bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 256: Activation of Cell Surface GRP78 by Anti-GRP78 Autoantibodies Accelerates Lesion Development by Promoting Endothelial Cell Activation
Authors: Crane Elizabeth D , Austin Richard C
Published: March 2022

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is a molecular chaperone localized to the endoplasmic reticulum (ER) in healthy cells that functions to assist in the correct folding of newly synthesized proteins and to prevent aggregation of folding intermediates. In addition, GRP78 is also present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a membrane signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies isolated from the serum of cancer patients. We have demonstrated previously that high levels of anti-GRP78 autoantibodies accelerate lesion development in apoE
-/-
mice. However, the role of anti-GRP78 autoantibody activation of cell surface GRP78 on endothelial cells and how this contributes to atherogenesis is unknown. The objective of this study is to identify factors that mediate surface GRP78 expression on endothelial cells and to determine the mechanism by which surface GRP78 activation on endothelial cells contributes to atherogenesis. Here we demonstrate that induction of ER stress by tunicamycin increased surface GRP78 expression in cultured human aortic endothelial cells. We also show that activation of surface GRP78 on endothelial cells by anti-GRP78 autoantibodies significantly increases gene expression of the adhesion molecules ICAM-1 and VCAM-1. Pretreatment of these cells with a calcium chelator or an inhibitor of NF-B activation attenuated the anti-GRP78 autoantibody-induced promotion of ICAM-1 and VCAM-1. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation, an important initiating event in atherogenesis. These findings further our understanding of the role of anti-GRP78 autoantibodies and the activation of surface GRP78 in endothelial cell function and lesion development. Furthermore, inhibiting the interaction of anti-GRP78 autoantibodies with surface GRP78 could present a novel therapeutic strategy to modulate lesion growth and thereby reduce the risk for atherosclerosis and cardiovascular disease.
Abstract 266: Disturbed Flow Promotes Endothelial Cell Injury via the Induction of Developmental Genes
Authors: Mahmoud Marwa , Kim Rosemary , de Luca Amalia , Gauci Ismael , Hsiao Sarah , Evans Paul C
Published: March 2022

Introduction:
Atherosclerosis is influenced by blood flow patterns which exert wall shear stress (WSS) on endothelial cells (EC). Low oscillatory WSS promotes atherosclerosis by inducing EC apoptosis and activation, whereas high unidirectional WSS is protective. We used microarray technology coupled to computational fluid dynamics to study the transcriptome of EC at regions of the porcine aorta exposed to low, oscillatory or high, unidirectional WSS. The study revealed differential expression of multiple genes that co-ordinate embryonic development. We hypothesised that this gene set includes WSS-sensitive regulators of EC survival.

Methods and Results:
EC were isolated from distinct anatomical regions of the porcine aorta using collagenase (n=6), and quantitative RT-PCR revealed elevated expression of developmental genes (GATA4, HAND2, TWIST1, FZD5, BMP2, SLIT2, PDGFRA, and FBN2) at the inner (low WSS) compared to the outer curvature (high WSS) (all p<0.05). Similarly, en face staining demonstrated that expression of GATA4 protein in EC was higher at the inner compared to the outer curvature of the murine aortic arch (p<0.05). Studies using cultured porcine aortic EC (n=7) or human umbilical vein EC (n=7) revealed that GATA4, HAND2, TWIST1, FZD5, BMP2, SLIT2, PDGFRA, and FBN2 were induced in HUVEC or PAEC exposed to low, oscillatory WSS for 72h using either the orbital shaking (210 rpm; +/-WSS 4 dyn/cm2) or IBIDI™ (+/-5 dyn/cm2 at 2Hz) systems (all p<0.05). The rate of caspase-3 activation was significantly higher in EC exposed to low, oscillatory WSS compared to high, unidirectional WSS (3.1% vs 0.7%; p<0.0001; n=10). Silencing of GATA4, FZD5 and BMP2 significantly reduced apoptosis in EC exposed to low, oscillatory WSS (1.2%, 0.6% and 1.6%; p<0.05).

Conclusions:
We conclude that WSS influences EC viability through the induction of GATA4, FZD5 and BMP2; molecules that have a well-defined role in embryonic development. Further work is required to define the molecular mechanisms that underlie the induction of apoptosis by these molecules. Our observations illuminate the molecular mechanisms that regulate the focal nature of vascular injury and atherosclerosis.
Abstract 269: Xanthine Oxidase Inhibition by Febuxostat Attenuates Experimental Atherosclerosis in Mice
Authors: Nomura Johji , Busso Nathalie , Matsui Chieko , Tsujimoto Syunsuke , Shirakura Takashi , Tamura Mizuho , Kobayashi Tsunefumi , So Alexander , Yamanaka Yoshihiro
Published: March 2022

Objective:
Atherosclerosis is a chronic inflammatory disease of the vascular wall. Multiple mechanisms are implicated in the inflammatory process, including oxidative stress. Xanthine oxidase (XO), which is converted from xanthine dehydrogenase (XDH), is a major enzymatic source of reactive oxygen species (ROS) and has been reported to be involved in atherosclerosis, but the underlying mechanisms remain unclear. We have examined whether XO contributes to atherosclerosis in mice and whether XO mediates cholesterol crystal (CC)-induced inflammation.

Approach and Results:
ApoE
-/-
mice fed a high cholesterol diet for 12 weeks (ApoE
-/-
) developed atherosclerotic plaques, increased ROS generation, increased tissue activities of XO/XDH, and endothelial dysfunction. Immunohistochemical analysis revealed that XO/XDH were increased in macrophages in atherosclerotic plaques and in aortic endothelial cells. Febuxostat, a highly potent XO/XDH inhibitor, significantly attenuated plaque formation, decreased ROS generation, and improved endothelial dysfunction in ApoE
-/-
mice without affecting plasma cholesterol levels. Additionally, in aorta from febuxostat-treated group, MCP-1 mRNA was significantly decreased whereas eNOS mRNA was increased. Furthermore, we examined the effects of febuxostat on CC-induced inflammation in macrophages. Febuxostat significantly inhibited CC-induced ROS formation as well as MCP-1, IL-1α, IL-1β and IL-6 production and its effects could be recapitulated by ROS scavengers.

Conclusions:
These results demonstrated that increased XO leads to ROS formation and contributes to the progression of atherosclerosis via endothelial dysfunction and macrophage inflammation, and that febuxostat may be a potential therapeutic agent to target inflammation in atherosclerosis.
Abstract 272: Targeted Activatable Cell-Penetrating Peptides Identify Atherosclerotic Plaques in Mice with Noninvasive Magnetic Resonance Imaging
Authors: Schwanz Heidi A , Hua Ning , Slocum Connie L , Kramer Carolyn D , Whitney Michael , Jiang Tao , Genco Caroline A , Tsien Roger Y , Hamilton James A
Published: March 2022

Atherosclerotic plaque progression from stable lesions to advanced plaques that can spontaneously disrupt is promoted by numerous enzymes, including thrombin and extracellular matrix metalloproteases (MMPs). Assessing the activity of these enzymes in vivo by noninvasive MRI could be a valuable tool for the risk assessment of plaque disruption and a powerful method for monitoring the effectiveness of therapies. Novel thrombin- and MMP-targeted activatable cell-penetrating peptides (ACPPs) have a mechanism for activation and retention at sites of high enzymatic activity and show great promise for plaque detection due to incorporation of gadolinium (Gd), an MR active agent. In addition to the morphological information that is traditionally provided by MRI, both ACPPs also allow for a functional assessment of plaque. We hypothesized that these new targeted ACPPs would provide enhanced contrast at a smaller dosage and improve the sensitivity and specificity of plaque detection. The aorta of fat fed male ApoE knockout mice were imaged with a T1-weighted spin echo sequence before and after the administration of a substandard dose of non-targeted Gd-DTPA (2.5 μmol/kg, compared to the standard dose of 0.1-0.2 mol/kg). Administration of a comparable dose of thrombin- or MMP-targeted ACPP yielded a great increase in contrast around the aortic plaques (Figure 1). In conclusion, administration of the targeted ACPP achieved superior contrast and, therefore, demonstrates the potential to achieve high detection power of plaques with a much lower and safer dose of Gd.
Abstract 283: Role of AMP-Activated Protein Kinase α1 in Atherosclerosis
Authors: Zhang Miao , Zhu Huaiping , Ding Ye , Moriasi Cate , Liu Zhaoyu , Zou Ming-Hui
Published: March 2022

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy and has cardiovascular protective effects. The catalytic subunit of AMPK consists of α1 and α2 isoforms and AMPKα1 is highly expressed in vascular cells and macrophages. To investigate the potential role of AMPKα1 in atherosclerotic processes, we crossed AMPKα1 deficient (AMPKα1
-/-
) mice with atherogenic ApoE deficient (ApoE
-/-
) mice. ApoE
-/-
/AMPKα1
-/-
mice and control ApoE
-/-
mice were fed a Western diet for 10 weeks. Deficiency of AMPKα1caused a lessened lesion area and decreased monocyte/macrophage infiltration of the lesions without significant lipid profile changes. Further, hematopoietic deficiency of AMPKα1 by bone marrow transplantation resulted in less atherosclerotic lesions, suggesting that AMPKα1 in bone marrow-derived cells, possibly macrophages, plays an important role in atherosclerosis development. Indeed, AMPKα1 deletion dramatically increased monocyte/macrophage apoptosis and cell death. Monocyte to macrophage differentiation was also impaired in AMPKα1
-/-
mice. The impaired monocyte differentiation and increased macrophage death were associated with the inhibition of autophagy induced by AMPKα1 deficiency. Taken together, our results reveal a novel role of AMPKα1 in controlling differentiation and survival of monocytic cells, and progression of atherosclerosis.
Abstract 284: A Deficiency in the HDL Receptor SR-BI Attenuates Atherosclerotic Plaque Regression in ApoE-Hypomorphic Mice
Authors: Zhang Yi , Trigatti Bernardo
Published: March 2022

A deficiency in the HDL receptor, SR-BI, results in accelerated arterial plaque development in ApoE-R61 hypomorphic (“Hypo-E”) mice, however the effect of a lack of SR-BI on atherosclerotic plaque regression is unknown.To explore the role of SR-BI in the regression of arterial atherosclerosis in the Hypo-E mouse model, SR-BI-deficient/Hypo-E (SR-BI-deficient) or SR-BI-expressing/Hypo-E (control) mice were fed high-cholesterol, high-fat diets (HFD) for either 4 or 11weeks (w) to induce the formation of atherosclerotic plaques of similar size in the aortic sinus. The mice were then switched to a normal chow diet (NCD) for 11 w. As had been shown previously by others, atherosclerotic plaques in control mice regressed almost completely during the 11 w of NCD feeding. In contrast, atherosclerotic plaques in SR-BI-deficient mice did not undergo regression but rather continued to grow in size after the switch from HFD to NCD. Concomitantly, circulatory cytokines (IL-6 and TNF-alpha) and atherogenic lipoproteins were elevated by HFD feeding in both groups, and returned to basal levels in control mice, but remained elevated in SR-BI-deficient mice after the switch from HFD to NCD. Immunofluorescence analyses revealed that CD11c-positive dendritic cells were retained in the plaques of SR-BI-deficient Hypo-E mice, which exhibited apparently attenuated expression of CCR7. In a separate experiment SR-BI-deficient mice were fed HFD for 4 weeks and then switched to NCD without or with 0.5% probucol. In untreated group, atherogenic lipoproteins remained elevated and plaques continued to grow after the switch from HFD to NCD, tripling in size by 8 weeks. Probucol treatment resulted in reduced levels of VLDL and LDL cholesterol and halted further lesion growth but did not trigger plaque regression. Probucol treatment was, however, accompanied by a decrease in CD11c staining in atherosclerotic plaques when compared to mice fed HFD for 4 weeks alone or followed by a further 8 weeks of NCD. Probucol also decreased expression of VCAM-1 in coronary arteries. These findings suggest that atherosclerotic plaque regression is impaired in SR-BI deficient mice. Probucol was able to halt further plaque growth but did not trigger plaque regression in SR-BI deficient hypo-E mice.
Abstract 288: Circulating sRAGE Identifies Bicuspid Aortic Valve Patients and Associated Aortopathies in Patients Referred for Surgery
Authors: Branchetti Emanuela , Poggio Paolo , Grau Juan B , Shaw Richard E , Desai Nimesh D , Gorman Joseph H , Gorman Robert C , Bavaria Joseph E , Ferrari Giovanni
Published: March 2022

Background:
Thirty to 50% of Bicuspid Aortic Valve (BAV) patients require surgery for aortic valve replacement (AVR), ascending aortic replacement (AA), or both. To prevent adverse aortic events they are risk-stratified using imperfect criteria based on metric measurements of the ascending aorta. As a result, a significant number of aortic dissections occur outside of the parameters suggested by the guidelines. Advanced Glycation End products (AGE) are associated with valve and vascular remodeling and trigger the release of a soluble receptor (sRAGE). We hypothesize that circulating sRAGE may identify BAV patients and that its levels may be used as risk stratification tool for adverse aortic events.

Methods:
sRAGE was measured in 135 patients (BAV n=74; Tricuspid Aortic Valve (TAV) n=61) meeting inclusion criteria over 338 enrolled patients undergoing AVR and/or AA. Logistic regression and multivariate analysis were performed.

Results:
sRAGE identified BAV patients independently of age, gender, and common risk-factors for vascular disease (p<0.001). Within the BAV cohort, patients referred for AA and AVR had higher sRAGE values than patients undergoing AVR only (p=0.002). BAV patients younger than 60 years of age, presenting with both valve and aortic diseases (fast progressors), had higher sRAGE than older patients who only needed AVR (“slow progressors”). Histological analysis showed that sRAGE correlates with dysfunctional aortic microstructure and does not correlate with aortic diameter (R2=0.007, p=0.51) or diameter/BSA (R2=0.011, p=0.42).

Conclusions:
These results advocate for a blood test to diagnose BAV and to risk-stratify these patients for aortopathies independently of metric measurements.
Abstract 298: Effects of Long-Term Autophagy Inhibition With Chloroquine on the Development and Progression of Experimental Abdominal Aortic Aneurysms
Authors: Ramadan Azza , Wheatcroft Mark D , Quan Adrian , Singh Krishna K , Lovren Fina , Teoh Hwee , Al-Omran Mohammed , Leong-Poi Howard , Verma Subodh
Published: March 2022

Abdominal aortic aneurysms (AAA) are a significant cause of worldwide mortality and morbidity. Although several mechanisms have been put forth, the central determinants of progressive AAA development appear to involve heightened vascular smooth muscle cell (VSMC) apoptosis, with increased degradation of the extracellular matrix, in the setting of chronic inflammation. Recent evidence suggests that autophagy genes are deregulated in human AAA and that autophagy may modulate VSMC fate and plasticity.

We sought to determine if long-term autophagy inhibition with chloroquine attenuates AAA progression and mortality in an experimental model of AAA.

ApoE-/- mice were implanted with miniosmotic pumps containing either saline (n=15) or angiotensin II (Ang II; 1.44 mg/kg/day) (n=27). Mice from each group were subdivided and administered either the potent autophagy inhibitor chloroquine (50 mg/kg/day, i.p.) or saline for 8 weeks. Hemodynamics, abdominal aortic diameter (by ultrasound) and mortality were monitored. Necroscopy evaluations, histological and immunohistological staining for markers of autophagy, apoptosis and inflammation were conducted.

Kaplan-Meier estimates were compared between the four groups over the 8-week follow-up. Ang II infusion was associated with a cumulative mortality of 38%. Long-term chloroquine treatment did not alter Ang II-associated mortality (43%, p>0.05 vs. saline-treated group). When rupture-related AAA mortality was specifically evaluated, no difference between the Ang II groups was observed. Maximal luminal diameter of the abdominal aorta was similar between the Ang II-infused groups (Saline: 1.88±0.13 mm vs. Chloroquine: 1.97± 0.17 mm, p>0.05). Ang II infusion also significantly increased MAP, an effect that was unaffected by chloroquine treatment (Saline: 140±16 mmHg vs. Chloroquine: 135±11 mmHg, p>0.05).

Long term chloroquine administration does not alter the development and/or natural history of experimental AAA. These data suggest that a global and non-specific autophagy inhibition strategy is unlikely to represent a target for intervention for AAA. Whether cell- and/or gene-specific targeting of the autophagy machinery improves AAA outcomes remains to be determined.
Abstract 301: Cytochrome P450 1b1 is Essential for the Development of Aortic Aneurysm in ApoE Knockout Mice
Authors: Thirunavukkarasu Shyamala , Khan Nayaab S , Ghafoor Hafiz U , Jennings Brett L , Malik Kafait U
Published: March 2022

Previously we have shown that the development of hypertension and neointimal growth is dependent on cytochrome P450 (CYP)1B1 activity. This study was conducted to address the role of CYP1B1 in the development of angiotensin II (Ang II)-induced aortic aneurysms and associated pathogenesis. Sixteen week old male

ApoE
-/-
/Cyp1b1
+/+

and

ApoE
-/-
/Cyp1b1
-/-

mice were administered Ang II (750 ng/min/Kg) or its vehicle for one month using micro-osmotic pumps implanted subcutaneously. A separate group of

ApoE
-/-
/Cyp1b1
+/+

mice receiving Ang II were injected twice weekly with the selective inhibitor of CYP1B1 2,3',4,5'-tetramethoxystilbene (TMS) (300 μg/Kg) or its vehicle DMSO (i.p.). Ultrasound studies showed that Ang II infusion produced abdominal aortic aneurysms in the

ApoE
-/-
/Cyp1b1
+/+

mice that were prevented by simultaneous treatment of these mice with TMS, and in

ApoE
-/-
/Cyp1b1
-/-

mice. Histological and immunohistochemical analysis of aortic aneurysms revealed that Ang II increased degradation of collagen, elastin and actin, increased expression of matrix metalloproteinases MMP2 and MMP9 as well as markers of inflammation including macrophages and CD3
+
T cells and increased production of reactive oxygen species (ROS) in the

ApoE
-/-
/Cyp1b1
+/+

mice; these changes were minimized by treatment of these mice with TMS and in

ApoE
-/-
/Cyp1b1
-/-

mice. These data suggest that Ang II induced abdominal aortic aneurysms and associated pathophysiological changes in

ApoE
-/-

mice are mediated by CYP1B1 most likely via generation of ROS. Therefore, CYP1B1 could serve as a novel target for the development of agents to treat abdominal aortic aneurysms and associated pathogenesis.
Abstract 320: Apoptosis-Resistant CD34+ Cells Improve Collateral Circulation Post Femoral Artery Occlusion in Diabetic Mouse Model
Authors: Sen Sabyasachi , Jerry Joseph
Published: March 2022

Literature shows that EPCs contribute to increased collateral vessel formation following vaso-occlusion. However diabetes is associated with poor collateral vessel formation. We cultured human EPCs (defined as CD34+ cells) and exposed them to 5.5 mM (equivalent to 99 mg%, normal glucose, NG) and 20mM (equivalent to 360mg%, high glucose, HG) glucose, which resulted in significant EPC apoptosis within 48hrs. We noted HG exposure was associated with up-regulation of P53 and its downstream genes such as P21, PUMA and Caspase-3. We hypothesized that EPC apoptosis in HG is secondary to up-regulation of P53 and silencing or knocking out P53 may help neovascularization. We obtained mouse EPCs from P53 KO mice and observed that p53 null EPCs are more resistant to cell death in HG while retaining expression of essential endothelial genes. Next, we used Lenti and Adenovirus mediated siRNA methods to silence P53 in human EPCs (hEPCs) and P53 silenced hEPC showed better survival in HG. We developed unilateral femoral artery occlusion model to mimic peripheral vascular disease in streptozotocin induced type 1 diabetic or Leptin resistant type 2 diabetic mouse model. We delivered saline, P53 WT and P53 null mEPCs intra-muscularly around the femoral occlusion (n=10 in each group) and counted number of capillaries in a 20x microscopic field. We also quantified increased vessel formation by laser doppler assay of the ischemic and contralateral hind limbs upto 2 weeks, post EPC transplantation and conducted qRT-PCR and western blot of key endothelial genes such as eNOS, p-eNOS, VEGF-A, PECAM-1 and vWF from both quadriceps in all groups of mice.

Results:
The group that received P53null EPCs had the most favorable outcome. Preliminary results from transplantation of lenti p53 silenced human CD34+ cells also indicate increased vessel formation compared to non p53 silenced hCD34+ cell in NOD-SCID diabetic mice. Summary: Transplantation of P53 null EPCs in presence of hyperglycemia help improve collateral vessel formation post femoral artery occlusion compared to WT-EPC transplanted group.

Conclusion:
Our results indicate towards positive role of apoptosis resistant CD34+ cells in diabetic PVD by increasing collateral formation with significant clinical implications.
Abstract 333: Platelet and Endothelial Aggregation Receptor-1 Tempers Tubulogenesis via the Akt Pathway
Authors: Vandenbriele Christophe
Published: March 2022

Platelet endothelial aggregation receptor-1 (PEAR1) is a transmembrane receptor expressed on platelets and endothelial cells (ECs). We aim to unravel its function in ECs. qPCR analyses for PEAR1 in cultured human endothelial progenitors (blood outgrowth endothelial cells (BOECs)), in ECs from human umbilical cord (HUVECs and HUAECs) and in ECs freshly isolated from multiple vascular beds showed the lowest expression in BOECs and the highest in macro- and microvascular ECs from heart and liver. HUVECs and HUAECs showed intermediate PEAR1-expression. Staining of fast growing skin hemangiomas versus normal skin ECs revealed low expression of PEAR1 in rapidly proliferating tumor ECs.

Short hairpin based miRNA lentiviral PEAR1-knockdown (shPEAR1) doubled the hybrid EA.hy926 and HUVEC EC proliferation rate compared to control lentiviral-transduced ECs. This was associated with downregulation of HES1-expression (a downstream regulator of the NOTCH-pathway), with increased baseline Akt-P levels (PI3K-activity) in shPEAR1 cells and with decreased levels of the phosphatase PTEN (PI3K-antagonist). Expression of the nuclear proliferation suppressor p21CIP1 in shPEAR1 ECs was reduced by 60±2% (mean±SEM, qPCR and Western blot), whereas the band intensity for p34/CDC2 significantly rose on Western blots (increased mitosis). PEAR1-knockdown also enhanced EC migration via reduced expression of β1- and β3-integrins both in HUVECs and EAhy926 ECs. Both enhanced proliferation and migration contributed to increased tube formation in shPEAR1 HUAECs (in vitro matrigel tube formation assays). During HUAEC tube formation, PEAR1-expression rose 5-fold, associated with a 3-fold increase of PTEN. However, PTEN-levels dropped 1,5-fold during tube formation of shPEAR1 HUAECs, compatible with increased phosphorylation levels of Akt in shPEAR1 ECs.

Our study provides the first evidence that PEAR1-expression is variable in ECs from multiple vascular beds and that PEAR1 plays a suppressive role on proliferation, migration and tube formation of ECs through regulation of the PI3K/Akt/PTEN-pathway. Our data suggest a regulatory role for PEAR1 in the NOTCH-pathway via HES1. The upregulation of PEAR1 during EC maturation is compatible with this suppressive role.
Abstract 336: The Imbalanced Redox Homeostasis in Senescent Endothelial Cells is Due to Dysregulated Thioredoxin-1 and NADPH-Oxidase 4
Authors: Altschmied Joachim , Goy Christine , Jakob Sascha , Haendeler Judith
Published: March 2022

Senescent endothelial cells are not only present in aged vessels, but also in atherosclerotic lesions, where they contribute to endothelial dysfunction. One hallmark of cellular senescence is an imbalanced redox homeostasis. However, the underlying mechanisms how this imbalance occurs are not completely understood. Thioredoxin-1 (Trx-1) is one of the most important redox regulators in primary human endothelial cells. It has previously been shown that Trx-1, in addition to its oxidoreductase activity has anti-apoptotic properties. Cellular generators of reactive oxygen species (ROS) are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate whether and how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells, an ex vivo model mirroring the situation in aged or diseased vessels.

We treated primary human endothelial cells for two weeks with hydrogen peroxide to generate stress-induced premature senescence in these cells. Senescence induction was confirmed by an increase in senescence-associated beta-Galactosidase and nuclear p21, two established senescence markers. Moreover, cytosolic and mitochondrial reactive oxygen species formation were enhanced. Concomitantly, we observed decreased Trx-1 levels and elevated NOX4 levels, which could explain the imbalance in the cellular redox homeostasis. Moreover, the lysosomal protease Cathepsin D, known to degrade Trx-1 under short-term oxidative stress, was over-activated, providing a mechanistic basis for reduced Trx-1 protein levels. Inhibition of “over-active” Cathepsin D by the specific, cell-permeable inhibitor pepstatin A counteracted the increase in nuclear p21, ROS formation and degradation of Trx-1, thus leading to blockade of stress-induced premature senscence by stabilizing the cellular redox homeostasis. Moreover, aortic Trx-1 levels negatively correlated with NOX4 expression in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium and their wildtype littermates.

Our data show that loss of Trx-1 and upregulation of NOX4 may importantly contribute to the imbalanced redox homeostasis in senescent endothelial cells ex vivo and in vivo.
Abstract 354: Inducible Disruption of Endothelial Hyaluronan Synthase-2 Expression in Adult Mice Reveals a Direct Involvement of Glomerular Endothelial Surface Hyaluronan in Development of Albuminuria and Renal Injury
Authors: van den Berg Bernard , Boels Margien , Jansen Erik , Avramut Cristina , Meldner Sascha , van der Vlag Johan , Koster Bram , van Zonneveld Anton Jan , Grone Herman-Josef , Vink Hans , de Koning Eelco , Rabelink Ton
Published: March 2022

We demonstrated that infusion with hyaluronidase of C57Bl/6 mice for 4 wks to chronically reduce EC surface hyaluronan expression resulted in glomerular albumin passage and podocyte injury. However, with systemic hyaluronan degradation it precludes definitive conclusions on cellular-specific effects. Here we investigated the hypothesis that it is specifically the structure and integrity of the glomerular EC glycocalyx that determines whether or not albumin passage across the capillary wall occurs, and subsequently protects against development of kidney injury. To this end, we generated a conditional EC specific hyaluronan synthase2-knock out (HAS2–KO) mouse model. 8 Week old B6.VECad-creERT2.has2f/f.Rosa26-tdTomato were injected i.p. for 5 consecutive days with 2mg/0.2μL tamoxifen (control B6.VECad-creERT2.Rosa26-tdTomato) to induce an endothelial specific deletion of has2. Compared to ctrl, these mice show that 4 wks after induction, weight gain was significantly more increased (mean ± SEM; 2.9 ± 0.7 [n=8] vs. 5.2 ± 0.6g [n=11], p=0.02). With prominent endogenous albumin passage over the barrier, visualized as a DAB reaction product in electron microscopy after anti-albumin-HRP antibody incubation of 2%PFA fixated tissue slides, visible 2 wks after induction, a significant (p=0.036) increased ACR (36.0 ± 1.9 [n=8] vs. 57.7 ± 7.9 [n=11]) was found only at wk 4. During this period no significant change in BP (85 ± 2/114 ± 2 [n=8] vs. 90 ± 4/119 ± 4mmHg [n=10], diastolic/systolic) or in heart rate (712 ± 14 [n=8] vs. 747 ± 8beats/min [n=10]) was observed. Renal morphology revealed with comparable glomerular surface area (5342 ± 270 [n=3] vs. 5508 ± 250 μm2 [n=6]), significant increase of glomerular mesangial area (20.1 ± 0.9 [n=3]) vs. 38.4 ± 1.2% [n=6], p<0.01) and capillary surface area (16.6 ± 1.0 [n=3]) vs. 22.8 ± 0.7% [n=6], p=0.046). In conclusion, the data so far indicate that an intact glomerular EC-ESL is essential for glomerular integrity and prevention of albumin filtration is Key to preserving local quiescence of mesangial cells and podocytes. The conditional EC specific HAS2–KO mouse seems to be a valid model to assess the role of ESL in glomerular permeability barrier and vascular induced glomerular changes.
Abstract 357: The Role of X-Box Binding Protein 1 Splicing in Smooth Muscle Cell Proliferation and Neointima Formation
Authors: Zeng Lingfang , Li Yi , Yang Junyao , Hu Yanhua , Xu Qingbo
Published: March 2022

Rationale:
Smooth muscle cell (SMC) proliferation plays an important role in neointima formation in response to vascular injury. However, the underlying mechanism is still not well understood. As one of the key transducer in endoplasmic reticulum stress response, the X-box binding protein 1 (XBP1) has been demonstrated to contribute to endothelial cell proliferation, autophagic response and apoptosis, but less is known about its role in SMC growth.

Methods and Results:
In this study, we intend to study the role of XBP1 in SMC proliferation and its contribution to neointima formation in response to vascular injury. Platelet derived growth factor (PDGF) induced XBP1 splicing in SMCs via the interaction between PDGF receptor and inositol requiring enzyme 1 alpha (IRE1α). Transient over-expression of the spliced XBP1 (XBP1s) increased SMC proliferation, while knockdown of XBP1 or IRE1α abolished PDGF-induced SMC proliferation. XBP1s down-regulated calponin H1 gene expression at mRNA level through microRNAs targeting the 3’-terminal untranslated region. Reconstitution of calponin H1 via adenoviral transfer could ablate XBP1s-induced SMC proliferation. Proteomics analysis of secreted proteins revealed that over-expression of XBP1s in SMCs decreased the secretion of transforming growth factor β family. Addition of exogenous TGFβ3 abolished XBP1s-induced calponin H1 suppression and SMC proliferation. High levels of TGFβ3 and calponin H1 were detected in SMCs isolated from SM22-Cre/XBP1loxP mice. These SMCs showed retardation in proliferation. Importantly, in the femoral artery injury model, XBP1 deficiency in SMCs prevented the neointima formation.

Conclusion:
XBP1 splicing plays an important role in SMC proliferation via regulating calponin H1 expression. Targeting XBP1 splicing in SMC may provide a novel therapeutic strategy to intervene neointima formation.
Abstract 36: Uncovering Lipid-Independent Prothrombinase Function Using Human-Snake Chimeras of Blood Coagulation Factor V
Authors: Verhoef Daniel , Camire Rodney M , Reitsma Pieter H , Bos Mettine H
Published: March 2022

The prothrombinase complex, which converts prothrombin to the key regulatory enzyme thrombin, consists of the serine protease Xa (FXa) and cofactor Va (FVa; A1-A2-A3-C1-C2 domains). The latter dramatically enhances the rate of prothrombin catalysis and exclusively associates with FXa on negatively charged phospholipid surfaces through its lipid-binding C-domains. Interestingly, we have previously shown that the FVa and FXa homologues found in the venom of the Australian snake Pseudonaja textilis bypass the requirement for a membrane surface to achieve complexation. Here we investigated whether the C-domains of venom-derived P. textilis factor V (ptFV) drive its lipid-independent cofactor function and, if so, can sustain this unique property in the setting of human FV. Therefore, we swapped the C-domains of constitutively active B-domainless human FV (hFV) and ptFV and expressed and purified the chimeric variants hFV-ptC and ptFV-hC. Using a purified prothrombinase assay with the lipid-independent substrate prethrombin-1, FV cofactor activity was examined with the corresponding human or venom-derived P. textilis FXa species in the presence or absence of lipids. We hypothesized that the C-domains constrain ptFV in a conformation optimal for lipid-independent cofactor function. Surprisingly, ptFV-hC displayed full cofactor activity, irrespective of the availability of anionic membranes. Similarly, hFV-ptC functioned equivalent to hFV as no gain-of-function in the absence of lipids was observed. Subsequent lipid-titration experiments indicated that whereas both C-domain species promoted maximum rates of substrate conversion at identical phospholipid concentrations, the lipid-affinity of the ptFV C-domains was at least 10-fold reduced as compared to that of hFV. These results demonstrate that the P. textilis C-domains do not play a role in the unique capacity of ptFV to function in the absence of lipids, suggesting that they may be of little relevance to ptFV cofactor function. As such, our findings imply an alternative mode of macromolecular complex assembly for P. textilis venom FVa and FXa and their natural substrate prothrombin, thereby potentially challenging the current paradigm of human prothrombinase assembly and function.
Abstract 387: Ischemic Stroke Following Discontinuation of Antiplatelet Therapy in Essential Thrombocythemia: A Case Report
Authors: Chen Yuefeng , Cao Shanjin , Naik Geetha D , Kolanuvada Bangaruraju , Alaie Dariush , Petrillo Richard L
Published: March 2022

Introduction:
Essential thrombocythemia (ET) is a rare cause of ischemic stroke. Low-dose aspirin is currently recommended for the prevention of thrombotic events in ET. However, the benefit is controversial. Herein, we report a case of ischemic stroke induced by ET following discontinuation of antiplatelet therapy.

Case presentation: An 87- year-old African American male brought to ER with one episode of black stool (Guaiac positive). He had history of peripheral vascular disease, deep venous thrombosis, coronary artery disease, type 2 diabetes, and a stroke that happened 6 years ago. He had been on oral aspirin 81mg and clopidogrel 75mg daily which were discontinued after admission. Laboratory studies showed significantly elevated platelet count of 640 x 10^9/L on admission. Two days later, he developed one episode of transient slurred speech and right side body weakness. At that time, his platelet count was 736 x 10^9/L. Five days later he developed slurred speech, difficulty swallowing and right side hemiplegia. MRI brain demonstrated acute infarct in left basal ganglia and posterior limb of the left internal capsule. Platelet count was 859 x 10^9/L which continued to increase to over 1000 x 10^9/L after the stroke. Diagnosis of ET was confirmed with the detection of JAK2 V617F mutation in peripheral blood sample. Thrombolysis was not initiated due to new gastrointestinal bleeding although no source was identified with endoscopic study, but aspirin and clopidogrel were resumed, and hydroxyurea was added. Physical therapy was started.

Discussion:
Thrombotic complications are major causes of morbidity and mortality in ET likely due to persistently enhanced platelet activation. Low-dose aspirin is currently recommended in ET patients. However, no randomized trials have directly assessed the efficacy and safety of low-dose aspirin or other antiplatelet drugs for primary prophylaxis of thrombotic complications in ET, and only a non-significant risk reduction was reported for secondary prophylaxis of arterial thrombosis with antiplatelet drugs in ET. The case described here indicates that antiplatelet therapy is important in high risk ET patients, and caution should be exercised when discontinuing antiplatelet drugs in these patients.
Abstract 390: Macrophage p90RSK Activation Inhibits Efferocytosis via Phosphorylation of ERK5 S496 and Intensifies Atherosclerosis Plaque Formation
Authors: Heo Kyungsun , Akike Masashi , Taunton Jack , Fujiwara Keigi , Abe Jun-ichi
Published: March 2022

Backgrounds:
We previously reported that ERK5 activation promoted efferocytosis and M2 macrophage (MΦ) polarization in advanced plaque formation. Although we recently found the critical role of p90RSK activation and its inhibitory effect on the ERK5 transcriptional activity via S496 phosphorylation, it’s still unclear how MΦ p90RSK interplay with ERK5 in the atherosclerosis (AS) development.

Methods and Results:
We found that p90RSK activation was significantly increased in advanced atherosclerotic lesion. It has been reported that angiotensin II (AngII) type I receptor (AT1) of MΦ accelerates AS by impairing efferocytosis and we found 200 nM AngII activated p90RSK, but not ERK5. Interestingly, p90RSK inhibitor, FMK-MEA treatment significantly increased efferocytosis-related opsonins and M2-type genes mRNA levels, whereas AngII failed to inhibit these increases, suggesting a crucial role of p90RSK activation in regulating efferocytosis and inflammation. A significantly enhanced level of ERK5-S496 phosphorylation was observed in the peritoneal MΦ from macrophage-specific wild type-p90RSK transgenic (WT-p90RSK-MTg) mice. WT-p90RSK-MTg/LDLR-/- mice fed HCD accelerated AS and necrotic core formations compared with NLC/LDLR-/- mice, suggesting the pro-atherogenic effect of MΦ p90RSK. To determine the roles of p90RSK and ERK5 activation and its interplay in regulating MΦ proliferation, we studied effects of colony stimulating factor 1 (CSF-1), statins, and AngII on MΦ proliferation. First, we examined p90RSK activation and ERK5 transcriptional activity and found AngII and CSF-1, but not pitavastatin, activated p90RSK, while ERK5 transcriptional activity was increased by CSF-1 and pitavastatin, but not by AngII. However, only CSF-1, but not AngII and pitavastatin, promoted MΦ proliferation. Interestingly, CSF-1 lost its proliferative effect in MΦ isolated from ERK5-MKO or in those treated with FMK-MEA, suggesting that activation of both p90RSK and ERK5 are necessary, and only one of them is not sufficient, to promote MΦ proliferation.

Conclusion:
These data strongly support that the balance between p90RSK and ERK5 would be a key factor for the regulation of MΦ propagation and functions, and regulates AS formation.
Abstract 391: Device-Relevant Dynamic and Constant Shear Stresses Induce Prothrombotic Platelet- and Monocyte-Derived Microparticles
Authors: Meyer Andrew D , Kamucheka Robin M , Nair Prajeeda , Reddoch Kristin M , Montgomery Robbie K , Parida Bijaya K , Cap Andrew P , Mackman Nigel , Ramasubramanian Anand K
Published: March 2022

Cardiovascular blood-recirculating devices provide life saving cardiopulmonary support to critically ill populations. Unfortunately, common device-related thrombotic complications continue to result in significant mortality, neurological morbidity, and limb loss. Past studies have shown that increased wall shear stress from blood-recirculating devices activates circulating monocytes and platelets. Less well studied is the enhancement of thrombosis by platelet-derived (PMPs) and monocyte-derived microparticles (MoMPs). These are small (0.1-1 micron) cell-derived membrane vesicles released from activated cells. Studies using a biological stimulant demonstrate that MoMPs initiate clotting whereas PMPs enhance clotting. Few if any studies have defined the contributions of device-produced microparticles (MPs) to changes in thrombotic potential. Our hypothesis is the increased dynamic or constant shear stress generated by a blood-recirculating device promotes release of prothrombotic MPs. Using an Anton Paar MCR Rheometer we exposed whole human blood
in vitro
to both dynamic (70 dynes/cm
2
at 0.05hz) and constant (70 and 35 dynes/cm
2
) shear stress conditions for two minutes. After shear, MPs were collected with dual centrifugation and then quantified using a BD FACS Canto II flow cytometer with forward scatter photomultiplier tube (resolution=200nm). Then we pelleted and washed MPs to measure changes in thrombin generation with a Calibrated Automated Thrombogram (CAT). Dynamic shear stress generated 3841±1171 PMPs/μL and 3522±1090 MoMPs/μL, (MEAN±SE, n=6). Constant shear stress at 70 dynes/cm
2
and 35 dynes/cm
2
generated PMPs/μL of 4937±1655 and 1880±389 and MoMPs/μL of 6935±2029 (n=3) and 2262±1041 respectively (MEAN±SE, n=3 for each). All shear conditions generated an increase in MP concentration over static conditions of 644±120 PMPs/μL and 538 MoMPs/μL (p<0.01). Dynamic and constant sheared MPs increased the estimated thrombin potential and peak thrombin generation by at least 30% compared to static (p<0.05). This study is the first to demonstrate that device relevant constant and dynamic shear stresses can generate PMPs and MoMPs that promote thrombosis.
Abstract 399: Apolipoprotein AI Suppresses Diabetes-Associated Leukocytosis
Authors: Barrett Tessa J , Distel Emilie , Murphy Andrew J , Hazen Stanley L , Fisher Edward A
Published: March 2022

Objective:
Diabetes-associated hyperglycemia increases circulating neutrophil and monocyte counts in mice via the proliferation and expansion of bone marrow myeloid progenitors. Clinically, diabetic patients also have elevated leukocyte counts, with the differing subsets (inc. neutrophils and monocytes) phenotypically more inflammatory when compared to leukocytes from non-diabetics. An emerging concept is that there is a positive association of leukocyte counts with the increased risk of cardiovascular disease (CVD) in diabetics. Various reports have shown that apolipoprotein AI (apoA1) and high density lipoprotein (HDL) can suppress both myeloproliferation and reduce the inflammatory state of neutrophils and monocytes. Given that low apoA1/HDL is a typical feature of diabetic dyslipidemia this study aimed to establish whether raising apoA1/HDL levels could reduce hyperglycemia-driven leukocytosis in a model of diabetes.

Methods and Results:
C57Bl6 (WT) and hApoA1 (overexpressing) transgenic (TG) mice were given daily i.p. injections of STZ for 5 d to induce diabetes. The TG mice had circulating [hApoa1] of 400 mg/dL (vs. 120 mg/dL mApoA1 in WT) and [HDL-C] levels of 150mg/dL (vs 50 mg/dL in WT). Hyperglycemia induced leukocytosis in WT mice (total WBC 18.6 k/uL), however elevated hApoA1 attenuated this to 13.8 k/uL. This decrease included a significant reduction in the circulating neutrophil (3.5 vs 2.5 k/uL) and monocyte counts (1.3 vs 0.9 k/uL) in the diabetic hApoA1 TG mice. Attenuation of leukocytosis was explained by a reduction in the proliferation and expansion of the neutrophil and monocyte myeloid progenitors in the bone marrow of the diabetic hApoA1 TG mice. Furthermore, s.c. injections of hApoA1 into diabetic WT mice attenuated hyperglycemia-driven myeloid proliferation.

Conclusion:
This study provides the first evidence that apoA1, the major protein component of HDL, can potently suppress diabetes-associated leukocytosis by overcoming the myeloid proliferation observed in diabetes.
Abstract 402: Increased Levels of Extracellular DNA in Plasma Are Associated With Diabetic Microangiopathy
Authors: Le Jeune Sylvain , Charue Dominique , Baudry Camille , Bihan Hélène , Mourad Jean-Jacques , Tedgui Alain , Blanc-Brude Olivier
Published: March 2022

Introduction:
Diabetic hyperglycemia leads to severe glycation of blood and vascular components. Glycation end products are toxic and linked to chronic inflammation and vascular dysfunction, particularly in the microcirculation. Hyperglycemia mediates endothelial apoptosis and the formation of neutrophil extracellular traps (NETs), with genomic DNA released into the blood stream. Circulating DNA (cirDNA) levels may thus testify for vascular injury in type 2 diabetes.

Patients and methods:
Blood from type 2 diabetic patients and healthy volunteers was collected after signed and informed consent (collection Diabelyse DC-2011-1480). All volunteers were more than four months away from acute cardiovascular events or infection, free from neoplasia, coagulopathy or hemoglobinopathy, and had not smoked, consumed alcohol or coffee within 24 hours. Platelet-free plasma (PFP) was prepared within 4 hours, frozen and only thawed once. We quantified total extracellular DNA in plasma by fluorimetry after labeling with the intercalating probe Sytox Green™.

Results:
65 diabetic subjects were included (age= 62+/-14 yo; sex ratio= 0.86; BMI= 32.5+/-8.7; HbA1C levels= 8,4+/-1,9%). 58,1% of diabetic patients were treated with insulin. 28,6% patients presented macroangiopathy and 67,7% microangiopathy, including 57,6% with retinopathy (21,7% treated by laser) and 58,1% with nephropathy. 25,9% patients displayed microalbuminuria, 22,4% proteinuria and 35,4% renal insufficiency (creatinin clearance <60 ml/min/1,73m
2
). 52 matched non-diabetic volunteers were included.

Levels of cirDNA were significantly elevated in diabetic patients with microangiopathy (27,9+/-13,9 RFU) versus other patients (19,4+/-6,1 RFU; p=0,04), or healthy volunteers (15.6+/-5.2 ; p=3.10-7). We noted a 50% increase in cirDNA specific to retinopathy (p=0.026), and a trend associating elevated cirDNA with nephropathy (+40%, p=0.060), microalbuminuria or proteinuria (+45%, p=0.060). There was no change in cirDNA during renal insufficiency (p=0.63).

Conclusion:
CirDNA in plasma is particularly easy to quantify by simple and robust assays. Its measure could contribute to the early and specific diagnostic of microvascular injury in diabetic patients.
Abstract 405: Central IKKß Inhibition Prevents Air Pollution--Mediated Peripheral Inflammation and Exaggeration of Type 2 Diabetes
Authors: Liu Cuiqing , Sun Xia , Fonken Laura K , Xu Xiaohua , Bai Yuntao , Maurya Santosh , Morishita Masako , Harkema Jack , Ying Zhekang , Sun Qinghua , Nelson Randy , Rajagopalan Sanjay
Published: March 2022

Objective:
Prior experimental and epidemiologic data support a link between exposure to fine ambient particulate matter (<2.5 μm in aerodynamic diameter, PM
2.5
) and development of insulin resistance/Type II diabetes mellitus (Type II DM). We investigated the role of hypothalamic inflammation in PM
2.5
-mediated diabetes development and the underlying mechanism.

Approach and results:
KKay mice, which are susceptible to Type II DM, were assigned to either concentrated PM
2.5
or filtered air (FA) for 4-8 weeks via a versatile aerosol concentrator and exposure system (n=7-8 for each group). PM
2.5
exposure led to hyperglycemia and insulin resistance, which were accompanied by increased hypothalamic IL-6, TNFα, and IKKβ mRNA expression and increased microglial/astrocyte reactivity. Targeting the NFκB pathway with intra-cerebroventricular administration of an IKKβ inhibitor (IMD-0354, n=8 for each group), but not TNFα blockade with infliximab (n=6) for each group), improved glucose tolerance, insulin sensitivity, rectified energy homeostasis (O
2
consumption, CO
2
production, respiratory exchanging ratio and heat generation) and reduced peripheral inflammation in response to PM
2.5
.

Conclusions:
Central inhibition of IKKβ prevents PM
2.5
mediated peripheral inflammation and exaggeration of type II diabetes. These results provide novel insights into how air pollution may mediate susceptibility to insulin resistance and Type II DM.