bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 454: Childhood Determinants of Subendocardial Viability Ratio in Biracial (Black-White) Young Adults: The Bogalusa Heart Study
Authors: Fernández Alonso Camilo , Li Shengxu , Chen Wei , Srinivasan Sathanur R , Berenson Gerald S
Published: March 2022

Background:
non-invasive estimation of myocardial oxygen supply and demand as measured by Subendocardial Viability Ratio (SEVR) continues to gain importance in the early detection of coronary artery disease (CAD). While adult risk factor associations with SEVR have been documented, especially in patients with CAD, no information is available on the childhood predictors of SEVR in relatively young and healthy adults.

Methods:
this aspect was examined in 610 non-institutionalized individuals (68.7% whites, 54.6% females; aged 29.4 - 51.3 years) that participated in the Bogalusa Heart Study and had cardiovascular risk factors information available since childhood. SEVR was estimated in adulthood through applanation tonometry of the radial artery. The average value of childhood measurements was used as the childhood value, standardized to age, race and sex-specific z-scores. Multivariable linear regression analyses were used to estimate childhood risk factor associations with SEVR in adulthood.

Results:
Blacks and females had significantly lower mean levels of SEVR (p<0.001) compared to their counterparts. Body Mass Index (BMI) in childhood resulted as the only significant predictor of SEVR in white (β= -0.19, p<0.01), but not in black (β= 0.06, p=0.59) adults. No significant associations were observed in other childhood risk factors such as: high density lipoprotein cholesterol, low density lipoprotein cholesterol and blood pressure (both, systolic and diastolic)

Conclusion:
These findings support the hypothesis that the impact of risk factors on adult cardiovascular disease starts early in life and differs among white and black race groups. Further, the adverse effects of childhood obesity on coronary microcirculation in adulthood, as measured through SEVR, underscores the importance of primordial prevention of CV risk factors at an early age, especially obesity, to avoid regrettable complications in adulthood.
Abstract 455: The Effects of Diet on Occlusive Coronary Artery Atherosclerosis, Thrombosis and Myocardial Infarction in SR-BI/LDL Receptor Double KO Mice
Authors: Fuller Mark T , Trigatti Bernardo L
Published: March 2022

Objective:
Deficiency of the HDL receptor, SR-BI, in apolipoprotein E knockout or hypomorphic mice respectively results in spontaneous or diet-inducible occlusive coronary artery atherosclerosis, myocardial infarction, and early death. Here, we examine the effects of SR-BI deficiency on cardiovascular phenotypes in LDLR knockout mice fed different atherogenic diets.

Approach and Results
SR-BI/LDLR double knockout and control LDLR knockout mice were fed diets containing different amounts of fat and cholesterol and which either lacked or contained 0.5% sodium cholate. Double knockout mice fed diets high in cholesterol exhibited significantly reduced survival compared to LDLR knockout mice fed the same diets. In addition to increased aortic sinus atherosclerosis, we observed significant coronary artery atherosclerosis in double knockout mice, and diet dependent accumulation of platelets in coronary artery atherosclerotic plaques. This was accompanied by substantial myocardial fibrosis in double knockout mice fed high cholesterol diets. Double knockout mice also exhibited higher circulating cytokine levels, monocytosis with increased proportions of monocytes expressing high levels of Ly6C, and higher adhesion molecule expression in coronary artery endothelial cells compared to control LDLR knockout mice.

Conclusions:
Diet accelerated atherosclerosis and occlusive, platelet rich coronary artery disease in SR-BI/LDLR dKO mice is affected by amounts of cholesterol and cholate in atherogenic diets, and is accompanied by increased expression of VCAM-1 and ICAM-1 in coronary arteries and increased Ly6C+ monocytes in circulation. The increased VCAM-1 and ICAM-1 in coronary artery endothelial cells in SR-BI-deficient mice likely explains their increased susceptibility to atherosclerosis in coronary arteries.
Abstract 469: Air Pollution Promotes CD36-Dependent Accumulation of Oxidized Lipids
Authors: Rao Xiaoquan , Zhong Jixin , Maiseyeu Andrei , Harkema Jack , Sun Qinghua , Rajagopalan Sanjay
Published: March 2022

BACKGROUND:
The adverse effect of air pollution on cardiovascular disease such as atherosclerosis has long been proposed. We previously reported that exposure to fine particular matter <2.5 microns (PM2.5) increased oxidized lipids in the lung and atherosclerotic plaque burden in the aorta. However, it is currently unclear how signals emanating from the lung contribute to systemic and vascular effects given the fact that the vast majority of PM2.5 particles do not cross the lung barrier.

Methods and Results:
Using an in vivo whole body exposure system, ApoE-/- mice were exposed to filter air (FA) or concentrated PM2.5 for 6 months. Exposure to PM2.5 increased expression of CD36, a scavenger receptor responsible for oxidized lipid uptake, on macrophages. In addition, CD36-dependent phagocytosis was also enhanced in macrophages isolated from ApoE-/- mice exposed to PM2.5. There were increased CD36-expressing macrophages in the plaque of PM2.5-exposed ApoE-/- mice. In line with that, inhalation of PM2.5 increased 7-ketocholesterol, the dominant form of oxidized lipids, in thoracic aorta of ApoE-/- mice. Exposure to PM2.5 promoted the formation of foam cell as demonstrated by increased lipid droplets formation inside macrophages after incubation with oxLDL, while knockdown of CD36 by siRNA could at least partially block this effect. Consistently, macrophages isolated from CD36-/- mice were less sensitive to oxLDL-induced form cell formation, compared to wild-type macrophages.

CONCLUSIONS:
Air pollution accelerates atherosclerosis through promoting CD36-dependent accumulation of oxidized lipids. Our data suggest a role for CD36 in lipid dysregulation and foam cell formation in atherosclerosis.
Abstract 47: Point-of-Care Seeded Blood-Derived Endothelial Cells Rapidly Endothelialize Nitinol Stent in a Pig Model of Personalized Cell Therapy
Authors: Mills James S , Jantzen Alexandra E , Baker Katherine , Lin Fu-Hsiung , Truskey George A , Achneck Hardean E
Published: March 2022

Intimal hyperplasia after percutaneous intervention is especially problematic in peripheral arteries. In coronary arteries, drug-eluting stents (DES) have greatly reduced intimal hyperplasia; however, DES not only prevent smooth muscle cell proliferation but also inhibit endothelial cell (EC) healing. In an effort to rapidly endothelialize Nitinol vascular stents, we have developed a novel rapid seeding technology (QuickSeeding). 5000 micropores (~32.4 ± 0.4 μm) were laser-drilled circumferentially into Nitinol stent delivery systems to enable ECs to flow across the stent struts and thereby attach to the stent while in its compressed state within 5 min at the point-of-care. To evaluate EC adhesion after stent deployment, ECs were isolated from human umbilical cord blood progenitor cells and QuickSeeded, resulting in a uniform stent surface coverage of 55,000 ± 9,500 cells/cm2 (n=4). QuickSeeded stents were then deployed in tubing and tested in a flow circuit at 15 dynes/cm2, representing arterial flow shear stress. After 48 hr of flow, ECs had spread over the stent surface, demonstrated by PECAM staining of cell junctions. To assess EC function, total nitrite was measured as a marker of NO production and increased from 0.41 ± 0.13 nmol under static conditions to 7.83 ± 1.97 nmol after 24 hr of flow (p<0.05, n=5). These results demonstrate a flow-dependent increase in NO secretion. To test this technology in a large animal model, ECs were grown out from peripheral blood circulating progenitor cells of 4 pigs, fluorescently labeled, and seeded onto 4 stents. The 4 stents were then percutaneously implanted into the carotid arteries of the original 4 pigs. Each pig received an identical uncoated stent in the contralateral artery as control. Fluorescent microscopy and SEM showed that control stents were devoid of ECs but QuickSeeded stents were endothelialized after 2 days. Functionality of ECs adhering to stent struts was confirmed with a metabolic assay after explantation.
Abstract 477: The Genetic Interval Sle1 is Sufficient to Accelerate Atherosclerosis in LDLr-/- Mice
Authors: Wilhelm Ashley J , Rhoads Jillian P , Major Amy S
Published: March 2022

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by T and B cell activation and increased circulating autoantibodies. While kidney disease and infection are common causes of early death, patients surviving more than a year after diagnosis have a strikingly increased risk of atherosclerosis resulting in heart attack or stroke. Despite identification of this increased risk more than 40 years ago, mechanisms for SLE-accelerated atherosclerosis remain elusive. The B6.Sle1.2.3 mouse model of SLE contains three genetic loci that confer SLE susceptibility: Sle1, Sle2 and Sle3. We have shown that bone marrow transplant from B6.Sle1.2.3 into LDLr
-/-
mice results in increased atherosclerosis accompanied by more CD4
+
T cells in the lesions, pointing to T cell dysregulation as a contributing factor. While all three loci are necessary for fully penetrant lupus nephritis, whether one particular interval is responsible for accelerated atherosclerosis is unknown. We have shown that Sle3, the interval associated with T cell dysregulation, is not adequate to accelerate atherosclerosis. Therefore, the purpose of the current study was to test the hypothesis that Sle1, which is linked to chronic lymphocyte activation, is sufficient to modulate atherosclerosis in LDLr
-/-
mice. LDLr
-/-
mice were irradiated and received bone marrow from Sle1, Sle3 or Sle1.2.3 mice. Following a 16 week recovery period, mice were placed on Western diet for 8 weeks. At sacrifice, spleens were collected for flow cytometry and atherosclerotic lesion area in the aortic root was evaluated. LDLr.Sle1 and LDLr.Sle1.2.3 mice exhibited similar increases in the percentage of splenic CD4
+
T cells and activated T cells compared to LDLr.Sle3 controls. Interestingly, both LDLr.Sle1 and LDLr.Sle1.2.3 mice had a 30% increase in atherosclerotic lesion area compared to controls, along with increased numbers of CD4
+
T cells in the lesions. These changes were independent of serum cholesterol and triglyceride levels. Our results indicate that the genetic interval Sle1 is sufficient to cause accelerated atherosclerosis in LDLr
-/-
mice and that this increase is related to aberrant lymphocyte homeostasis and activation.
Abstract 48: Successful Rejuvenation of Mesenchymal Stem Cells From Amputated Limbs of Vascular Patients for Therapeutic Applications
Authors: Brewster Luke , Robinson Scott , Griffiths Sarah , Rajabalan Ajai , Wang Ruoya , Li Haiyan , Peister Alexandra , Copland Ian , McDevitt Todd
Published: March 2022

Background:
Mesenchymal stem cells (MSC)from patients with critical limb ischemia(CLI) are inferior to healthy donor MSCs (hMSC). Yet cellular therapy may play a role in CLI, and CLI is a relevant setting to test cell rejuvenation techniques. Pooled human platelet lysate (PL) is a novel serum for cell rejuvenation free of xenogens common in fetal bovine serum (FBS).

Objective:
To rejuvenate with PL or FBS MSCs cultured from the bone marrow of amputated limbs and compare the biologic function in the setting of CLI (iMSC) or CLI and diabetes (idMSC) to that of hMSC.

Methods:
iMSCs (n=4) and idMSCs (n=4) were cultured from the bone marrow of ischemic and ischemic and diabetic amputation specimens and compared to hMSCs (n=4). MSCs were confirmed by FACS and differentiation assays, and doubling time carried out to senescence of comparison groups (passage 9). Secretome analyses (HGF, VEGF, FGFb, MCP-1, IL-6/8) were performed by ELISA after culturing MSCs in basal media + 0.25% HSA. Endothelial cell (EC) proliferation and migration was compared to negative control after stimulation with MSC conditioned media. PKH-stained ECs were co-cultured with these MSCs, and MSC effect on EC invasion quantified under fluorescent imaging. All experiments were performed in quadruplicates with replicates. Statistical analyses were performed with students’ t-test, and ANOVA with Bonferroni or Tukey correction using a significance of P<.05.

Results:
MSCs were successfully cultured in both FBS and PL and confirmed by FACS and differentiation assays. Doubling time was delayed in iMSCs and idMSCs compared to hMSCs by passage 7, but PL significantly lessened this effect compared to FBS. Therapeutic levels of HGF, VEGF, and FGFb were identified in the secretome (25-100pg/10,000 MSCs), and conditioned media stimulated EC proliferation over negative control (PBS). EC invasion was significantly stimulated by co-culture with all MSCs compared to ECs alone, and there were no differences between hMSCs and iMSCs or idMSCs on EC proliferation, migration, or invasion.

Conclusion:
iMSC and idMSCs rejuvenated in cell culture from early passages (p<7) have similar biologic profiles to that of hMSCs. Streamlined culture rejuvenation paradigms may enable autologous cellular therapy in CLI patients.
Abstract 480: Increased Expression of Dipeptidyl Peptidase-4 in Atherosclerosis: A Role for TLR4/MyD88 Signaling
Authors: Zhong Jixin , Rao Xiaoquan , Oghumu Steve , Braunstein Zachary , Satoskar Abhay , Rajagopalan Sanjay
Published: March 2022

OBJECTIVE:
Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed peptidase with high levels of expression on innate and adaptive cell populations. DPP4 expression is upregulated in a number of conditions associated with inflammatory activation of macrophages. In this study we attempted to understand the mechanisms by which DPP4 is regulated in atherosclerosis.

METHODS AND RESULTS:
We compared DPP4 expression on patients with stable established cardiovascular disease (SCVD, n=20) and compared them with control subjects without atherosclerosis using flow cytometry. Compared to healthy controls, SCVD patients had increased DPP4 expression on peripheral monocytes (4.22 ± 0.44 vs. 5.76 ± 0.44, in control vs. SCVD, p=0.037) and T cells (27.46 ± 2.67 vs. 48.06 ± 1.96, control vs. SCVD, p<0.0001). DPP4 expression on T cells and monocytes correlated with plasma non-HDL level (R
2
=0.153, p=0.036 and R
2
=0.244, 0.012 respectively). In cultured peripheral blood mononuclear cells (PBMCs) stimulated with LPS, DPP4 expression increased 4-fold compared to control cells. Increased DPP4 with LPS was abrogated by Toll-like receptor 4 (TLR4)-specific siRNA knockdown. Macrophages deficient for MyD88, an adaptor protein for Toll-like receptor signaling, displayed reduced expression of DPP4. Consistent with that, MyD88
-/-
mice demonstrating improved post-prandial glycemia to oral glucose challenge.

CONCLUSIONS:
DPP4 expression is up-regulated in atherosclerosis and involves TLR4-MyD88 dependent activation. Our result suggest a role for this pathway in metabolic dysregulation and inflammation in atherosclerosis.
Abstract 483: EGR1 Mediates Suppression of PKD1 Expression, an Action Antagonized by Pioglitazone in Differentiated Angiotensin II-Treated THP-1 Cells
Authors: Charolidi Nicoletta , Torsney Evelyn , Pirianov Grisha , Laing Ken , Nohturfft Axel , Cockerill Gillian W
Published: March 2022

Aims:
Perixosome proliferator-activated receptor γ (PPARγ) agonists have been shown to inhibit Angiotensin II-induced experimental abdominal aortic aneurysms. Since increased macrophage infiltration in the vascular wall is a hallmark for initiation of aneurysmal pathology, we set out to explore the effects of the PPARγ agonist pioglitazone on Angiotensin II-treated, phorbol-ester-differentiated monocytes, with the scope to identify molecular changes that may be responsible for the infiltrating phenotype of macrophages.

Approach and Results:
Using microarray-based expression profiling of phorbol-ester-stimulated THP-1 monocytic leukemia cells, we found that Angiotensin II (Ang II) or Ang II and pioglitazone were able to cause a number of aneurysm-related gene changes. Among those, polycystic kidney disease 1 (PKD1) was significantly up-regulated. Mutations that lead to changes in PKD1 expression are the main cause of autosomal dominant polycystic kidney disease, which is linked to increased incidence of aneurysms. Online-software analysis of the PKD1 proximal promoter revealed a putative Early Growth Response 1 (EGR1) binding site, which we confirmed by chromatin immuneprecipitation (ChIP) and quantitative real time (RT) PCR. Further analyses of publicly available ChIP-sequencing data (ENCODE project) revealed that this putative binding site in the PKD1 proximal promoter overlapped with a conserved EGR1-binding peak present in 5 other cell lines. Quantitative RT-PCR showed that EGR1 suppressed PKD1, while Ang II significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in THP-1 cells that were lenti-virally transduced with EGR1 short-hairpin RNA, reduced EGR1 levels led to significant up-regulation of PKD1, especially after treatment with pioglitazone.

Conclusion:
PKD1 is an EGR1-target gene that is suppressed during monocytic differentiation at the time of EGR1 expression. Ang II treatment up-regulates the expression of PKD1 and pioglitazone mediates the opposite effect by displacement of EGR1 from their shared binding site in the PKD1 proximal promoter through a mechanism that requires further investigation.
Abstract 489: Peak Wall Stress and Rupture Risk of Abdominal Aortic Aneurysms Are Not Influenced by Age
Authors: Lindquist Liljeqvist Moritz , Hultgren Rebecka , Gasser Christian , Roy Joy
Published: March 2022

OBJECTIVES:
Older patients with Abdominal Aortic Aneurysms (AAA) face higher perioperative mortality and, according to a recent meta-analysis, a higher risk of rupture. The relationship between age and AAA biomechanics, however, has not been studied previously. Our aim was to investigate age dependent differences in peak wall stress (PWS) and peak wall rupture risk (PWRR = wall stress / wall strength), as estimated by finite element modeling (FEM).

METHODS:
80 patients, 52-88 years old with AAA diameters between 40 and 60 mm, who had undergone computed tomography angiography (CT-A), were included. PWS and PWRR were estimated, by FEM, based on patient demographics and CT-A-derived AAA geometry, including the intraluminal thrombus (ILT). Three diameter matched age groups (≤65, 66-75 and ≥76 years old) were compared with respect to ILT-volume, PWS and PWRR with ANOVA and Kruskal-Wallis test. Correlation was tested with Spearman’s rank correlation coefficient.

RESULTS:
All age groups had similar diameters (53.2 vs 52.8 vs 54.6 mm, P=0.44). No associations between age and biomechanical parameters were found. Differences in PWS (200.9 vs 199.0 vs 198.3 kPa, P=0.98) and PWRR (0.39 vs 0.37 vs 0.38 P=0.74) between age groups were small and non-significant. No correlation existed between age and PWS (r=0.018, P=0.88) or PWRR (r=0.031, P = 0.78). In the whole cohort, women had a significantly higher PWRR than men had (0.48 vs 0.35, P<0.0001).

CONCLUSIONS:
Biomechanical parameters estimated by FEM do not differ between different age groups. However, as previously shown, women have a higher rupture risk which may explain the correlation between age and risk of rupture found in recent studies, since more women are found in the higher age groups.
Abstract 494: Proinflammatory Role of Stem Cells in Abdominal Aortic Aneurysms
Authors: Ryer Evan J , Schworer Charles M , Garvin Robert P , Tromp Gerard , Bernard-Eckroth Kamell R , Franklin David P , Elmore James R , Kuivaniemi S H
Published: March 2022

Background:
The pathogenesis of abdominal aortic aneurysm (AAA) formation includes inflammation, vascular smooth muscle cell apoptosis, extracellular matrix degradation and oxidative stress. It is well established that multi-potent stem cells have an important role in cardiovascular health and disease but the role of stem cells in AAA formation remains controversial. We sought to describe the presence of stem cells in human AAA tissue. Additionally, we investigated the differentiation of stem cells within the aneurysmal aorta.

Methods:
Infra-renal aortic wall specimens (8 AAA patients; 5 males and 3 females; ages ranging from 55 to 78 years) were collected from patients undergoing AAA repair operations. Non-aneurysmal infra-renal aortic control samples were collected at autopsies (N=5; 3 males and 2 females; donor ages ranging from 53 to 78 years). Using immunohistochemistry, we compared the abundance of STRO1+, cKit+, and CD34+ cells in aortic tissue. Using double immunofluorescence staining, we evaluated stem cell differentiation into smooth muscle cells (SM22), fibroblasts (FSP1), and macrophages (CD68).

Results:
Both the media and adventitia of infra-renal AAA samples demonstrated a significantly greater number of STRO1+, c-Kit+ and CD34+ cells when compared with matched non-aneurysmal control aortic tissues. Furthermore, double immunofluorescence staining identified that AAA stem cells express the macrophage marker CD68 but not the smooth muscle cell marker SM22 or the fibroblast marker FSP1.

Conclusions:
Stem cells are significantly elevated in infra-renal AAA tissue compared to matched control aortic tissue. Our data also demonstrate that AAA stem cells express macrophage surface antigens but not smooth muscle cell or fibroblast markers. These finding suggest an inflammatory/immune role of stem cells during AAA pathogenesis.
Abstract 501: Noninvasive Photodynamic Therapy of Murine Atherosclerosis Using Macrophage-Targeted Nanoparticles
Authors: Kessinger Chase W , El Mahgraby Amr , Erdem S S , Jetzfellner Thomas , Mauskapf Adam , Ntziachristos Vasilis , McCarthy Jason R , Jaffer Farouc A
Published: March 2022

Objectives:
Cerebrovascular disease leading to stroke remains a leading cause of death and disability worldwide. Photodynamic therapy (PDT) of atherosclerotic plaque macrophages may be a promising approach to stabilize rupture-prone carotid lesions, but a noninvasive PDT method for carotid atherosclerosis is lacking. Here we developed a near-infrared (NIR) absorbing macrophage-targeted photosensitizing nanoparticle to determine whether plaque macrophages can be successfully ablated using noninvasive PDT.

Methods:
CLIO-CyAm7-Chlorin is a dextran-based nanoparticle that contains a NIR photosensitizer (Chlorin, abs 650nm) for PDT, and a discrete NIR fluorochrome (CyAm7, ex/em 750/767nm) for fluorescence imaging of nanoparticle distribution. Noninvasive PDT optimization was performed in vivo via a PDT ablation study of hepatic macrophages in C57BL6 mice (n=65). Varying doses, exposure times, and laser power were explored. A separate group of ApoE-/- mice (n=16, 8wk HCD) injected with CLIO-Chlorin-CyAm7 or control CLIO-CyAm7 (10mgFe/kg) received noninvasive PDT of aortic root atheroma. Mice were sacrificed 1 day post-PDT for histology. Computational simulations of noninvasive PDT for human plaques were studied at multiple PDT wavelengths and light fluence.

Results:
Noninvasive PDT optimization showed that increasing doses of CLIO-CyAm7-Chlorin and laser exposure time had strong linear correlations with increasing liver injury (r2=0.88 and 0.65, respectively). Mice injected with control CLIO-CyAm7 showed no damage of the liver by tunel or H&E, independent of dose or time of PDT. In ApoE-/- mice, CLIO-CyAm7-Chlorin and CLIO-CyAm7 localized to inflamed aortic root plaques, and was confirmed to be macrophage localized on fluorescence imaging. CLIO-CyAm7-Chlorin/PDT showed a 6-fold increase in tunel (+) cells in aortic root plaques compared to control CLIO-CyAm7/PDT treated animals (p<0.01). Computational simulations revealed that further NIR-shifting of the photosensitizer would enable noninvasive PDT up to 1-2 cm deep, potentially enabling noninvasive PDT of human carotid plaques.

Conclusions:
CLIO-CyAm7-Chlorin is a multifunctional NIR nanoparticle that allows noninvasive macrophage-targeted PDT of atherosclerosis.
Abstract 507: Homotypic Interactions and Networking Among Vascular Smooth Muscle Cells Are Mediated by a Transforming Growth Factor-ß-Cadherin-11 Axis
Authors: Balint Brittany , Yin Hao , Pickering J Geoffrey
Published: March 2022

Formation and stabilization of arteries requires that smooth muscle cells (SMCs) coalesce into sheets and layers of co-aligned cells. However, the molecular basis by which SMCs form a stable collective is largely unknown. To elucidate key steps in forming an ordered SMC collective, we tracked differentiation-competent human SMCs as they were induced to shift from individual behavior to collective patterning. Transmission electron microscopy identified the acquisition of well-developed adherens junctions during this transition. As well, immunofluorescence microscopy revealed both cadherin-2 and -11-containing intercellular junctions, the latter not previously identified in vascular SMCs. Furthermore, these junctions underwent striking elongation during collectivization (3-fold elongation, p<0.001), effectively strapping cells to each other. Adherens junction maturation was associated with down-regulation of phosphorylated SMAD2 and could be reversed by addition of transforming growth factor-beta (TGFß)1 (p<0.001). Time-lapse video microscopy revealed that collectivization of SMCs was partially inhibited by a blocking antibody to cadherin-2 (p=0.021) and strikingly inhibited by a cadherin-11 blocking antibody (p<0.001). Imaging of Fura2-loaded SMCs revealed coordinated calcium transients among stably contacting SMCs (p<0.001), but these were inhibited when cadherin-11 was blocked (p<0.001). Immunostaining of small and medium sized arteries in the mouse hindlimb identified OB-cadherin in both endothelial and SMC layers.

Conclusions:
Cadherin-11 is present in the artery wall and mediates the acquisition of stable and functional homotypic interactions among contacting SMCs. This TGFß-regulatable phenomenon of collectivization could be critical to vascular development, repair, and regeneration.
Abstract 523: Persistent Disrupted Mitochondrial Adaptation in Primary Vascular Cells from a Diabetic Model
Authors: Keller Amy C , Knaub Leslie A , McClatchey P. Mason , Connon Chelsea A , Geary Kate E , Bouchard Ron , Walker Lori A , Reusch Jane E
Published: March 2022

People with diabetes (DM) have an excess burden of cardiovascular disease (CVD). Impairments in vascular contractility predict poor CVD outcomes, and mitochondria are essential for arterial contraction and relaxation. Mitochondrial dysfunction is observed with DM and insulin resistance. Our laboratory has reported failed vascular mitochondrial adaptation to exercise in DM rats. We hypothesize that repairing vascular mitochondrial function could restore contractility in DM. We employed primary aortic smooth muscle cells (SMC) from a lean, insulin resistant DM rat, Goto-Kakizaki (GK), and the Wistar (W) control to test mitochondrial adaptation to metabolic stress. SMC were exposed to low glucose (LG, 5 mM glucose) or high glucose (HG, 25mM) treatments for 1, 4 and 24 hours. Mitochondrial respiration, superoxide and dynamics (fission and fusion) were measured. Respiration in W SMC exposed to HG was unchanged compared to the LG control, but was decreased after 4 hours of HG treatment in the GK SMC (p≤0.05). GK SMC have significantly increased whole cell production of H
2
O
2
as compared to W SMC, but not mitochondrial superoxide after 4 hours of HG treatment. In contrast, W SMC exposed to HG significantly had increased superoxide relative to LG control (p<0.05). No differences in total mitochondrial complex expression were seen in baseline W and GK SMC comparisons; phosphorylated eNOS and PGC-1α, direct upstream regulators of mitochondrial biogenesis, were increased and decreased, respectively, in the GK SMC as compared to the W SMC (p<0.05). Preliminary quantitative immunohistochemistry shows a significant mitochondrial biogenesis and fusion response to the initial hour of HG treatment in the W SMC; conversely, GK SMC showed a decrease in mitochondria after 4 hours of HG treatment. Fis 1 was significantly decreased at 1 hour in W and increased at 24 hours in the GK (p<0.05). Significant changes in fusion included decreased OPA1 and mfn1 in W SMC and increased OPA1 and decreased mfn1 in the GK. Overall, these data reveal that distinct mitochondrial adaptation to a hyperglycemic state is persistent in DM cells ex vivo. Primary SMC can be used as a model to define signaling events determining these changes and development of therapeutic targets to repair contractility.
Abstract 53: An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma
Authors: Huang Ying , DiDonato Joseph A , Levison Bruce S , Parks John S , Fisher Edward A , Smith Jonathan D , Hazen Stanley L
Published: March 2022

High density lipoproteins (HDL) are a heterogeneous group of particles formed predominantly around the major structural protein, apolipoprotein A1 (apoA1). Contemporary findings suggest functional measures of HDL/apoA1 may provide improved clinical relevance. Moreover, recent studies indicate apoA1 in human atheroma is dysfunctional, extensively oxidized by myeloperoxidase (MPO)-generated oxidants, and that in vitro oxidation of apoA1 by MPO impairs its cholesterol acceptor function. Using phage-display affinity maturation and recombinant immunoglobulin engineering we developed a high affinity monoclonal antibody (mAb) that specifically recognizes apoA1/HDL modified by the MPO/H
2
O
2
/halide system. An oxindolyl alanine (2-OH-Trp) moiety at tryptophan 72 of apoA1 serves as the mAb recognition site. Mutagenesis studies confirm a critical role for apoA1 Trp72 in MPO-dependent inhibition of ABCA1-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. While apoA1 containing a 2-OH-Trp
72
group (oxTrp72apoA1) is in low abundance within the circulation, it is remarkably abundant within human atherosclerotic plaque, accounting for 20% of apoA1 in aortic lesions. Functional characterization of immune-purified oxTrp72apoA1 from human atherosclerotic plaque and plasma reveals a lipid-poor lipoprotein virtually devoid of cholesterol acceptor activity. Recovered oxTrp72apoA1 demonstrated impaired capacity to promote HDL biogenesis in vivo, and potent pro-inflammatory activities including NF-κB activation and VCAM-1 surface expression on endothelial cells. Elevated oxTrp72apoA1 levels among sequential subjects presenting to a preventive cardiology clinic (n=627) were associated with increased cardiovascular disease risk, including after adjustments for traditional risk factors, apoA1 and MPO [Odds ratio (95%confidence interval), 4.2(1.5-13.0)]. Circulating oxTrp72apoA1 levels provide a window into monitoring the generation of an abundant pro-atherogenic apoA1 form within the artery wall.
Abstract 530: Impaired Ability of Pulmonary Microvascular, but Not Macrovascular, Endothelial Cells From Patients With Pulmonary Arterial Hypertension to Respond to High Shear Stress
Authors: Szulcek Robert , Fontijn Ruud , van Bezu Jan , Vonk Noordegraaf Anton , van Hinsbergh Victor , Bogaard Harm Jan , van Nieuw Amerongen Geerten P
Published: March 2022

Rationale:
We hypothesize that severe pulmonary vascular remodelling in pulmonary arterial hypertension (PAH) results from sustained endothelial injury caused by increased shear stress (SS) in pre-capillary endothelial cells. Therefore we tested whether human pulmonary arterial and microvascular endothelial cells (HPAEC and MVEC resp.) from PAH patients are capable to adapt to high levels of SS.

Methods & Results:
HPMVEC were isolated from PAH patients and controls (lung tumor patients). Static controls were compared to cells under physiological low (LSS, 2.3 dyn/cm
2
) and pathological high SS (HSS, up to 25 dyn/cm
2
). Different flow profiles (laminar, bifurcation-like) were applied and electrical resistance of the monolayers was recorded as a measure of cell proliferation and barrier function. Under static culture conditions, PAH cells showed a decreased and irregular distribution and organization of VE-cadherin and CD31 at the cell periphery, indicating weakened adherens junctions. PAH and control HPMVEC formed similar endothelial barriers with a baseline resistance of 7767 vs. 8102 ohms, and responded to challenge with the hyper-permeability inducer thrombin (1 U/mL) with a specific maximal drop in resistance (to 2109 vs. 2083 ohms). Control HPMVEC re-aligned in flow direction, whereas HPMVEC, but not HPAEC from PAH-patients showed a delayed alignment response. Furthermore, application of HSS to control and PAH HPMVEC caused severe injury when bifurcation-like flow patterns were used. In contrast HPMVEC in laminar flow regions were able to withstand HSS challenge. Known shear-response genes were not affected (normal induction of KLF2, thrombomodulin, eNOS, SMAD6/7 and downregulation of MCP1). Protein analysis showed reduced expression of PECAM1 as part of a shear sensing complex (PECAM1/VEGFR2/VEcadherin) and its downstream signaling molecule Tiam1. Partial depletion of PECAM1 in control HPMEC mimicked the impaired realignment response.

Conclusion:
HPMVEC, but HPAEC from PAH-patients have defects in the shear sensing complex accompanied by a hampered ability to adapt to pathological HSS, with severe endothelial damage at bifurcations.
Abstract 539: P66Shc Adaptor Protein Protects From Short-Term Ischemic Myocardial Injury via Intracellular Salvage Pathways
Authors: Akhmedov Alexander , Montecucco Fabrizio , Braunersreuther Vincent , Camici Giovanni G , Jakob Philipp , Reiner Martin F , Paneni Francesco , Galan Katia , Pelli Graziano , Vuilleumier Nicolas , Belin Alexandre , Vallée Jean-Paul , Mach Francois , Lüscher Thomas F
Published: March 2022

Background:
Formation of reactive oxygen species (ROS) contributes to many pathophysiological processes. Although ROS production is also involved in some physiological processes, the imbalance between their generation and removal, i.e. oxidative stress, plays a major role in myocardial injury caused by ischemia/reperfusion (I/R). The mammalian Shc locus encodes three Shc isoforms: p46Shc, p52Shc and p66Shc. The p66Shc is not involved in mitogenic signals as p46Shc/p52Shc, but it functions as a critical mediator of intracellular oxidative signal transduction. Various studies relate p66Shc to cardiovascular disease; however, few data are available on the role of p66Shc in myocardial I/R.

Methods and Results:
8-12-week-old male p66Shc deficient (p66Shc-/-) mice and corresponding C57Bl/6 wild-type (WT) control mice were subjected in vivo to different durations of I (30, 45 and 60 min) followed by 24h of R. Infarct size was assessed morphologically. After 30 min of ischemia (I), p66Shc-/- mice developed markedly larger infarcts as compared to WT. This effect was confirmed by in vivo silencing of p66Shc prior to I/R. Both genetic deletion and silencing of p66Shc displayed increased post-ischemic levels of serum cardiac troponin I. However, the observed effect on infarct size was limited to 30 min of I since by increasing ischemia duration to either 45 or 60 min infarct size did no longer differ between p66Shc-/- and WT mice. Moreover, differently from WT, infarct size in p66Shc-/- was not significantly larger with increasing duration of I (from 30 to 60 min). On the molecular level the observed effect was linked to the inhibition of phosphorylation of protein kinase Akt and transcription factor STAT3 - two key members of prosurvival pathways RISK and SAFE, respectively. Inhibition of STAT3 activation further led to mitochondrial swelling and cellular apoptosis in cardiac tissue of p66Shc-/- mice.

Conclusions:
Our data suggest that genetic deletion of p66Shc leads to an increased sensitivity to myocardial infarction with larger infarcts with shorter, but not prolonged I, and that prosurvival pathways are involved. Therefore, activation of p66Shc may provide resistance to I and represent a novel therapeutic target in the early phase of myocardial infarction.
Abstract 553: Relaxin Attenuates Angiotensin II--Induced Proinflammatory Signaling in Vascular Smooth Muscle Cells
Authors: Vukelic Sasa , Lassegue Bernard , Griendling Kathy
Published: March 2022

Relaxin 2 (RLX) was traditionally considered a reproductive hormone. However, recent studies indicate that it also has potent short-term vasodilatory and long-term arterial remodeling and anti-fibrotic effects. Based on these findings, a recombinant form of relaxin, Serelaxin, was successfully used in treatment of the acute heart failure. In the present study, we tested the hypothesis that RLX can attenuate the Angiotensin II (Ang II) mediated vascular inflammation. Ang II, the principal hormone of the renin-angiotensin system, can induce secretion of the Monocyte Chemoattractant Protein-1 (MCP-1) and Interleukin 6 (IL-6). Release of these proinflammatory mediators is an initial step in vascular inflammation, an important early component in the development of cardiovascular disease. Utilizing ELISA, we observed that RLX pretreatment inhibited Ang II-induced secretion of MCP-1 and IL-6 by 70 (p<0.05) and 87% (p<0.05), respectively, in human aortic smooth muscle cells. The same effect was achieved in isolated mouse aortas. We next studied the effect of RLX on Ang II-induced activation of the NFκB pathway and ROS signaling. RLX pretreatment inhibited Ang II-mediated induction of NADPH oxidase 5 expression and production of H2O2 as measured by the sensor HyPer. RLX also prevented P65 phosophorylation and nuclear translocation in response to Ang II. We conclude that RLX inhibits secretion of IL-6 and MCP-1 likely through interference with ROS-mediated NFκB activation. Via this mechanism, RLX may thus represent a therapeutic treatment of pro-inflammatory cardiovascular conditions such as acute heart failure, peripheral arterial disease, and aortic aneurysms.
Abstract 566: Broad-Spectrum Chemokine Inhibition Blocks Inflammation-Induced Pathological Angiogenesis but Preserves Hypoxia-Driven Physiological Angiogenesis
Authors: Ridiandries Anisyah , Prosser Hamish , Hawkins Clare , Bursill Christina
Published: March 2022

Introduction:
Angiogenesis is critical for survival and in the regenerative response to tissue hypoxia. An imbalance in its regulation causes excessive angiogenesis, exacerbating inflammatory diseases such as cancer and atherosclerosis. Chemokines from the CC-chemokine class are increasingly implicated in the regulation of inflammatory pathological angiogenesis but, in contrast, have a minimal role in hypoxia-driven physiological angiogenesis. Inhibition of CC-chemokines may therefore differentially regulate angiogenesis depending on the pathophysiological context. The ‘35K’ protein uniquely inhibits the CC chemokine class and does not affect chemokines from other classes.

Aim:
To determine the effect of CC-chemokine inhibition with 35K on inflammation-induced angiogenesis and hypoxia-driven angiogenesis using human coronary artery endothelial cells (EC) in in vitro angiogenic functional assays.

Results:
Matrigel tubule formation was strikingly inhibited (95.8%, p<0.001) by 35K (50nM) under the inflammatory stimuli of macrophage conditioned media. The addition of 35K also reduced inflammation-induced proliferation by 88.3% (p<0.001). Furthermore, we found the addition of 35K suppressed inflammation stimulated EC migration speed by 15.8% (p<0.05). By contrast, in hypoxia, 35K (50nM) reduced tubule formation by 45.2% (p<0.01), indicating a relative preservation of tubulogenesis in hypoxia compared to inflammation. Consistent with this, in hypoxia, 35K reduced proliferation by 43.8% (p<0.05). Moreover, the speed of EC migration was completely preserved under hypoxic conditions following the addition of 35K. Mechanistic studies supported our functional observations. Angiogenic growth factor, VEGF, was inhibited (53.4%) by 35K (200nM) under inflammatory conditions yet was augmented in hypoxia (31.5%). 35K also decreased key angiogenesis transcription factors, p65 (37.3%) and HIF-1α (87.3%) in inflammation.

Conclusion:
Broad-spectrum chemokine inhibition by 35K blocks inflammation-induced angiogenic processes, whilst preserving hypoxia-driven angiogenesis. This may present as a therapeutic strategy to reduce pathological angiogenic disease, without the severe side effects of current therapies.
Abstract 572: Serotonin and Selective Serotonin Reuptake Inhibitors in Platelet Function and RhoA Activation
Authors: Oliver Kendra H , Hamm Heidi E , Carneiro Ana M
Published: March 2022

Platelet hyperactivity leads to thrombotic disease, underlying cardiovascular (CV) pathologies including stroke and myocardial infarction. Clinical data suggest that selective serotonin reuptake inhibitors (SSRIs) have hemostatic effects, signifying a potentially novel mechanism to treat CV disease. SSRIs target the serotonin transporter (SERT), which maintains dense-granule serotonin (5HT) in platelets. Serotonin is also a platelet agonist, acting through 5HT2A receptors, yet the full role of 5HT in platelet physiology is not well understood. We hypothesized that SERT KO mice, mimicking chronic SSRI treatment, would exhibit a bleeding phenotype due to reduced platelet function. Consistent with previous reports, we demonstrated that SERT KO mice have decreased whole blood 5HT. There were comparable concentrations of the 5HT metabolite 5HIAA, suggesting that overall synthesis and metabolism of 5HT is unchanged. We next demonstrate that there is a bleeding phenotype in the SERT KO mice through a significantly increased tail-bleed time and increased thrombin time (TT) in whole blood. Additionally, SERT KO mice have reduced platelet retraction, a platelet function attributed to decreased RhoA activation. To determine if sub-chronic SSRI treatment alters platelet function, we treated both wild type and a hyperactive platelet (KI) mice with citalopram in drinking water for 6 days, one day exceeding the turnover rate of platelet production (15mg/kg/day). Activation of RhoA was examined to determine if reduced serotonylation leads to reduced platelet function. Collectively, these data suggest that genetic or pharmacological inhibition of SERT activity could lead to reduced platelet function. These findings may be due to a decrease in intracellular 5HT, as 5HT is still present for 5HT2A activation as determined by the presence of the metabolite. Future experiments will examine the mechanism of SSRI-mediated platelet activity attenuation, which may include reduced serotonylation of important signaling molecules.
Abstract 588: Optimization of Radiolabeled Liposomes for in vivo Atherosclerosis Imaging by SPECT
Authors: Ogawa Mikako , Uchino Ryuji , Kawai Ayumi , Magata Yasuhiro
Published: March 2022

Objectives:
Molecular imaging enables non-invasive
in
vivo
detection of vulnerable plaques. Last year, we reported an atherosclerosis imaging agent,
111
In-labeled liposome. The liposome was modified with phosphatidylserine (PS), since macrophages recognize PS to phagocytize apoptotic cells in the plaque. PS modification was successful and we could visualize atherosclerotic plaques by SPECT. However, the too quick blood clearance seemed to have reduced PS-liposome accumulation to the plaque. Therefore, in this study, we PEGylated the liposome to retard the blood clearance.

Methods:
PS liposomes were prepared by lipid-film hydration method. The liposomes, sizes in 100 or 200 nm, were PEGylated with PEG2000 or PEG5000 in 1 or 5% w/w. The liposomes were radiolabeled by encapsulating
111
In-NTA using active-loading method. For
in
vitro
uptake study, the liposomes were incubated with mouse peritoneum macrophages. The in vivo biodistribution studies were done in ddY mice.
En
face
autoradiograms were obtained with ApoE-/- mice 2 hr after the liposome injection. For SPECT imaging, WHHL rabbits were used.

Results:
The
in
vitro
uptake levels to macrophages were decreased by PEGylation. 5% PEG5000 decreased the uptake to the half of non-PEGylated one, but 1% PEG2000 affected less than 5%.
In
vivo
results showed slower blood clearance in PEGylated liposomes (12 %dose/g for 5%PEG5000, 2.0 %dose/g for 1%PEG2000, and 1.0 %dose/g for non-PEGylated liposome at 60 min after the injection). PS modification improved target-to-nontarget ratios, and 5%PEG2000 added 200 nm liposome showed the highest uptake to the region. The aorta was successfully visualized by SPECT at 48hr after the
111
In-labeled 5%PEG2000-PS200 liposome injection.

Conclusions:
PEGlylation prolonged the blood clearance, but decreased the uptake to macrophages. However, the effect was larger for blood clearance and decrease in macrophage uptake was acceptable for
in
vivo
imaging.
Abstract 590: LDL Particle Number is Associated With Incident Atherosclerotic Cardiovascular Disease Among Persons With a 10-Year Risk of <7.5%
Authors: Melander Olle , Shiffman Dov , Caulfield Michael P , Louie Judy Z , Rowland Charles M , Devlin James J , Krauss Ronald M
Published: March 2022

Background:
The 2013 AHA/ACC guidelines on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular disease (ASCVD) risk recommend that individuals with LDL-C between 70 and 190 mg/dL should receive statin therapy if their 10-year risk of ASCVD is >7.5%. LDL particle number is not included in the calculation of 10-year risk, but it has been reported to be to be associated with ASCVD. Therefore we asked whether LDL particle number was associated with ASCVD events among those whose 10-year risk does not exceed the 7.5% threshold for statin therapy.

Methods:
We determined LDL particle number by ion mobility in a prospective, population-based study of 4755 men and women from the Malmö Diet and Cancer Study Cardiovascular Cohort. Among the 1928 participants who at baseline had <7.5% 10-year ASCVD risk, LDL-C <190mg/dL, and were free of ASCVD and diabetes, Cox proportional hazards models were used to analyze the association between LDL particle number and incident ASCVD.

Results:
During a median of 16.4 years of follow-up, 89 participants had a first incident ASCVD event. LDL particle number was associated with ASCVD events (P=0.003) after adjustment for age, sex, LDL-C, HDL-C, triglycerides, systolic and diastolic blood pressure, use of antihypertensive medication, and smoking. Participants in the top LDL particle number tertile had more than two-fold increased risk of ASCVD (HR = 2.50; 95%CI 1.32 to 4.74) in the fully adjusted model when compared with the bottom tertile. LDL-C, HDL-C, and triglycerides were not associated with incident ASCVD in these 1928 participants (P>0.25) in models that did or did not include LDL particle number.

Conclusion:
Among those who do not meet the 2013 AHA/ACC statin treatment guidelines, those in the top LDL particle number tertile were at elevated risk of ASCVD events, a risk that was independent of traditional risk factors, including standard lipid measurements.