaginous areas of diabetic blood vessels, correlating with an increase in its type A receptor (ETA), osteochondrogenic transcription factor Runx2, and receptor for advanced glycation end-products (RAGE). Importantly, blocking the biological activity of ETA with its antagonists effectively inhibited matrix calcification of SMCs in culture associated with high glucose at levels of hyperglycemia. Addition of the ETA antagonists, atrasentan and BQ-123, to SMC cultures resulted in an inhibition of SMC calcification up to 71%, whereas ETB antagonism with BQ-788 resulted in only a slight reduction (~20%). Additionally, SMCs treated with atrasentan expressed higher mRNA levels of myocardin, a master transcription factor co-activator of SMCs, and lower expression levels of Runx2, suggesting a preservation of SMC phenotype in their original fate decision through ETA antagonism. Further exploration of ET1-ETA signaling through addition of ET1 to SMCs cultures showed a dose-dependent increase in SMC calcification, occurring only in high glucose cultures. Finally, blocking RAGE signaling with a neutralizing antibody reduced not only high glucose-accelerated SMC calcification but also the calcification enhanced by exogenous ET1. Taken together, our results showed a correlation of ET1-ETA with diabetic vascular osteochondrogenesis and its critical role in high glucose-associated SMC calcification. A possible interplay between the ET1-ETA and RAGE pathways is currently under investigation.