bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 196: Matrix Metalloproteinases Regulation in Thrombus Resolution Is Independent of TGF-β in a PAI-1 Ko Mouse Model of Venous Thrombosis
Authors: Obi Andrea T , Wakefield Thomas W , Ballard-Lipka Nicole E , Farris Diana M , Myers Daniel D , Henke Peter K , Diaz Jose A
Published: March 2022

Introduction:
Plasminogen activator inhibitor (PAI-1) is the primary regulator of urokinase (u-PA) and tissue (t-PA) plasminogen activators. Observations in mice with impaired fibrinolysis have demonstrated an alternate pathway of thrombus resolution by matrix metalloproteinase (MMP)- 9 and 2. TGF-β is a potent inducer of TIMP-1 expression and is found in both thrombus and vein wall post-thrombosis.

Hypothesis:
PAI-1 deficiency and resulting hyperfibrinolytic state will improve thrombus resolution and result in reciprocal downregulation of MMP-2 and MMP-9 via TGF-beta induction of TIMP-1, resulting in decreased vein wall fibrosis.

Methods:
Inferior vena cava (IVC) ligation was performed in wild-type (WT) mice (N=194) and PAI-1 deficent mice (N=164). IVC, thrombus and blood samples were collect at 2, 6 and 14 days post procedure. The IVC and thrombus were assessed for thrombus weight and samples collected for zymography (MMP-2 and MMP-9), ELISA (TIMP-1, TGF-β) and histology.

Results:
PAI-1 deficient mice had similar thrombus weight (TW) versus WT at 2 days, but smaller at days 6 (p<0.0001) and 14 (p=0.0085). In thrombus and vein wall (VW), active MMP-9 was decreased in acute thrombosis (p<0.0001), and MMP-2 was decreased during late thrombus resolution (p<0.05). TIMP-1 levels in VW were elevated across all time points (p<0.05), whereas VW TGF-beta was not different compared to WT. Neutrophils were decreased at day 2 (p = 0.0309) and monocytes decreased at day 14 (p = 0.0028).

Conclusions:
(1) PAI-1 deficient mice exhibit a hyperfibrinolytic phenotype with normal thrombotic response to IVC ligation, but improved thrombus resolution when compared to WT mice. (2) MMP-9 and MMP-2 were suppressed, confirming a reciprocal relationship between plasmin and MMP activation as mediators of thrombus resolution. (3) TIMP-1 production was elevated in vein wall independent of TGF-beta. (4) Decreased neutrophils at day 2 correlated with decreased MMP-9 and decreased monocytes at day 14 correlated with decreased MMP-2, suggesting a plasmin-induced inhibition of leukocyte recruitment. (5) Final histology showed no difference in vein wall fibrosis or thickness at chronic time points, indicating that improved thrombus resolution does not lessen vein wall fibrosis.
Abstract 20: Evidence of UGT2B7 as a Pharmacogenetic Determinant of Variability in Serum Fenofibric Acid Levels in Human Subjects
Authors: Arafah Azher , Guillemette Chantal , Tsai Michael Y , Boreen Christopher R , Straka Robert J
Published: March 2022

BACKGROUND:
Genetic polymorphisms affecting the drug metabolizing gene [UDP-glucuronosyltransferases or UGTs-(specifically
UGT2B7
)] have been reported by our group as a significant source of variability in the lipid-lowering response to fenofibrate. Fenofibrate’s lipid-lowering response displays exceptionally high inter-subject variability which may represent one source of the mixed results from outcome based clinical trials such as ACCORD and FIELD. Given the association between lipid response and fenofibric acid serum concentrations, the purpose of this study was to investigate the pharmacokinetic significance of the
UGT2B7
promoter SNP (A-327G SNP (rs7662029)) on serum fenofibric acid concentrations in humans.

METHODS:
Using a genotype guided approach, we prospectively recruited subjects (>18 years) whose genotype was either AA or GG for
UGT2B7
A-327G to receive 28 days once daily fenofibrate (145mg). Steady-state area under the serum fenofibrate concentration vs. time curve (AUC
0-24
) was calculated from 8 determinations on day 28. Fasting lipid profiles where collected at baseline and after fenofibrate administration.

RESULTS:
49 (39% male) individuals with a mean (±SD) age of 33 (±11.8) years completed the study. This included 26 with AA and 23 with GG for
UGT2B7
A-327G. The mean (±SD) AUC
0-24
was 218.4±98 μg*hr/mL for AA and 300±131 μg*hr/mL for the GG individuals (
p
=0.017). Overall, associations between higher AUC
0-24
and percent change in lipid fraction from baseline, were confirmed for TG (r
2
=0.26, p-value= 0.0002), total cholesterol (r
2
=0.12, p=0.014), LDL-C (r
2
=0.17, p= 0.0032,), non-HDL-C (r
2
=0.11, p=0.019) but not HDL-C (NS). Adjusted for BMI,
UGT2B7
A-327G explained 22% of the fenofibric acid AUC
0-24
, with AA individuals having 27.3% lower AUC
0-24
compared to GG individuals.

CONCLUSIONS:
We confirm
UGT2B7
A-327G to be a significant determinant of serum fenofibric acid concentrations and hence a potential important source of fenofibrate’s variability in lipid response and/or clinical outcomes.
Abstract 210: Crossveinless 2 Regulates Bone Morphogenetic Protein 9 in Human and Mouse Vascular Endothelium
Authors: Yao Yucheng , Jumabay Medet , Ly Albert , Wang Anthony H , Abdmaulen Raushan , Bostrom Kristina I
Published: March 2022

The importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced in a staged fashion by the activin-like kinase receptor 1 (ALK1) after stimulation by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation by ALK2, ALK1, BMP-4 and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression and proliferation of ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in a thickened, abnormal endothelium with increased markers of endothelial differentiation. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.
Abstract 215: Inflammation-Induced Thrombogenic Phenotype in the Microvasculature: The Roles of Enhanced Platelet Production and Reactivity
Authors: Senchenkova Elena Y , Yildirim Alper , Komoto Shunsuke , Granger D Neil
Published: March 2022

It is well recognized that different pathological conditions associated with inflammation are also accompanied by altered platelet function and enhanced thrombus development. Experimental colitis (induced by dextran sodium sulfate, DSS) and angiotensin II (AngII)-induced hypertension (HTN) are two examples of pathologically distinct inflammation models that exhibit enhanced platelet adhesion in venules and accelerated thrombus formation in arterioles. Whether these responses represent alterations in platelet production, life-span, and/or function remains unclear. In this study, we compared the changes in platelet production and reactivity (to thrombin) that occur in murine models of DSS colitis and AngII-induced HTN. Mature (TO-) and immature (TO+) platelets in blood were quantified using thiazole orange (TO) staining with flow-cytometry. The in vivo biotinylation method was used to determine the life-spans of TO+ and TO- platelets. Platelet aggregation velocity was measured using a low-angle light scattering technique. Thrombus formation, induced by light/dye injury, was monitored in cremaster muscle arterioles and platelet adhesion was quantified in the venules. Our findings indicate that both models of inflammation promote platelet adhesion and enhanced thrombus formation in the microvasculature. The enhanced thrombosis normally detected in both DSS-induced colitis and AngII-induced hypertension models was not evident in interleukin-6 (IL-6) deficient mice and in WT mice treated with an IL-6 receptor blocking antibody. DSS colitis was accompanied by an increased appearance of both TO+ and TO- platelets, increased platelet reactivity (aggregation) to thrombin, and no change in platelet turnover. The AngII HTN model exhibits an increase in platelet turnover, an increased number of TO- (but not TO+) platelets, and no change in platelet aggregation, suggesting that an altered endothelial cell phenotype likely makes a larger contribution to the accelerated arteriolar thrombosis in this model. The divergent responses in these two IL-6 dependent models of microvascular thrombosis may reflect the relative importance of platelets and microvascular endothelial cells as molecular targets for IL-6 mediated thrombogenic responses.
Abstract 219: Tumor Necrosis Factor α Drives Lung Endothelial Cell Apoptosis During Ischemic Acute Kidney Injury
Authors: White Laura E , Cui Yan , Hassoun Heitham T
Published: March 2022

Introduction:
Ischemic acute kidney injury (AKI) alters the innate immune response, activating cellular and soluble mediators which perpetuate microvascular barrier dysfunction and distant organ injury. We have previously identified that lung TNFR1 activation is a critical pathway to programmed cell death in an
in vivo
lung injury model during ischemic AKI.

Objectives:
Hypothesizing that lung endothelial cells (ECs) are a key target for programmed cell death, we investigated lung EC-specific transcriptional and functional pathways to TNFR1-dependent apoptosis during ischemic AKI.

Methods:
Male Sprague-Dawley rats (∼250gm) underwent 60 minutes of bilateral renal pedicle occlusion (IRI) or sham laparotomy (sham) and were sacrificed at 24hrs. Rat lung microvascular ECs (RLMVECs) grown to confluence were treated with serum from rats obtained following sham or kidney IRI +/- Etanercept, a TNFα/TNFR1 signaling inhibitor, or vehicle. RLMVECs underwent 1) custom RT-PCR analysis for pro-apoptotic, pro-inflammatory, and TNF superfamily candidate genes, 2) caspase-3 activity assay, and 3) PARP activity assay during 2,4, or 24 hours of ischemic AKI. *p<0.05 vs. sham, paired t-test; n≥4/group.

Results:
Compared to sham, RLMVECs exposed to IRI serum displayed a pro-apoptotic transcriptome during ischemic AKI with activation of TNF superfamily (TNFR1, TNFR2, TNFα) and apoptosis genes (Fasl, Dapk1, Bcl10) over a time course. RLMVECs demonstrated phenotypic programmed cell death with increased caspase-3 activation (100±1.12 vs. 106.5±0.79*) and PARP activity (10±3.47 vs. 61.75±20.45*) at 24 hours during ischemic AKI. Treatment with Etanercept ameliorated activation of the pro-inflammatory EC transcriptome (E-Selectin, ICAM-1, IL-6 and RhoB) and also attenuated programmed cell death with no increase in caspase-3 activity (100±0.88 vs. 101.98±0.48, p=0.096) or PARP activity (10±2.33 vs. 10.91±2.15, p=0.783) compared to vehicle during ischemic AKI.

Conclusions:
Ischemic AKI produces distinct changes in the pulmonary EC phenotype with activation of pro-inflammatory and pro-apoptotic pathways driven by TNFα/TNFR1 signaling. Further investigation of potential mechanisms of kidney-lung crosstalk during AKI may identify potential therapeutic targets for this deadly disease.
Abstract 225: Prognostic Markers in Young Patients with Premature Coronary Heart Disease
Authors: van Loon Janine E , de Maat Moniek P , Deckers Jaap W , van Domburg Ron T , Leebeek Frank W
Published: March 2022

Introduction.
So far little is known on the prognosis of young patients with coronary heart disease (CHD) and possible predictors of recurrent events. Therefore, we aimed to evaluate the survival and prognostic implications of traditional cardiovascular risk factors and inflammatory and prothrombotic markers in patients with premature CHD.

Methods.
We obtained follow-up data from 353 young patients with a first cardiac event (men ≤ 45 years and women ≤ 55 years). Baseline characteristics on traditional cardiovascular risk factors were collected at the time of the first event, and levels of C-reactive protein (CRP), von Willebrand Factor (VWF), and fibrinogen were measured one to three months after the first event to exclude an acute phase response. We performed age and sex adjusted Cox’ regression analyses to assess the relationship between these markers and recurrent events (presented as hazard ratios (HR) with 95% confidence intervals). We used three different endpoints: all cause mortality, recurrent cardiac event (myocardial infarction or revascularisation procedure), and any recurrent event (cardiac event, cerebrovascular event or all cause mortality).

Results.
During a total follow-up time of 1483 person years (mean 4.2 years), 42 patients had a recurrent cardiac event (12%), 11 patients died (3%), and two patients had a cerebrovascular event (<1%). Traditional risk factors were not more frequent among patients with a recurrent event than among patients without a recurrent event. However, CRP was associated with an increased risk of any recurrent event (1.27 [1.01-1.59] per unit increase in lnCRP). Also, both CRP and fibrinogen were associated with all cause mortality. The HR for all cause mortality was 5.00 [1.04-24.04] for CRP levels above the 50
th
percentile and 5.04 [1.05-24.2] for fibrinogen levels above the 50
th
percentile. VWF levels were not associated with recurrent events.

Conclusions.
Fifteen percent of young patients with a first cardiac event have a recurrent event or die after a median follow-up of 4.2 years. Elevated levels of CRP and fibrinogen are associated with the risk of recurrent events in these young patients.
Abstract 23: Identification of a Novel Intracellular Cholesteryl Ester Hydrolase (CES3) in Human Macrophages: Compensatory Increase Following CES1 Silencing
Authors: Bie Jinghua , Wang Jing , Ghosh Shobha
Published: March 2022

Removal of free cholesterol (FC) from lesion-associated macrophage foam cells represents the major mechanism for reducing the lipid burden of atherosclerotic plaques and intracellular cholesteryl ester (CE) hydrolysis is recognized as the rate-limiting step in this process. We have earlier reported cloning of human macrophage CE hydrolase (gene symbol CES1) that associated with cytoplasmic lipid droplets and enhanced cellular CE mobilization. Macrophage-specific transgenic expression of CES1 attenuated diet-induced atherosclerosis and lesion necrosis in ldlr
-/-
mice. Several enzymes have since been identified as intracellular CE hydrolases in human macrophages and the objective of the present study was to determine the relative contribution of CES1 to the total CE hydrolytic activity in human macrophages. Immune-precipitation by CES1-specific antibody resulted in 70-80% decrease in enzyme activity indicating that CES1 is responsible for >70% of the total CE hydrolytic activity (Panel A). To examine the effects of CES1 depletion/knockdown on FC efflux, human monocyte/macrophages (THP1) were stably transfected with 4 different CES1-specific shRNA vectors (p81-p84) to generate THP1-shRNA cells; cells transfected with pRSL vector were used as controls. Despite significant reduction in CES1 expression both at mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel carboxylesterase, CES3 (Panel B). Human macrophage CES3 was identical to the reported CES3 except it lacked exon 8 and, in wild type THP1 cells it was only expressed at 29.2±6.1 percent of CES1. Transient over-expression of this novel variant of CES3 led to an increase in CE hydrolytic activity (>4 fold), mobilization of intracellular lipid droplets and reduction in cellular CE content (108.1±7.65 vs 160.9±7.00, P=0.0005), establishing CES3 as a bona fide CE hydrolase. Reciprocal backup circuits, where deficiency of a gene leads to an increase in another gene with similar function, have been described and this study provides first evidence of functional compensation in intracellular CE hydrolysis whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. To gain insight into the mechanisms underlying the observed increase in CES3 following CES1 deficiency
in silico
analyses of CES3 promoter was performed and several NF-kB binding sites were identified. Based on our earlier demonstration of a decrease in NF-kB activation by CES1 over-expression, it is speculated that conversely, deficiency of CES1 increases NF-kB activation leading to compensatory increase in CES3 expression.
Abstract 230: Protein Kinase D1 Mediates Vegf-induced Vascular Permeability In Vitro And In Vivo By Promoting Endothelial Cell Stress Fiber Formation
Authors: Zhao Jinjing , Yang Ying , Jin Felix Q , Liu Microbian , Wong Chelsea , Mastrangelo Michael A , Jin Zheng Gen
Published: March 2022

Background

Vascular hyperpermeability is critical components of many human diseases, including inflammation, cancer and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a potent vascular permeability factor. Better understanding of the molecular mechanisms by which VEGF induces vascular permeability may provide novel therapeutic targets for these diseases. We and others have recently demonstrated that protein kinase D1 (PKD1), a newly discovered serine/threonine protein kinase, is implicated in VEGF signaling. We hypothesized that PKD1 plays a crucial role in VEGF-induced vascular permeability in vivo and vitro by promoting endothelial cell stress fiber formation.

Methods and results

Using Tie2-Cre transgenic mice and PKD1 flox mice, we generated endothelium-specific PKD1 knockout (PKD1-ecKO) mice. PKD1-ecKO mice were viable and born at the expected Mendelian ratio. Comparing with wild type mice, PKD1-ecKO mice exhibited the dramatic reduction of VEGF-induced vascular permeability in lung and ear with Miles assay, expressed as Evans blue leakage. Selective pharmacological PKD inhibitor also significantly attenuated in vivo vascular permeability induced by VEGF intravenously injection in mice. In cultured endothelial monolayer, down-regulation of PKD1 by siRNA, GFP-PKD1 dominant negative mutant plasmid or PKD inhibitor significantly reduced VEGF-induced endothelial permeability, measured by bovine serum albumin leakage through endothelial monolayer cultured on transwell system. Moreover, we found that the inhibition of PKD1 diminished F-actin stress fiber formation and the dismantlement of endothelial adherens junctions that lead to intercellular gap formation and leakage in response to VEGF. We further observed that PKD1 acted as a downstream mediator of Rho activation in VEGF-induced stress fiber formation. Specifically, Rho inhibitor Y27632 blocked VEGF-induced PKD1 phosphorylation, while down-regulation of PKD1 expression and activation did not affect VEGF-stimulated Rho activation in endothelial cells.

Conclusion

PKD1 is required for VEGF-induced vascular permeability in vivo and in vitro through promoting F-actin stress fiber formation and disrupting endothelial adherens junctions.
Abstract 235: Epigenetic Mediator PCAF Moderates Arteriogenesis
Authors: Bastiaansen A J , Ewing M M , Nossent A Y , Peters H A , de Vries M R , van Oeveren-Rietdijk A M , de Boer H C , van Zonneveld A J , Quax P H
Published: March 2022

Introduction:
Collateral artery formation or arteriogenesis is a highly regulated process involving multiple factors and cell types, with a pronounced role for the inflammatory system. In the current study we focus on a transcriptional co-activator P300/CBP-associated factor (PCAF). PCAF, also known as lysine acetyltransferase (KAT)2B, has histone acetylating activity and promotes inflammatory gene transcription. The functional role of PCAF in the process of arteriogenesis is unknown.

Methods and results:
To assess the role of PCAF in arteriogenesis we used a murine hindlimb ischemia model. We demonstrated that PCAF mRNA expression after the surgical induction of hindlimb ischemia is reduced in both adductor and calf muscle of wildtype (WT) mice. In vitro, the lack of PCAF resulted in a reduced inflammatory response in whole blood, vascular smooth muscle cell and splenocyte stimulation assays. In vivo, the PCAF
-/-
phenotype showed a hampered blood flow recovery after double ligation of the femoral artery, measured by laser Doppler perfusion imaging (ratio 0.42±0.08 in PCAF
-/-
vs 0.71±0.08 in WT at day 7) and by immunohistological analysis of collateral arteries. Also pharmacological inhibition of PCAF by local administration of garcinol reduced blood flow recovery after single femoral artery ligation (ratio 0.76±0.07 with garcinol vs 1.07±0.06 in control at day 7).

PCAF deficiency influences arteriogenesis locally by disturbing the regulation of pro-arteriogenic genes in the ischemic adductor muscle. Moreover PCAF
-/-
mice showed a different post-ischemic regulation of circulating inflammatory cells. In particular, PCAF
-/-
mice showed reduced numbers of circulating monocytes (0.12*10E6/mL in PCAF
-/-
vs 0.37*10E6/mL in WT) and fewer monocytes were detected around the collateral arteries already one day after surgery.

Conclusions:
We conclude that PCAF regulates arteriogenesis by modulating the post-ischemic inflammatory response.
Abstract 236: Significant Mechanical Puncture Resealing Properties of a Novel (Nano-Composite) Compliant Hemodialysis Access Graft Compared with PTFE Conduit Currently in Clinical Use
Authors: Chong Debra S , Batra Ramesh , Lindsey Ben , Seifalian Alex M , Hamilton George
Published: March 2022

Aims:
The compliance, non-thrombogenicity and biodurability of POSS-PCU graft have been reported in
in vitro
studies.
In vivo
studies reveal high patency rate at 9 months (to be published data). A further application of this graft could be in vascular access, where currently PTFE (polytetrafluoroethylene) is the prosthetic conduit of choice. Resilience to puncture with large guage (15-17Ch) dialysis needles is vital for a graft being used as vascular access conduit. We performed ex-vivo tests to compare this specific property between POSS-PCU and PTFE grafts.

Methods:

A flow circuit containing human blood was used to mimic the arterial system using a phantom pulse generator. Lengths of test graft (8 - 10 cm) were connected to the circuit and submerged in a 2 litre isotonic (0.9%) saline bath. The pulse generator was set at 80 beats/min. A single 15 Ch dialysis needle was used to puncture the full wall thickness of the graft. Samples of saline bath were taken at distances of 2.5cm and 5cms from the puncture site at 5 minute intervals, for a period of 30 minutes. The concentration of red blood cells (cells/litre) were calculated at each time and location point for each graft being compared.

Results:

Macroscopically, the POSS-PCU graft ’self-sealed’ within seconds after the initial puncture whereas the PTFE puncture site had not sealed by 30 minutes. There was about 15 times lower concentration of red blood cells seen in the samples with POSS-PCU graft compared with PTFE at both 2.5 and 5cm. POSS-PCU as compared to PTFE leaked less red blood cells over a total of 30 minutes. The results were statistically significant (p<0.0001) when analysed with the Friedman test.

Conclusions:
A significantly better resilience to needle puncture of POSS-PCU over PTFE graft suggests an improved alternative for vascular access surgery. This may result in safer earlier needling and possibly also reflect better long term durability.
Abstract 242: Toll-like Receptor 3 Activation Is Involved in Venous Thrombosis Development
Authors: Lemarié Catherine A , Aghourian Meghedi N , Wu Jianqiu , Blostein Mark D
Published: March 2022

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. Inflammation may have a role in both the genesis and resolution of venous thrombi. Although septic thrombophlebitis occurs, most deep venous thrombosis are sterile. Recently, the role of toll-like receptor (TLR) signaling in modulating sterile inflammation has become better defined in experimental injury models. TLR3, for example, is activated by viral long double stranded RNA and endogenous RNA released by cellular apoptosis and necrosis, and induce a proinflammatory cellular response. Short single-stranded RNA, such as those used in RNA inhibition, stimulate TLR3 in endothelial cells and inhibit neoangiogenesis. Thus, we hypothesize that TLR3 might be involved in the inflammatory development of venous thrombosis. Intravenous injection of polyinosine polycytidylic acid (polyI:C), a synthetic double-stranded RNA analog, increase the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle (p<0.05). TLR3 stimulation with polyI:C is also associated with an increased fibrin, neutrophil and macrophage content (p<0.05). Using a specific fluorescent probe for RNA, we found that FeCl3 induces RNA release and thus increases RNA content in the thrombus. We next investigated the effect of RNase on thrombus development. Intravenous injection of RNase decreased thrombus size, as well as fibrin, neutrophil and macrophage content in the thrombus compared to vehicle (p<0.05). To verify the direct implication of TLR3, thrombosis was induced on TLR3 deficient mice using the FeCl3 model. Results showed that fibrin content after FeCl3-induced thrombosis is comparable between the groups. However, the occlusion of the IVC was only partial (80%) in the TLR3 deficient mice, whereas it was complete in the wild type animals. Taken together, these results strongly suggest that RNA release and TLR3 stimulation after endothelial cell injury participate in thrombus formation by inducing a pro-inflammatory response leading to the recruitment of neutrophils and macrophages.
Abstract 247: Thoracic Aortic Aneurysm and MicroRNA Expression in Bicuspid and Tricuspid Aortic Valve Patients: miR143/145 and miR133a/b Correlate with VSMC Phenotypic Change and CTGF Expression
Authors: Branchetti Emanuela , Sainger Rachana , Milewski Karianna , Poggio Paolo , Grau Juan B , Gorman Robert C , Bavaria Joseph E , Ferrari Giovanni
Published: March 2022

Introduction.
Altered microRNA (miRs) expression plays a role in vascular remodeling by regulating smooth muscle cells (VSMC) phenotype and Extracellular matrix (ECM) remodeling. Recently, miR 29 has been linked to thoracic aortic aneurysm (TAA) development by affecting the structural integrity of the vascular wall. TAA occurs in patients with a Bicuspid Aortic Valve (BAV) earlier and more rapidly than in those with a Trileaflet Aortic Valve (TAV). We have recently observed that TAV-TAA aorta is characterized by accumulation of VSMC with synthetic phenotype, ECM disorganization and cellular loss. In contrast BAV-TAA aorta shows accumulation of contractile VSMC. In addition, CTGF, a molecular determinant of VSMC phenotype, is elevated in non-dilated BAV aorta and in both BAV and TAV aneurismal tissues.

Hypothesis.
We hypothesized that a selective miRs expression may correlate with CTGF levels and VSMC phenotype in the ascending aorta of BAV and TAV patients with and without aneurysm.

Methods.
A bioinformatics analysis followed by miR143/145, 133a/b and 29a expression tested by quantitative real-time PCR was performed in aortic tissues collected from 16 BAV and TAV patients with and without TAA (n=4 patients/group).

Results.
We identified a direct correlation between altered miRs expression, CTGF levels and VSMC phenotype in BAV and TAV patients with and without aneurysm: miR-143 was significantly downregulated in TAV-TAA tissues where we observed an accumulation of synthetic VSMC, while was upregulated in BAV non dilated aorta. miR-143/145 expression was upregulated in BAV non dilated and dilated tissues, where we observed an accumulation of contractile VSMC, while was not significantly changed in TAV-TAA tissues. miR-133a/b, recently described as suppressors of CTGF expression, were downregulated in BAV and TAV aneurismal tissues and in BAV non dilated aorta where CTGF levels are strongly upregulated. miR29a, recently reported as a regulator of aortic wall structure, was downregulated in TAV-TAA while no significantly changed in BAV non dilated and dilated tissues.

Conclusions.
The characterization of miR143/145 and miR133a/b in the tissue of BAV and TAV patients, and their ability to regulate CTGF and VSMC phenotype is a novel risk stratification tool for patients at high risk to develop TAA.
Abstract 251: Semiautomated Measurement of Ascending Aortic Aneurysm Versus Manual Measurement: Assessment of Variability
Authors: Entezari Pegah , Kino Aya , S Galizia Mauricio , Yaghmai Vahid , C Carr James
Published: March 2022

Purpose:
To evaluate inter method and intra observer variability of a novel semi-automated method for measurement of ascending aorta aneurysm (AAA) in ECG gated dual source CT angiogram (DSCTA).

Material and Method:
Under an IRB approved protocol, multiplanar reformatted images of 40 patients with AAA were measured in DSCTA images. Transaxial maximum diameter of inner wall to inner wall was measured at five anatomic locations of thoracic aorta: annulus, sinus, sinotubular junction (STJ), mid ascending aorta at the level of right pulmonary artery (MAA), and proximal aortic arch immediately proximal to innominate artery (PROX) (Figure 1). Measurements were performed by conventional manual method and the novel semi-automated segmentation software and compared. Inter method and intra observer variability were evaluated according to Intra Class Correlation (ICC). Time required for post processing of the aorta with both methods was recorded in all the cases.

Results:
Mean maximum diameters in semi-automated method were: annulus 31.10 mm, sinus: 41.95 mm, STJ: 38.05 mm, MAA: 41.88 mm, and PROX: 37.60 mm. Manual method showed mean maximum diameter of 30.87 mm, 42.05 mm, 36.87 mm, 41.18 mm, and 36.42 mm at annulus, sinus, STJ, MAA, and PROX, respectively.

There was an excellent inter method and intra observer agreement between two methods at all five points (ICC > 0.9). The post processing time was 8’2’’±1’35” for the semi-automated method while this time was 19’± 20” for the manual method.

Conclusion:
The novel semi-automated tool demonstrated an excellent agreement with conventional manual method in measurement of AAA in ECG gated DSCTA with the advantage of significant reduction in the post processing time.
Abstract 262: HIV-1--Infected Subjects with Low Viremia Have Dysfunctional HDL Compared with Healthy Subjects
Authors: Kelesidis Theodoros , Currier Judith S , Yang Otto O
Published: March 2022

Introduction:
Dysfunctional HDL may be a novel biomarker of atherosclerotic disease. Using a cell based assay in a small pilot study we have previously demonstrated for the first time that dysfunctional HDL is present in HIV-1-infected persons despite low viremia. However, the cell-based assays of functional properties of HDL are not applicable to large scale clinical studies. It has not been shown in the setting of a case control study whether HIV-1-infected subjects have dysfunctional HDL compared to healthy subjects.

Objectives:
To determine whether HIV-1-infected subjects with low viremia (viral load < 50 copies/ml) have dysfunctional HDL compared to healthy subjects using a novel cell free assay

Methods:
Using a novel assay based on the effect of HDL on the oxidation of dihydrorhodamine 123 (DHR) (J Lipid Res. 2011 Dec; 52(12):2341-51) we determined oxidative (functional) properties of different HDL samples isolated from plasma from 50 HIV-1 infected patients with low viremia on antiretroviral therapy and from 50 healthy volunteers matched for age and race. The DHR oxidation rate (DOR) in the presence of different samples of HDL was normalized by the DOR value of a control sample (HDL isolated from pooled plasma samples of healthy subjects) and the relative DOR was calculated.

Results:
The average relative DOR values from 50 HIV-1-infected patients was 1.46 ±0.29 versus 0.87 ±0.15% in 50 healthy subjects (p<0.0001)(Figure). These results indicate that HIV-1-infected subjects had approximately 60% more dysfunctional HDL compared to healthy subjects.

Conclusions:
HIV-1-infected subjects with low viremia have largely dysfunctional HDL compared to healthy subjects. Use of our novel fluorometric method may allow determination of the role of HDL functional phenotype in the development of atherosclerosis in vivo in the setting of clinical studies.
Abstract 267: Redox-Dependent Transcriptional Regulation: A Potential Mechanism for Collateral Growth Impairment During Chronic Oxidative Stress
Authors: DiStasi Matthew R , Labarrere Carlos A , McClintick Jeanette N , Li Lang , Miller Steven J , Unthank Joseph
Published: March 2022

Vascular risk factors characterized by oxidative stress are correlated with impaired collateral growth through mechanisms largely unknown. Spontaneously hypertensive rats (SHR) with increased oxidative stress were used to investigate potential mechanisms mediating impaired collateral compensation. Microarray analysis of global gene expression was performed with control and collateral mesenteric artery samples (n=4) from SHR and their normotensive controls (WKY) 24 hours after model creation. In SHR control arteries, several mechano-sensitive and redox-dependent transcription regulators were down-regulated including Jun (-5.2X, P=0.02), Egr-1 (-4.1X, P=0.01), and NFkB1 (-1.95X, P=0.04). Collateral arteries of WKY and SHR exhibited a fundamentally different gene expression pattern (<7% overlap), especially for molecules regulating cell growth and proliferation and gene expression. Predicted binding sites for NFkB and AP-1 were present in the genes altered in WKY but not SHR collaterals. Immunostaining demonstrated increased NFkB nuclear translocation in WKY but not in SHR collaterals (see figure). Increased NFkB nuclear translocation was also seen in collaterals of SHR pretreated with the anti-oxidant apocynin (Apo), which we have previously shown to restore a normal redox status and collateral growth. Based upon these results, we propose redox-dependent modulation of mechano-sensitive transcription factors as a mechanism to explain, at least in part, the fundamental differences in collateral gene expression between WKY and SHR and the impairment of collateral growth during chronic oxidative stress.
Abstract 27: Galectin-3 Binding Protein: A New Factor Promoting Venous Thrombosis
Authors: Diaz Jose A , Hawley Angela E , Wrobleski Shirley K , Shea Evelyn , Myers Daniel D , Wakefield Thomas W
Published: March 2022

Galectin-3 binding protein (Gal-3BP) was detected in procoagulant microparticles from mice and patients with venous thrombosis (VT), in prior studies from our laboratory. However, the mechanistic role of Gal-3BP and Galactin-3 (Gal-3) in the pathophysiology of thrombosis is unknown. Our hypothesis is that Gal-3BP and Gal-3 are critical to thrombus formation.

Methods:
Mice
: Using our inferior vena cava ligation mouse model of VT, microparticle samples from thrombosed and non-thrombosed wild type mice were analyzed for Gal-3BP. We then either neutralized Gal-3BP, using an antibody (Anti-M-Gal-3BP), or utilized Gal-3 knock-out (Gal-3 KO) mice, compared to mice given saline as controls (NaCl). We evaluated thrombus weight (TW), inflammatory cell counts, and the cell source of the molecules Gal-3BP and Gal-3 using western blot.

Patients:
Microparticles and plasma samples from patients who were positive or negative for VT were tested for Gal-3BP using ELISA.

Results:
Mice
: Two days after thrombosis, blocking Gal-3BP significantly reduced both vein wall inflammatory cells (
p
=0.0369) and TW (
p
=0.0024), compared to NaCl. In Gal-3 KO mice, TW was significantly smaller versus controls (
p
=0.0061). We found Gal-3BP on microparticles and platelets while Gal-3 was on monocytes.
Patients
: Gal-3BP was significantly elevated in microparticles (
p
=0.0495) and in plasma (
p
=0.0400), versus patients negative for VT.
Conclusions:
We demonstrated a clear link between Gal-3BP and VT, in both mice and patients. This is the first time that Gal-3BP has been shown to be involved with thrombus formation, making it a promising biomarker and potential target for therapeutic interventions of VT.
Abstract 270: Decrease of BNP Concentrations in Blood Perfusing Extremities of Patients with Advanced Peripheral Arterial Disease
Authors: Kurklinsky Andrew K , Heublein Denise M , Stockland Andrew H , Bjarnason Haraldur , Misra Sanjay , Mcphail Ian R , Friese Jeremy L , Andrews James C , Oderich Gustavo S , Burnett John C
Published: March 2022

Rationale:
The role of cardiac natriuretic peptides (NPs) in peripheral arterial disease (PAD) or thromboangiitis obliterans (TA) is unknown. Recent studies in mice suggested that BNP may be produced outside the heart in the ischemic limbs contributing to peripheral angiogenesis.

Objective:
Determine if synthesis or consumption/degradation of NPs occur in the ischemic legs of patients with PAD or TA.

Methods:
32 patients with critical limb ischemia (CLI) or claudication (PAD n=31; TA n=1; 20 males; mean age 72.9 years) underwent diagnostic angiography or revascularization. We determined differences of concentrations of BNP, NT-proBNP, and CNP in the blood of proximal veins and arteries of both legs. Concentrations differences were then compared between the more ischemic target and the contralateral self-control legs.

Results:
Values are means ± SD. P values <0.05 were considered significant.

In the PAD group, in the target extremity, concentration step-ups of NT-proBNP (11±68; p=0.40) and CNP (0.45±6.22; p=0.71) were not significant from the arterial to venous blood. In the contralateral self-control legs, NT-proBNP and CNP concentrations drops were also not significant (-37±160, p=0.7; -0.89±4.09, p=0.23). Venous BNP concentrations were significantly lower than arterial both in the target (-66±153, p<0.001) and self-control legs (-59±107, p<0.001). BNP concentrations drops were greater in the CLI than claudication cases driving the overall significance. In the patient with TA, whose only identifiable PAD risk factor was smoking, arterial concentrations of NPs were generally low (NT-proBNP 81; BNP 46; CNP 8.2), but higher than venous concentrations (80; 41; 6.7).

Conclusion:
Significantly lower venous BNP concentrations suggest possible BNP consumption/degradation in the ischemic legs. Concentrations of NT-proBNP, that has a longer half-life than BNP, were more stable suggesting no significant local production or conversion to BNP. CNP concentrations were stable. Recognizing the angiogenic properties of NPs and lack of evidence of peripheral production, studies are warranted in humans with PAD to determine the potential angiogenic properties of exogenous NPs supplementation in what could be considered a local NPs deficiency state.
Abstract 290: Key Intestinal Genes Involved in Lipoprotein Metabolism Are Downregulated in Insulin-Resistant Males
Authors: Couture Patrick , Tremblay Andre J , Guay Valerie , Lemelin Valery , Lamarche Benoit
Published: March 2022

Recent studies indicate that insulin-resistance (IR) is associated with elevated levels of triglyceride-rich lipoproteins (TRL), including apoB-48-containing TRL of intestinal origin, in both the fasted and post-prandial states. This is of interest because elevated levels of intestine-derived lipoproteins are associated with increased cardiovascular disease risk. However, the mechanisms underlying the overaccumulation of apoB-48-containing TRL in IR have not been fully characterized in human. Therefore, the objective of this study was to examine the relationships between apoB-48-containing TRL kinetics and expression of key intestinal genes involved in lipid and lipoprotein metabolism in nondiabetic, IR males compared with insulin-sensitive (IS) males matched for age and waist circumference. A total of 30 nondiabetic caucasian males were chosen for extremes of insulin sensitivity (14 IR and 16 IS subjects) and matched for age and waist circumference. In vivo kinetics of TRL apoB-48 were assessed using a primed-constant infusion of L-[5,5,5-D3]leucine for 12h in the fed state in all subjects. Expression of key intestinal genes involved in lipid and lipoprotein metabolism was compared by real-time PCR quantification in duodenal biopsy specimens from IR and IS participants taken in the fasted state. TRL apoB-48 pool size (PS) and production rate (PR) were increased by 105% (P<0.0001) and 45% (P=0.03), respectively in IR subjects compared with IS subjects. Expression of the nuclear transcription factors SREBP-2, SREBP1-C and HNF4α was significantly reduced by 31%, 28% and 25%, respectively in IR subjects. Similarly, many key genes involved in cholesterol, fatty acid, triglyceride and lipoprotein metabolism were significantly downregulated in IR individuals. In IR participants, intestinal expression of HMG CoAR, ACC1, SCD1, DGAT2, apoB and MTP was significantly reduced compared with IS subjects. Intestinal MTP expression was inversely correlated with TRL apoB-48 PS (r=-0.50, P=0.005) and PR (r=-0.32, P=0.07). These data suggest that IR is associated with intestinal overproduction of lipoproteins and downregulation of key intestinal genes involved in lipid/lipoprotein metabolism. Downregulation of these genes in the fasted state could represent an adaptive response to IR state.
Abstract 307: Influence of Dendritic Cells on Cholesterol Absorption and Excretion: Implication in Atherogenesis
Authors: Deswaerte Virginie , Huby Thierry , Saint-Charles Flora , Proschogo Nicolas , Beliard Sophie , Pirault John , Lesnik Philippe , Jessup Wendy
Published: March 2022

Atherosclerosis represents the chronic pathophysiological process implicated in the majority of cardiovascular (CV) diseases and constitutes one of the leading causes of death in the world. The development of atherosclerotic lesions is characterized by an accumulation of extracellular and intracellular lipids in the arterial wall. These phenomena are associated with a strong local immuno-inflammatory response characterized by the recruitment of various populations of leucocytes including monocytes, macrophages and dendritic cells (DCs).

DCs are central to the regulation of immunity and the polarization of the immune response. We recently demonstrated that the longevity/depletion of DCs directly impacts plasma cholesterol levels, which is the main risk factor for atherosclerosis.

To gain insight into the cellular and molecular mechanisms underlying the inverse relationship between DC numbers and cholesterolemia, we have evaluated the impact of DCs on cholesterol homeostasis, using CD11c-hBcl2/apoE KO mice, in which specific DC-expression of the anti-apoptotic transgene hBcl2 increase their longevity and numbers. Firstly, we quantified DC populations in the liver and in the intestine, which are the main organs involved in cholesterol metabolism. Secondly, we measured the rates of dietary cholesterol absorption, tissue cholesterol content and sterol and bile salt excretion. Thirdly, we analysed the expression of genes associated with cholesterol metabolism in the liver and the intestine.

We found an increase of DC numbers in the liver and in the intestine in the CD11c-hBcl2 apoE KO mice relative to control apoE KO mice. This increase of DC numbers was associated with reduced intestinal cholesterol absorption and fecal bile acid excretion, but also with greater fecal excretion of sterols. Finally, there were no differences in the cholesterol content of the intestine and the liver. Our results suggest that the decrease in plasma cholesterol level in CD11c-hBcl2 apoE KO mice relative to apoE KO mice could be due to both a decrease of intestinal cholesterol absorption and an increase in sterol excretion. The role of intestinal DCs in dietary cholesterol absorption and excretion is presently under investigation but the present study reveal that DCs are central to the atherosclerotic process, because they are implicated in cholesterol metabolism.
Abstract 309: The Role of TLT-1 in Atherosclerosis Lesion Formation
Authors: Gonzalez Marieli , Reyes Fiorella , Marrero Deborah , Washington A Valance
Published: March 2022

Platelets have a well documented role in atherosclerotic lesion formation. They play a role from the initiation of the lesion to final occlusion of an atherosclerotic vessel. While it is known that regulating aggregation is important to control the thrombus that leads to occlusion, the platelet’s role in promoting lesion formation is less defined. Molecules stored in the platelet a-granules such as p-selectin and cytokines, which promote the inflammatory side of platelet function, have been shown to affect lesion progression. These molecules, however, only provide an outline of the platelet function during this process. and thus our understanding of how platelets participate in the progression of atherosclerosis remains a gap in our knowledge. The Triggering Receptor Expressed Myeloid cells (TREM)-like transcript (TLT)-1 is a receptor found in the α-granules of both megakaryocytes and platelets. Antibodies to TLT-1 have been shown to inhibit platelet aggregation and accordingly studies using the treml1
-/-
mouse have demonstrated that TLT-1 null mice have reduced platelet aggregation and extended tail-bleeding times. Subsequent studies using the treml1
-/-
mouse demonstrated a predisposition to hemorrhage derived from an inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms. Additionally, apoE null mice have significantly higher levels of the soluble form of TLT-1 (sTLT-1) in their plasma compared to wild type mice suggesting that sTLT-1 may play a role in the progression of atherosclerotic lesions. Therefore, we hypothesized that removal of TLT-1 receptor will delay the formation of atherosclerotic lesions in an apoE deficient genetic background. To address our hypothesis, we have created an apoE/treml1 double null mouse and investigated the development of lesions in the aortic sinus. Our preliminary data show an unexpected trend where the lesions in apoE/treml1 double null mice have exacerbated compared to apoE or treml1
-/-
mice suggesting that TLT-1 may actually have a anti-inflammatory effect during the progression of atherosclerosis. The status of the current research will be presented here.
Abstract 31: In Vivo Targeting of Inflammation Associated Myeloid-Related Protein 8/14 via Gadolinium Immunonanoparticles
Authors: Maiseyeu Andrei , Badgeley Marcus A , Kampfrath Thomas , Mihai Georgeta , Deiuliis Jeffrey A , Liu Cuiqing , Sun Qinghua , Parthasarathy Sampath , Simon Daniel I , Croce Kevin , Rajagopalan Sanjay
Published: March 2022

Background:
Myeloid-related protein 8/14 complex (Mrp-8/14) is a highly expressed extracellularly secreted protein, implicated in atherosclerosis. In this study, we evaluated the feasibility of targeting Mrp in-vivo through synthetic immuno-nanoprobes.

Methods and Results: Anti-Mrp-14 and nonspecific IgG-conjugated gadolinium nanoprobes (aMrp-NP and IgG-NP respectively) were synthesized and characterized. Apolipoprotein E deficient (ApoE-/-) or ApoE-/-/Mrp14-/-(DKO) animals were fed a high-fat diet for 20 weeks followed by assessment of in-vivo pharmacokinetics and vascular targeting via magnetic resonance imaging (MRI). Bone marrow-derived myeloid progenitor cells were isolated from both ApoE
-/-
and DKO mice and differentiated to macrophages (BMDM). BMDM were treated with LPS, with or without Mrp8, Mrp14, or Mrp8/14 and conditioned media (CM) was collected and used for in-vitro studies. Mrp-activated cells secreted significant amounts of pro-inflammatory cytokines which was abolished by pretreatment with aMrp-NP. We show in vitro that aMrp-NP binds endothelial cells previously treated with CM containing Mrp8/14. MRI following intravenous delivery of aMrp-NP revealed prolonged and substantial delineation of plaque in ApoE
-/-
but not DKO animals with favorable pharmacokinetics (Figure 1). Flow-cytometric analysis of aortic digesta revealed aMrp-NP presence in Ly-6G+, CD11b+, CD11c+ and CD31+ cells in ApoE
-/-
but not in DKO animals.

Conclusion:
Targeted imaging with aMrp-NP demonstrates enhancement of plaque with binding to inflammatory cells and reduction in inflammation. This strategy has promise as a theranostic approach for atherosclerosis