bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 104: Cilnidipine Attenuated Angiotensin II-induced Abdominal Aortic Aneurysms in Apolipoprotein E-deficient Mice via Its Anti-oxidative Stress Effect
Authors: Kakio Yuki , Uchida Haruhito A , Umebayashi Ryoko , Wada Jun
Published: June 2021

Objective:
Chronic angiotensin II (AngII) infusion promotes development of abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Previous studies have shown that local inflammation and increased oxidative stress plays an important role on formation of AAAs. In addition, voltage-dependent N-type Ca
2+
channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by AngII in mice. Last year, we reported that cilnidipine, an N/L-type calcium channel blocker, attenuated AngII-induced AAAs in apolipoprotein E deficient mice. The purposed of this study was to determine the mechanism of cilnidipine reducing AngII-induced AAAs

Methods and Results:
Male apolipoprotein E deficient mice (8-12 weeks old) were fed a normal laboratory diet. Mice were infused subcutaneously with either cilnidipine or vehicle by osmotic minipumps. Three days later, mice were also infused subcutaneously with either AngII (1,000 ng/kg/min, n = 14-16) or saline (n = 4) by osmotic minipumps for 4 weeks. AngII increased systolic blood pressure. Cilnidipine decreased blood pressure in AngII-infused mice, but had not effect during saline infusion. Cilnidipine mildly restored plasma renin activity that were suppressed by AngII infusion. Cilnidipine mildly decreased plasma aldosterone concentrations that were increased by AngII infusion. Cilnidipine significantly reduced the incidence of AngII-induced AAAs (cilnidipine: 38 %, Vehicle: 88 %; P < 0.05). Elastica van Gieson staining demonstrated degeneration of elastic lamina by AngII was suppressed by cilnidipine administration. Immunohistochemical staining of CD68 revealed that cilnidipine administration attenuated accumulation of CD68 positive macrophage by AngII. In addition, cilnidipine reduced oxidative stress by AngII in 8-hydroxy-2’-deoxyguanosine and 4-Hydroxy-2-nonenal staining.

Conclusion:
Cilnidipine attenuated AngII-induced AAAs in male apolipoprotein E deficient mice by its anti-inflammation and anti-oxidative stress effect.
Abstract 108: Alcohol-related Diagnoses are Associated with High Mortality in Patients with Peripheral Arterial Disease
Authors: Abdel-Aal Ahmed Mohamed , Mendu Anuradha , Stewart Scott H , Yedlapati Siva Harsha
Published: June 2021

Background:
Moderate alcohol consumption is associated with lower risk of peripheral vascular disease (PVD). However, in heavy drinkers the evidence is limited, and there is little data on the effects of heavy drinking in patients admitted with PVD. We sought to investigate the prevalence and association of alcohol-related diagnoses with in-hospital mortality in patients presenting with PVD.

Methods:
We performed a cross-sectional analysis of the 2011 Nationwide Inpatient Sample (NIS). Using logistic regression methods appropriate for the NIS sample design, we estimated mortality associated with alcohol-related diagnoses in PVD patients and evaluated factors that might modify the association between alcohol and death.

Results:
PVD accounted for 158,683 (0.5%) of adult in-patient admissions with an in-hospital mortality rate of 4.6%. Of PVD admissions, a total of 4089 (2.6%) had concomitant alcohol-related diagnosis. Alcohol-related diagnoses were associated with increased mortality in PAD patients after controlling for factors associated with alcoholism, including age, sex, liver disease, hypertension, diabetes, renal failure, arrhythmias, drug abuse, gastro-intestinal bleed, and smoking [adjusted-odds ratio 1.7; 95% CI (1.3 - 2.4); P-value lt 0.001]. None of the factors included in the regression analyses interacted with alcohol abuse in predicting mortality, possibly due to lack of power.

Conclusion:
Alcohol-related diagnoses were associated with a modest increase in the risk for death in individuals presenting with PAD. This risk was not accounted for by common alcohol-related comorbidities. As a component of global efforts to limit hospital deaths from PAD, future research should identify the factors underlying this association.
Abstract 116: Revascularization but Not Supervised Exercise Therapy Prevents Progression of Fibrosis in the Gastrocnemius of Patients With Peripheral Artery Disease While Improving Limb Function
Authors: Ha Duy , Casale George , Luis Alicia , Harkins Kevin , Huber Reagan , Chengalasetty Tanmayee , Hickman Ruby , Hanna Mina , Swanson Stanley , DeSpiegelaere Holly , Pipinos Iraklis
Published: June 2021

Introduction:
Patients with peripheral artery disease (PAD) develop myofiber degeneration and fibrosis in their ischemic lower extremities, along with limb dysfunction. Walking performance improves with revascularization and exercise therapy, but effects on the myopathy are unknown. We previously showed fibrosis progresses with PAD and positively correlates with expression of vascular transforming growth factor-beta 1 (TGF-β1), a cytokine that stimulates collagen deposition.

Hypothesis:
We hypothesize that revascularization (RVS) and supervised exercise therapy (EXE) improve limb function in association with improved TGF-β1 dependent fibrosis.

Methods:
Gastrocnemius biopsies were collected from PAD patients (Fontaine Stage II; N=56) at baseline and 6 months after RVS (N=20), EXE (N=19), or no intervention (CTL; N=17). TGF-β1 expression was measured as grey scale units (gsu) by quantitative fluorescence microscopy of paraffin-embedded gastrocnemius sections. Collagen abundance was measured as optical density by quantitative multi-spectral bright-field microscopy of Masson Trichrome stained paraffin sections. Six Minute Walking Distance (SMWD), in meters, and Peak Walking Time (PWT), in seconds, were determined at baseline and 6 months. Relationships among TGF-β1, collagen, and limb function were assessed.

Results:
TGF-β1 expression and collagen density increased in CTL and EXE but not RVS patients. SMWD and PWT increased among RVS patients. PWT but not SMWD increased among EXE patients. SMWD and PWT were unchanged among CTL patients. The data are summarized in Table 1.

Conclusions:
RVS and EXE improved walking performance of patients with PAD, but only RVS prevented progression of fibrosis in the gastrocnemius of these patients. Over the same 6-month period, fibrosis increased in CTL muscle but was not sufficient to alter walking performance. The data suggest that benefits to the PAD leg may be greater with RVS compared to EXE.
Abstract 117: Development of a Hybrid Cryogel-coated Prosthetic Vascular Graft for Delivery of Targeted Gene Therapies
Authors: Huynh Cindy , Shih Ting-Yu , Samant Amruta , Pathan Saif G , Nelson David W , Mooney David J , Pradhan-Nabzdyk Leena , LoGerfo Frank W
Published: June 2021

Objectives:
Anastomotic neo-intimal hyperplasia (ANIH) remains a limiting factor in the long-term success of prosthetic vascular grafts. Understanding of the molecular mechanisms involved in the host response to prosthetic material has set the stage for using this material to deliver siRNA and other modulators of the ANIH-associated transcriptome response, along with antithrombotic agents. To create practical and effective drug delivery from a prosthetic material, we have combined a platform of polyethylene terephthalate (PET) fabric with an applied cryogel carrying the biologic agents, resulting in a bioactive prosthetic graft system (BPGS).

Methods:
Hybrid grafts were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun, knitted, or woven PET. Arg-Gly-Asp peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Human aortic endothelial cell (HAoEC) adherence and proliferation were assessed with a resazurin-based assay, and confocal microscopy was used to visualize cell interaction with the graft material. Heparin activity of the material in buffer solution was measured using an anti-Xa assay.

Results:
SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. HAoEC incorporated, adhered, and proliferated over 7 days for all materials (Figure 1). Anti-Xa assay confirmed continued activity of heparin from all grafts over 7 days.

Conclusion:
We have created a BPGS with a biocompatible cryogel polymer coating that allows for cell adherence and growth, with antithrombotic activity. This system allows application of the gel and biologic agents to PET prior to implantation, and provides flexibility in combining bioengineering and targeted gene therapy approaches to create an improved prosthetic graft adaptable to evolving strategies.
Abstract 119: Impaired Cardiac Sestrin2 Signaling in Response to Ischemic Insults with Aging
Authors: Quan Nanhu , Sun Wangqing , Wang Lin , Chu Dongyang , Chen Xu , Cates Courtney A , Li Ji
Published: June 2021

Introduction:
A novel stress-inducible protein, Sestrin2, declines in the heart with aging. AMP-activated protein kinase (AMPK) has emerged as a pertinent stress-activated kinase shown to have substantial cardioprotective capabilities against myocardial ischemia/reperfusion (I/R) injury. We have identified the interaction between Sestrin2 and AMPK in the ischemic heart.

Hypothesis:
The ischemic AMPK activation modulated by Sestrin2-AMPK complex in the heart is impaired in aging that sensitizes heart to ischemic insults.

Methods:
Young C57BL/6 mice (4-6 months) and aged C57BL/6 mice (24-26 months) were subjected to left anterior descending coronary artery occlusion for different time points of ischemia and ischemia/reperfusion (I/R) in order to determine the signaling activity in the left ventricle. The
ex vivo
working heart system was used for measuring substrate metabolism in the perfused hearts.

Results:
The protein expression level of Sestrin2 in aged heart was significantly lower than that of Sestrin2 in young heart (p<0.05). Intriguingly, ischemic AMPK activation was blunted in the aged hearts as compared with young hearts (p<0.05), the AMPK downstream glucose uptake and the rate of glucose oxidation were significantly impaired in the aged hearts during I/R (p<0.05 versus young hearts). The myocardial infarction size was significant bigger in the aged hearts (p<0.05 versus young hearts). Respective left ventricle lysates were immunoprecipitated with Sestrin2 antibody and the immunoblots revealed that Sestrin2 forms a complex with AMPK and AMPK upstream LKB1 during I/R, moreover, the binding affinity between Sestrin2 and AMPK is decreased in the aged hearts during I/R (p<0.05 versus young hearts). Furthermore, Sestrin2 knock out hearts demonstrate impaired ischemic AMPK activation and higher sensitivity to I/R-induced injury as compared to wild type hearts (p<0.01).

Conclusions:
Sestrin2 is a stress-induced scaffold protein that mediates the activation of AMPK in the ischemic myocardium via an interaction with upstream LKB1. Moreover, lower level of Sestrin2 in aging leads to blunted ischemic AMPK activation and increased sensitivity to ischemic insults.
Abstract 121: Effects of Tethering Branches on Limb Flexion-induced Deformations of the Human Femoropopliteal Artery
Authors: Poulson William , Kamenskiy Alexey , Deegan Paul , Lomneth Carol , MacTaggart Jason
Published: June 2021

Introduction:
High failure rates of femoropopliteal artery (FPA) interventions are commonly attributed to severe mechanical deformations that occur with limb movement. Recent data demonstrate large variability in FPA bending and torsion. We hypothesize that FPA branch tethering significantly influences arterial deformations.

Methods:
Under fluoroscopic guidance, nitinol markers were endovascularly deployed into 22 limbs of 11 human cadavers. Dilute contrast was injected into the FPA, and thin-section CT images were acquired with the limbs in straight (180°) and acutely bent (60°) postures. Image segmentation and 3D reconstruction were used to measure bending and torsion of the FPA by tracking spatial movement of the artery and markers. FPA bends were measured as radii of inscribed spheres, while torsion was calculated by measuring the angle of twist between each pair of consecutive markers in the straight and bent leg configurations. FPA branches were mapped and Pearson correlation
r
was used to assess whether bending and torsion values correlated with the distance to the nearest branch.

Results:
At the adductor hiatus the FPA demonstrated a bending radius of 3.43±9.14 mm with 15.03±9.95 °/cm twist, while below the knee the most acute bending radius was 2.21±7.03 mm and the maximum twist was 32.03±10.68 °/cm. Bending and torsion below the knee were more severe than at the adductor hiatus (p<0.01). On average, large branches were observed 6.82± 8.26 mm in proximity to the bend and 5.29± 5.16 mm to the twist in the adductor hiatus and 7.96± 6.52 mm and 6.31± 6.06 mm to the bend and twist below the knee. There was no correlation (
r
=-0.22, p=0.32) between bending and branch proximity at the adductor hiatus or in the below the knee arterial segments (
r
=-0.11, p=0.30). Similarly, no correlation was observed between twist and branch proximity (
r
=0.02, p=0.91 at the adductor hiatus;
r
=-0.02, p=0.87 below the knee).

Conclusions:
Limb flexion-induced deformations of the FPA demonstrate severe bending and torsion at the adductor hiatus and below the knee; however, neither bending nor torsion appear to be affected by the proximity of the FPA branches. These data suggest that tethering branches have minimal effect on limb flexion-induced bending and torsion of the FPA.
Abstract 124: Association Between Low Bone Mineral Density and Risk of Peripheral Vascular Disease: A Systematic Review and Meta-analysis
Authors: Upala Sikarin , Sanguankeo Anawin
Published: June 2021

Background:
Previous studies have suggested that osteoporosis and coronary heart disease have shared common risk factors. Some studies also suggested that low bone mineral density (BMD) also increased risk of peripheral vascular disease (PVD) or stroke. However, few longitudinal studies did not find significant association between these two conditions. We performed a systematic review and meta-analysis to explore association between low BMD and risk of PVD or stroke from prospective studies.

Methods:
We comprehensively searched the databases of PubMed/MEDLINE, EMBASE, and Cochrane Databases from their dates of inception to December 2015. The inclusion criteria were published prospective observational studies of PVD, stroke, BMD, osteoporosis, or fractures. Two authors independently assessed the quality of the articles and extracted the data. The primary outcome of interest was risk of new PVD or stroke events comparing between low BMD and normal BMD groups.

Results:
From 36 full-text articles, five observational studies involving 3,287 participants were included in the meta-analysis based on the random-effects model. There was increased risk of new peripheral vascular disease or stroke events in groups with low BMD compared with normal BMD controls with pooled hazard risk (HR) of 3.10 (95% CI: 1.32-7.23). In the subgroup analysis of fracture as a risk factor, there was no significant difference in risk of PVD or stroke in group with pooled HR of 1.17 (95% CI: 0.78-1.75).

Conclusions:
Low BMD is associated with increased risk of PVD and stroke from the meta-analysis of prospective observational studies. Clinicians should be aware of the close association between these two conditions. Early identification of PVD in patients with low BMD should be performed.
Abstract 143: Enrichment of Dimethylation of Lysine 4 on Histone 3 and Resident Vascular Stem Cell Transition to Vascular Smooth Muscle Cells
Authors: Hakimjavadi Roya , Burtenshaw Denise , Fitzpatrick Emma , Di Luca Mariana , Casey Gillian , Redmond Eileen M , Cahill Paul A
Published: June 2021

Data support a role for stem cell-derived vascular smooth muscle cells (SMC) in arteriosclerosis. Epigenetics play a critical role in SMC differentiation where histone proteins associated with the myosin heavy chain (Myh11) promoter are post-translationally modified by dimethylation of lysine 4 on histone 3 (H3K4me2). Studies report that ‘de-differentiated’ SMC do not exhibit loss of H3K4me2 at the Myh11 promoter even when mRNA levels decrease thereby allowing this modification to be used to track differentiated SMC.

Our aim was to determine the level of H3K4me2 and H3K27me3 methylation of differentiated SMCs and compare to resident vascular stem cells and stem cell-derived SMC.

Murine adventitial Sca1
+
stem cells (APCs), rat medial Sox10
+
multipotent vascular stem cells (MVSCs) and CD44
+
bone-marrow derived mesenchymal stem cells (MSCs) were all examined for methylation of lysine 4 (H3K4me2) and lysine 27 (H3K27me3) on histone 3 associated with the Myh11 promoter, before and after SMC differentiation and compared to fresh aortic differentiated SMC and sub-cultured de-differentiated SMC
in vitro
by Chromatin Immunoprecipitation (ChIP) assay. Cells were also examined for Myh11 expression, stemness (telomerase activity) and multipotentcy.

Differentiated rat and murine SMC were enriched for H3K4Me2 at the Myh11 promoter, compared to H3K27me3. Phenotypically de-differentiated sub-cutured rat and murine SMC were enriched for H3K4Me2 when compared to H3K27me3, but to a much lesser extent when compared to differentiated aortic SMC. In contrast, resident APCs and MVSCs, and bone-marrow derived MSCs, were all enriched for H3K27me3, concomitant with significant telomerase activity and multipotent differentiation capacity. The levels of enrichment of H3K27me3 dropped significantly after SMC differentiation with TGF-βeta1 concomitant with a significant enrichment of H3K4me2 to levels that mimicked the level of enrichment in de-differentiated SMC when compared to aortic differentiated SMC.

De-differentiated SMC exhibit reduced enrichment of H3K4me2 at the Myh11 promoter region when compared to differentiated aortic SMCs, but mimic the level of enrichment of H3K4me2 observed following resident vascular stem cell differentiation to SMCs.
Abstract 153: Molecular Mimicry Between Malondialdehyde and Group A Streptococcus Contribute to the Natural Selection of Conserved Innate Immune Response
Authors: Gonen Ayelet , Cole Jason , Oliveira Romulo , Diehl Cody J , Byun Young Sup , Stewart Chelsea , Buffalo Cosmo , Copps Jeffry , Olson Joshua , Que Xuchu , Montano Erica , Binder Christoph , Sotirios Tsimikas , Ghosh Partho , Nizet Victor , Witztum Joseph L
Published: June 2021

Innate immunity utilizes evolutionarily conserved pattern recognition receptors (PRRs) to provide an early and effective response against Pathogen-Associated Molecular Patterns (PAMPs) on microbial pathogens and/or against Danger Associated Molecular Patterns (DAMPs) on endogenous modified-self structures. Atherosclerosis is a chronic inflammatory disease in which lipid peroxidation is greatly increased leading to the generation of OxLDL, which contains a variety of proinflammatory oxidation-specific neoepitopes (OSE), such as phosphocholine (PC) containing oxidized phospholipids (OxPL). Our group has shown that OSEs are DAMPs, to which has evolved a concerted innate immune response mediated by PRRs. For example, CD36, CRP and IgM E06 all recognize the PC of OxPL, but also the PC on apoptotic cells as well as the PC on the cell wall of
S. pneumonia
(but not as part of a lipid). Accordingly, we postulated that both endogenous DAMPs and exogenous PAMPs provide natural selection for PPR responses to PC.

Malondialdehyde (MDA) is another prominent OSE target of three different PRR’s: SR-A, CFH, and the IgM NAb E014. Thus, we hypothesized that EO14 should also recognize an epitope/mimotope on an infectious pathogen. We screened a pathogen library with E014 and discovered it avidly bound to group A streptococcus (GAS). Because it was known that CFH also bound to GAS, and specifically to protein M, the major virulence factor of GAS, we used GAS with and without protein M to show that E014 specifically bound to protein M. Using a series of recombinant protein M fragments, we identified a 125 aa sequence required for binding. Using a synthetic peptide array to generate 15 aa-length overlapping peptides, we identified a 24 aa mimitope that E014 bound. We subsequently showed immunological cross reactivity between GAS, Protein M, the mimotope, and MDA in vitro and in vivo in mice and humans. Further, compared to immunization of mice with protein M, immunization with MDA-LDL provided partial protection against lethal infection with GAS. These data support the hypothesis that OSE are important targets of innate immunity and both oxidative events and pathogens have contributed to the natural selection of potent, shared innate immune responses to oxidation-specific epitopes.
Abstract 156: Deletion of the Lymphocyte Adaptor Protein LNK Promotes Aortic Dissection
Authors: Laroumanie Fanny , Saleh Mohamed A , McMaster William , Kirabo Annet , Madhur Meenakshi S
Published: June 2021

Background:
The immune response plays a key role in the development of aortic dissection. In particular, interferon gamma (IFNγ), produced by the T cell subsets, Th1 (CD4
+
) and Tc1 (CD8
+
), is elevated in human aortic dissection samples. Interestingly, a polymorphism in the gene
SH2B3
that encodes LNK has been associated with several cardiovascular diseases in humans. LNK is an intracellular adaptor protein that has been shown to negatively regulate T cell activation and cytokine signaling. We hypothesized that LNK deficiency promotes aortic dissection through enhanced Th1/Tc1 responses.

Methods:
Angiotensin II (Ang II; 1000 ng/kg/min) was infused for 14 days into LNK
-/-
or WT mice in the absence or presence of an initial intraperitoneal injection of saline equal to 10% of their body weight. Kaplan-Meier survival curves were generated and flow cytometry, RT-PCR, histology were used to assess aortic inflammation and remodeling. Naïve CD4
+
T cells from WT and LNK
-/-
mice were polarized
ex vivo
with Th1 polarizing cytokines and expression of IFNγ and T-bet were evaluated.

Results:
LNK
-/-
mice infused with Ang II exhibited an accelerated rate of aortic dissection or rupture compared to WT mice and this was exacerbated by saline injection prior to Ang II infusion (Figure). LNK
-/-
mice treated with salt plus Ang II exhibited increased aortic inflammation compared to WT mice. Aortic expression of IFNγ, T-bet and MMP9 were increased, while the expression of the procollagens type I and III were reduced in LNK
-/-
mice compared to WT mice.
Ex vivo,
naïve CD4
+
T cells from LNK
-/-
mice expressed more T-bet and IFNγ compared to corresponding naïve T cells from WT mice when cultured under Th1 polarizing conditions.

Conclusion:
Loss of LNK enhances the Th1/Tc1 responses and promotes adverse aortic remodeling leading to development of aortic dissection and rupture. Targeting LNK and the Th1/Tc1 cell subsets could be a potential therapeutic strategy for the management of aortic dissection.
Abstract 159: Synthetic High Density Lipoprotein - a Potential Therapy for Sepsis
Authors: Guo Ling , Morin Emily E , Yuan Yaxia , Zhan Chang-Guo , Gong Ming-Cui , Li Zhenyu , Standiford Theodore J , Schwendeman Anna , Li Xiang-An
Published: June 2021

Introduction:
Sepsis is the leading cause of death in critically ill patients with mortality rates exceeding 30%. The ability of endogenous HDL to neutralize and facilitate elimination of lipopolysaccharides (LPS) is known and synthetic HDLs (sHDL) have been administered in sepsis models with varying success. In the current study we optimized composition of sHDL, apolipoprotein A-I (apoA-I) mimetic peptide-phospholipid particles, to maximize their protective effect against endothelial dysfunction in sepsis, and examined changes in HDL and apoA-I levels in patients with sepsis to select sHDL doses for evaluation in a sepsis animal model.

Methods/Results:
We first measured HDL cholesterol (HDL-C) and apoA-I in patients at time of intake to the intensive care unit, observing HDL to be 45% lower in septic patients compared to non-septic patients (p<0.01), with HDL levels in non-surviving septic patients being significantly lower compared to those in surviving septic patients (p<0.01). Next, we tested a new generation of sHDL, ETC642, for its efficacy on cecal ligation and puncture (CLP)-challenged mice. We administered sHDL to mice 2h post CLP and found that sHDL treatment significantly increased plasma HDL-C and decreased BUN, plasma IL-6 and IL-10 levels. More importantly, sHDL therapy improved 7d survival rate greater than 2-fold over PBS treated mice (p=0.01). To understand the mechanisms underlying sHDL protection, we investigated the effect of sHDL on endotoxin-induced inflammatory response
in vitro
. sHDL not only neutralized LPS/LTA but also suppressed TLR4-/TLR2-NF-kB signaling in macrophages, and inhibited LPS/LTA/TNF-α induced endothelial cell activation.

Conclusions:
In this study, we show that HDL-C levels are markedly decreased in septic patients, which is associated with poor prognosis. We demonstrate that sHDL treatment increases HDL levels and effectively protects against CLP-induced septic death. We further established that sHDL therapy provides multiple protective mechanisms through LPS/LTA detoxification, suppressing inflammatory signaling in macrophages and inhibiting endothelial activation. Together, our findings suggest that targeting the vasculature via sHDL can be an effective platform for sepsis therapy.
Abstract 167: Doxycycline Inhibits Revascularization After Hindlimb Ischemia
Authors: Ankri- Eliahoo Galit , Fu George , Kenagy Richard , Cox Timothy , Tang Gale L
Published: June 2021

Objectives:
p27
kip1
, a gene affecting human response to arterial injury, affects collateralization after hindlimb ischemia (femoral artery ligation), possibly by regulating matrix metalloproteinase 2 (MMP2). We hypothesized that MMP2 mRNA expression would increase significantly more in ko mice after hindlimb ischemia and that MMP inhibition would affect p27
-/-
(ko) collateralization less than p27
+/+
(wt).

Methods:
Ko and wt mice had their femoral and collateral arteries harvested seven days after hindlimb ischemia for RNA isolation and qRT-PCR. Ko and wt mice were also fed chow without or with doxycycline (a broad spectrum MMP inhibitor) and subjected to hindlimb ischemia. The mice were followed with weekly footpad laser Doppler perfusion imaging for 28 days, and then sacrificed for microCT scanning. Vascular smooth muscle cells (VSMC) isolated from either wt or ko aortae were used in Boyden chamber migration assays without and with an MMP2 specific inhibitor.

Results:
MMP2 mRNA expression increased significantly more in ko collaterals than wt collaterals (312% vs. 50%, respectively). However, blood flow recovery in ko and wt mice treated with doxycycline decreased to a similar extent (27±2.5% and 33±1% decrease respectively), although ko mice still revascularized better than wt mice (
p
< 0.01). Gracilis collateral diameters increased less after doxycycline treatment in both groups compared to mice fed regular chow (78.5±12 and 80±7 vs 98±18 and 106±17 μm, respectively). The bridge collateral diameters increased less in ko mice fed doxycycline (66±32 vs 158±18.3 μm), and no bridge collaterals were detected in wt mice fed doxycycline. Wt mice fed regular chow had rare bridge collaterals. Ko VSMC migration was less affected by MMP2 inhibition than wt (29% less migration vs 48% less migration,
p
<0.01).

Conclusions:
MMP2 mRNA expression increased significantly more in p27 ko collaterals after hindlimb ischemia. The non-specific MMP inhibitor doxycycline inhibited revascularization after hindlimb ischemia, independent of p27 expression. p27 ko mice still revascularized better than wt mice after doxycycline treatment, suggesting that p27 has additional effects other than regulating MMP expression in the collateralization response.
Abstract 183: Collagen Type V (Col V) Contributes to the Development of Aortic Dissections and Aneurysms
Authors: Phan Noel M , Park Arick , Zhou Ting , Scott David , Wang Qiwei , Tang Paul , Greenspan Daniel S , Liu Bo
Published: June 2021

Background:
Negative remodeling of extracellular matrix (ECM) is a key pathological feature associated with aortic dissections and aneurysms. Col V is a minor fibrillar collagen that incorporates into growing fibrils with Col I, and is involved in regulating the geometry and tensile strength of ECM. Col V normally exists as α
1
(V)
2
α
2
(V)
1
heterotrimers but may present as α
1
(V)
3
homotrimers in pathological tissues. To experimentally increase the existence of α
1
(V)
3
homotrimer, we deleted the
Col5a2
gene, which encodes the α2(V) chain.
Col5a2
+/-
mice showed increased compliance and reduced tensile strength in the skin and aorta. In the present study, we tested whether
Col5a2
deficiency affects the pathogenesis of aortic dissections and aneurysms.

Methods and Results:
Six-month-old male
Col5a2
+/-
and
Col5a2
+/+
mice on an
Ldlr
null background were infused with Angiontensin II (AngII). Mortality for
Col5
a2
+/-
mice within the first 7 days was 45% vs 0% for WT (
P
<0.045, n=9), with 60% due to aortic arch rupture and 40% due to dissection. Among those that survived the 28 day treatment, 7/ 7
Col5a2
+/
-
mice developed abdominal aortic aneurysm, defined as greater than or equal to 50% increase in the maximal external suprarenal diameter over infrarenal aorta, whereas 5/9 WT showed aneurysm dilatation. Compared to WT, aneurysm dilatations were significantly increased in
Col5a2
+/
-
mice (132.1 ± 8.5% vs 90.12 ± 9.6%,
P
<0.017). Histological analyses revealed marked disarray and loss of elastic fibers in
Col5a2
+/-
aortas and no difference in levels of fragmentation. Moreover, medial layer integrity was deteriorated in
Col5a2
+/-
mice and was associated with increased infiltration of macrophages.

Conclusions:
Collectively, our data suggest that haploinsufficiency for the α2(V) chain leads to media layer changes and increased susceptibility to aortic arch ruptures, dissections, and larger aortic aneurysms. The α2(V) chain may thus represent a novel candidate marker/target for the identification and treatment of aortic dissection and aneurysm.
Abstract 188: I X2 X2
Authors: Zhou Ning , Stoll Shaunrick , Qiu Hongyu
Published: June 2021

Background:
An increase in aortic stiffness is a fundamental component of hypertension. However, the molecular mechanism involved is unclear. Our hypothesis is that the increased aortic stiffness in hypertension is partially due to the activation of serum response factor (SRF) /myocardin in vascular smooth muscle cells (VSMCs).

Methods:
4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured by a Millar catheter and by Doppler imaging echocardiography, respectively. VSMCs were isolated from thoracic aorta (TA), then cultured and tested at passages 2 to 4. Real time PCR and western blot were used to detect the gene and protein expression.

Results:
Aortic pressure was higher in SHR than WKY (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg,
p
<0.01). Aorta stiffness reflected by arterial stiffness index (ASI) was 2.5-fold higher in SHR vs WKY (
p
<0.01). Compared to WKY, VSMCs from SHR TA showed a 12-fold increase in SRF, a nuclear transcriptional factor, and 2.7-fold increase in myocardin, a smooth muscle specific coactivator of SRF, at both mRNA and protein levels (All,
p
<0.01 vs. WKY TA VSMCs). Myocardin nuclear translocation was also markedly increased in TA VSMCs from SHR by 12-fold versus WKY, (
p
<0.01). SRF/myocardin downstream target genes, α-smooth muscle actin (α-SMA) and SM22 were increased by 3-fold in TA VSMCs in SHR vs WKY (
p
<0.01).
In vitro
, CCG-100602, a specific inhibitor of SRF/myocardin interaction, not only strikingly repressed the increase of the expression in SRF, myocardin and the downstream targets α-SMA and SM22, but also blocked the nuclear translocation of myocardin in TA VSMCs from SHR (
p
<0.01 vs untreated SHR VSMCs). However, such effects were not observed on WKY TA VSMCs.
In vivo
, 2-week treatment with CCG-100602 significantly reduced MAP and ASI by 19% and 63% in SHR, respectively (
p
<0.01 vs untreated SHR), while aortic MAP and ASI in WKY rats remain unchanged upon the treatments.

Conclusion:
The activation of myocardin/SRF in aortic VSMCs presents a novel mechanism of increased aortic stiffness in SHR.
Abstract 190: The Effect of Particle Size on Apolipoprotein A-I Exchange in Reconstituted and Human-derived High Density Lipoprotein Particles
Authors: Borja Mark S , Ortega-Guerrero Lorena G , Oda Michael N
Published: June 2021

Objective:
The ability of high density lipoprotein (HDL) to promote cholesterol efflux from ATP binding cassette transporter A1 (ABCA1) is inversely associated with HDL particle size. The exchangeability of HDL’s primary protein constituent, apolipoprotein A-I (apoA-I), is positively correlated with cholesterol efflux capacity. However, it is unknown whether HDL particle size similarly affects the exchangeability of apoA-I. In this study, we investigated the effect of HDL particle size on the exchangeability of apoA-I on reconstituted (rHDL) and human-derived plasma HDL (pHDL) particles.

Method and Results:
Discoidal rHDL particles of well-defined size (17.0, 12.2, 9.6, 8.4 and 7.8 nm) were prepared from POPC and apoA-I by cholate dialysis and purified by size exclusion chromatography. Human HDL was isolated by subclass (HDL2b, 2a, 3a, 3b, 3c) from the plasma of healthy volunteers by sequential density gradient ultracentrifugation. HDL-apoA-I exchange (HAE) was measured by electron paramagnetic resonance (EPR) and non-denaturing gradient gel electrophoresis (NDGGE) following incubation with either spin-labeled or fluorescently-labeled, lipid-free apoA-I. Size-dependent effects were observed with rHDL particles, with the largest rHDL particles exhibiting 3-fold higher HAE compared to the smallest particles. In contrast, the HAE of human-derived pHDL particles was relatively constant across all subclasses at constant apoA-I concentration.

Conclusions:
HDL-apoA-I exchange is size-dependent in rHDL, with HAE positively associated with rHDL particle size, but it is size-independent in human pHDL. Our findings suggest that additional factors such as remodeling enzymes may facilitate apoA-I exchange from human pHDL.
Abstract 191: Microparticles Released From Various Cell lines are ABCA1 Dependent
Authors: Hafiane Anouar , Johansson Jan O , Genest Jacques
Published: June 2021

The physiological functions of microparticles (MPs) released from cells remain poorly defined. Here, we study MPs released from various cell lines; baby hamster kidney (BHK) cells, human THP-1 and hepG2 cells also expressing ABCA1. To assess the role of MPs in cholesterol homeostasis we incubated cells with apoA-I or CS-6253 (CS) a novel apoE derived mimetic peptide and potential cholesterol efflux and HDL modulator. When incubated with BHK cells expressing ABCA1 under mifepristone and Mock cells in time dependent manner (0,5 to 24h) we found that MPs are ABCA1 dependent. Accordingly the dependence of MPs release on ABCA1 activity is confirmed in human THP-1 cells and hepG2 cells after blocking ABCA1 with probucol (10 μM, 24h). Given the rate-limiting step of ABCA1 in cholesterol homeostasis, we hypothesized that MPs may have a role in cholesterol transport from cells. To address this point we isolated MPs by FPLC from media collected from
3
H-cholesterol labelled cells that were exposed or not to (1 μM) apoA-I, or CS in time dependent manner. We demonstrate that MPs accounted for ~ 1/3 of the total cholesterol released to the medium. In addition, we found that cholesterol carried by MPs is redistributed among resident lipoproteins under conditions when
3
H-MPs were incubated in plasma (1h, 37°C), as shown by FPLC profiles. In addition, the cholesterol regulatory role in cellular release of MPs was tested after depletion of plasma membrane cholesterol by methyl-β-cyclodextrin (CDX, 10 mM, for 30 min). There was a significant decrease in cholesterol counts from MPs released in CDX treated macrophages THP-1 cells when compared to cells not treated relative to each condition: cells alone (16%, p<0.05), cells incubated with; apoA-I (48%, p<0.01), or CS (66%, p<0.0001), indicating that MPs require membrane cholesterol. MPs isolated from cells do not contain apoA-I or CS in contrast to HDL particles as confirmed by Western blotting analysis. MPs characterization by nanoparticle tracking analysis demonstrated vesicles that display (67.76±4 to 920±26nm) average size. We conclude that MPs generation was markedly decreased by blocking ABCA1 suggesting that ABCA1 is critical determinant for MPs formation, and potentially cell communication.
Abstract 193: HDL-Small RNA Intercellular Communication in Systemic Lupus Erythematosus
Authors: Michell Danielle L , Moore Jared L , Ormseth Michelle J , Landstreet Stuart R , Zhao Shilin , Sheng Quanhu , Wilhelm Ashley , Stein C. Michael , Major Amy , Vickers Kasey C
Published: June 2021

Extracellular small non-coding RNAs (sRNAs) are a new class of disease biomarkers and are transferred between cells by high-density lipoproteins (HDL) in a novel form of intercellular communication. In chronic inflammatory states and auto-immunity, HDL can become dysfunctional, likely through alterations in its diverse cargo, including changes to sRNA signatures. We have previously found that HDL-microRNAs (miRNA) are altered in systemic lupus erythematosus (SLE); however, miRNAs are just one type of sRNAs. As such we hypothesized that changes to HDL-sRNA cargo and cell-to-cell communication in SLE extend beyond miRNAs. To test this hypothesis, HDL was isolated from SLE and control (n=6-8) subjects by density-gradient ultracentrifugation followed by size-exclusion chromatography. High-throughput sRNA sequencing of HDL demonstrated that tRNA-derived sRNAs (tDRs) were the most abundant class of sRNAs on HDL and were significantly altered in SLE subjects compared to controls (26 up, 10 down). In addition, circulating levels of angiogenin, an RNaseIII enzyme capable of cleaving parent tRNAs into tDRs, was also significantly (
P
<0.05) increased in SLE plasma. To determine if tDRs are altered in CD4+ T cells in SLE subjects, real-time PCR was used to quantify candidate tDRs, and we found that tDR-GlyGCC levels were significantly increased 4.2-fold in SLE (
P
<0.01). Most importantly, we found that T cells exported tDR-GlyGCC to HDL. To determine if T cell exported tDR-GlyGCC is transferred to other cells by HDL,
ex vivo
studies were completed using Trans-PhotoActivatable-Ribonucleoside-CrossLinking-ImmunoPrecipitation high-throughput Sequencing (Trans-PAR-CLIPseq) to trace sRNAs from human CD4+ T cells to HDL and then to recipient CD14+ monocytes and CD19+ B-cells. Using this approach, we found a cassette of T cell originating sRNAs, including tDR-GlyGCC, transferred by HDL to recipient B cells and monocytes and loaded onto RNA-Induced Silencing Complexes (RISC) targeting genes associated with inflammation. Here, we demonstrate that HDL facilitates the intercellular transfer of sRNAs between immune cells and these sRNAs are altered in SLE. This altered communication may contribute to T cell imbalance and B cell activation observed in SLE.
Abstract 198: HDL Function and Genetic Regulation in a Nonhuman Primate Model of HDL Cholesterol Extremes
Authors: Predazzi Irene , Pamir Nathalie , Plubell Deanna , Letaw John , Curran Joanne , Raboin Michael , Clarke Jordyn , Hales Beck Lauren , Tavori Hagai , Fazio Sergio , Vinson Amanda
Published: June 2021

Recent data indicate that HDL function has effects independent of HDL cholesterol (HDL-C) levels that better predict cardiovascular risk. We aimed to characterize sterol efflux across a wide range of HDL-C, and to identify genetic determinants for both traits in a non-human primate model. To do this, we took advantage of rhesus macaque families that display spontaneous, extreme variation in HDL-C (range 13-106 mg/dL), but have total cholesterol, LDL-C, and triglyceride levels considered normal in humans. We hypothesized that genetic regulation of HDL metabolism is conserved between macaques and humans, and that sterol efflux is independent of HDL-C across the observed range of HDL-C values. We selected 16 macaque half-sib pairs matched for age-class and sex, based on maximum differences in HDL-C characterized in a larger sample (n=193), and conducted deep sequencing of exons and regulatory regions. We assessed predicted function for all variants in macaques found at 23 genes that regulate reverse cholesterol transport or are associated with HDL-C in humans, and that share ~93% identity with the corresponding human proteins. We assessed sterol efflux using J774 macrophages labeled with [
3
H]cholesterol and stimulated with a cAMP analogue in 16 sequenced macaques, plus 6 macaques selected from the <5
th
/>95
th
percentiles of HDL-C (n=22). We found 27 predicted functional variants located at regulatory or coding regions in macaques that were within 10 bp of known variants in humans associated with HDL-C levels, coded protein deficiencies, or familial alpha-dyslipoproteinemias. Further, we show that 4 of these variants in
CETP
,
LCAT
, and
ABCA1
are expected to inhibit normal protein function, as indicated by
in silico
prediction of changes in protein folding. Within these families, correlation between HDL-C and sterol efflux was 0.78 (P=1.65 X 10
-5
), suggesting that a significant portion of the variation in efflux capacity cannot be accounted for by HDL-C levels. We conclude that functional genetic variation in pathways of HDL metabolism is conserved between humans and macaques, and is likely to influence both HDL-C and efflux capacity. However, a significant proportion of efflux capacity operates independently of HDL-C.
Abstract 206: Randomized Double Blind Clinical Trial on the Effect of Oral Alpha-cyclodextrin on Serum Lipids
Authors: Amar Marcelo , Kaler Maryann , Mokhtari Vida B , Shamburek Robert D , Sampson Maureen , Levine Stewart J , Remaley Alan T
Published: June 2021

Despite numerous therapeutic advances, cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in developed countries. Statins, currently the most effective therapeutic agents for reducing CVD risk, only reduce cardiovascular events by approximately 30%. One possible complementary approach for lowering LDL-C involves the use of oral agents that complex or associate with dietary fats, preventing their intestinal absorption. Bile acid sequestrants are among the oldest lipid-altering drugs and have been known for decades to improve lipid levels and also improve glucose control. Alpha-cyclodextrin (a-CD), a cyclical polymer containing 6 glucose sub-units, is found in many foods, drinks and is sold as an over the counter food supplement. a-CD has a hydrophobic central cavity that binds lipids; one gram of a-CD binds as much as 9 grams of dietary fat and animal studies as well as clinical trials showed that it reduces dietary lipid absorption.

This is the first study that evaluated the effect of a-CD in healthy subjects and was designed as a single center, double-blinded, cross-over, placebo controlled clinical pilot study. We screened 103 subjects, males and females, and obtained 75 completed studies (42% male, 58% female, mean age 34 ±12 y.o., BMI 25±4 kg/m2). Eligible subjects were randomized into two arms and received placebo or a-CD (6 grams P.O. a day; two 1 gram tablets per meal) for 12-14 weeks with a one week of wash out period between arms. Several biochemistry and lipid parameters were analyzed and statistics were performed using paired t-test. a-CD was well tolerated and no serious adverse events related to a-CD were observed. Minor GI symptoms were the most common complaint but occurred in less than 7% of subjects on a-CD (vs 3% in placebo). No significant differences were detected on serum lipid and other lipid values but fasting serum glucose (-13% a-CD vs placebo), insulin (-7% a-CD vs placebo), HbA1C (-6% a-CD vs placebo) were decreased. Further positive alterations were present in individuals with higher glucose levels. In conclusion, in healthy subjects, a dose of 6 grams a day of a-CD was able to improve plasma glucose related indexes, in the population studied, but had no significant effect in lipid or lipoprotein plasma levels.
Abstract 21: A Natural Repertoire of T Cells Recognizing ApoB-100 is Generated Early in Life and is Progressively Depleted During Atherosclerotic Disease
Authors: Wolf Dennis , Gerhardt Teresa , Miller Jacqueline , McArdle Sara , Kimura Takayuki , Jenkins Marc , Ley Klaus
Published: June 2021

Background:
A large body of evidence implicates a role for T cell driven auto-immunity in atherosclerosis. T cells in the atherosclerotic plaque specifically respond to auto-antigens, including ApoB-100, the main protein in low-density lipoprotein (LDL). However, existence, function, and location of auto-reactive T cells in mice have not been demonstrated.

Methods and Results:
We have previously identified several peptides derived from mouse ApoB-100 that bind with high affinity to the I-A
b
MHC class II molecule of C57BL/6 mice. Immunization with these peptides conferred atheroprotection. We designed a novel fluorochrome-labeled P6:I-A
b
multimer to detect T cells specifically recognizing this complex by flow cytometry. Surprisingly, we detected small numbers of P6:I-A
b+
CD4
+
T cells in young C57Bl/6 mice that reside in peripheral lymph nodes, indicating the existence of a small natural repertoire of P6:I-A
b
auto-reactive T cells. This repertoire of T cells was increased in atherosclerosis-prone A

poe
-/-

and

Ldlr
-/-

mice and showed signs of previous antigen-exposure in 4 week old animals. T cells recognizing P6:I-A
b
were undetectable directly after birth, but expanded rapidly within the first 28 days in lymph nodes. The majority of P6:I-A
b+
T cells expressed the defining transcription factors of T
H
1, T-bet, T
H
17, ROR-gamma T, or of T-regulatory cells, FoxP3. Feeding of Apoe
-/-
mice with a western diet induced a further skew towards the T
H
1 and T
H
17 lineage, but also resulted in a progressive loss of antigen-specific cells over time. In Apoe
-/-
mice fed with a western diet for 1 year, but not in Apoe
-/-
mice fed with a standard chow diet, auto-reactive T cells disappeared. Mechanistically, we found enhanced expression of exhaustion markers like ICOS-1 or PD-1 in antigen-specific T cells likely due to persisting antigen-exposure in this model.

Conclusion:
Our findings indicate that T cells specifically recognizing a peptide derived from ApoB-100 do not expand during the natural course of disease, but instead exist in atherosclerosis-prone animals in early life. Chronic exposure to antigen induces a progressive loss of auto-reactive T cells.