bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumour: Fibroblast Interactions Promote Invadopodia-Mediated Migration and Invasion in Oral Squamous Cell Carcinoma
Authors: Kato Koroku , Miyazawa Hiroki , Kawashiri Shuichi , Lambert Daniel W.
Published: November 2022

Objectives. In the progression of cancer, interactions between cancer cells and cancer-associated fibroblasts (CAFs) play important roles. Cancer cell invasion is facilitated by filamentous actin (F-actin)-rich membrane protrusions called invadopodia, and the relationship between CAFs and invadopodia has been unclear. We used oral squamous cell carcinoma (OSCC) to investigate CAFs’ effects on the formation of invadopodia, and we assessed the expressions of invadopodia markers and CAF markers ex vivo and their relationship with clinical parameters and survival. Materials and Methods. We examined the effect of culture with normal oral fibroblast (NOF)-derived and CAF-derived conditioned medium on the migration and invasion of two OSCC-derived cell lines using Transwells in the absence/presence of Matrigel. Immunoblotting and immunocytochemistry were conducted to assess the expressions of the invadopodia markers tyrosine kinase substrate 5 (Tks5) and membrane type 1 matrix metalloproteinase (MT1-MMP). We also used immunohistochemistry to examine patients with OSCC for an evaluation of the relationship between the CAF marker alpha smooth muscle actin (αSMA) and the expression of Tks5. The patients’ survival was also assessed. Results. Compared to the use of culture medium alone, NOF-CM and CAF-CM both significantly increased the OSCC cells’ migration and invasion (

p
<
0.05

), and they significantly increased the expressions of both Tks5 and MT1-MMP. After the depletion of Tks5, the OSCC cells’ migration and invasion abilities decreased. The expression of Tks5 and that of αSMA were correlated with poor survival, and a high expression of both markers was associated with an especially poor prognosis. Conclusions. These results indicate that the formation of invadopodia is (i) important for OSCC cells’ migration and invasion and (ii) regulated by the interaction of OSCC cells and stromal fibroblasts.
In-Line and Off-Axis Hybrid Digital Holographic Imaging Method Based on Off-Axis Hologram Constraints
Authors: Wang Fengpeng , Fan Feifan , Yuan Deren , Wang Xinghua
Published: November 2022

We put forward a novel hybrid iterative algorithm to improve the imaging quality of digital holography. An off-axis hologram is added to the iteration process via interference and inverse interference process and becomes part of the constraints. A frequency domain filter varying with the number of iterations is used to improve the competitive advantage of low frequency information in the early iterations, while retaining the high frequency information. In practical applications, an additional iterative process is used after averaging filtering to suppress the influence of the imperfect consistency between the reconstructed reference wave and the actual reference wave. Numerical simulations and experiments show that image reconstruction may be significantly improved compared to the conventional method.
HMGA1 As a Potential Prognostic and Therapeutic Biomarker in Breast Cancer
Authors: Wang Tian , Zhou Ting , Fu Fangfang , Han Yingyan , Li Yan , Yuan Ming
Published: November 2022

Background. High-mobility group AT-hook1 (HMGA1) protein plays an important role in various diseases. However, the contribution of HMGA1 in breast cancer remains to be tapped. Methods. The expression of HMGA1 was analyzed in The Cancer Genome Atlas (TCGA) and TIMER database, and immunohistochemistry was performed in 39 breast cancer (BC) patients. The correlation between HMGA1 expression and prognosis was evaluated using Kaplan–Meier plotter (KM plotter) in patients with breast cancer. Then, cBioPortal and bc-GenExMiner were requisitioned to analyze the contribution of HMGA1 expression to clinical features. In order to reveal the function of HMGA1 in breast cancer cells, enrichment analysis was performed using the clusterProfiler R software package. Moreover, CCK8 assay, EdU assay, and Cell Cycle Assay were performed to assess the proliferation, and transwell assay was used to evaluate cell migration and invasion. Flow cytometry was used to explore the role of HMGA1 on cell apoptosis. After that, the effect of HMGA1 on signaling pathways in BC cells was detected by western blot. Results. HMGA1 was highly expressed in a variety of tumors tissues, including BC. High HMGA1 expression was correlated with poor prognosis in BC patients. Meanwhile, HMGA1 expression was increased in molecular phenotypes with poor prognosis (ER-, PR-, and HER2+) and associated with high-grade group, lymph node metastasis, and NPI (Nottingham Prognostic Index). Further, function analysis revealed HMGA1 was enriched in DNA replication and cell cycle pathways in breast cancer. Moreover, knockdown of HMGA1 caused apoptosis, inhibited proliferation, migration, and invasion of MCF-7 and MDA-MB-231 cells, in which the oncogenic signaling pathway of PI3K/AKT/MMP9 played a critical role. Conclusions. HMGA1 was important for breast cancer progression and was a critical prognostic indicator, prompting a potential therapeutic target of breast cancer.
Supersymmetric Dyons, Superstrings, and Rotating Wormholes
Authors: Olszewski E.
Published: November 2022

We construct supersymmetric dyon solutions based on the ‘t Hooft/Polyakov monopole. We show that these solutions satisfy

κ

-symmetry constraints and can therefore be generalized to supersymmetric solutions of type I

S
O

32

string theory. After applying a

T

-duality transformation to these solutions, we obtain two

D
3

-branes connected by a wormhole, embedded in an

M
5

-brane. We analyze the geometries of each

D
3

-brane for two cases: one corresponding to a dyon with vanishing spin and the other corresponding to a magnetic monopole with nonvanishing spin. In the case of the vanishing spin, the scalar curvature is finite everywhere. In the case of the nonvanishing spin, we find a frame dragging effect due to the spin. We also find that the scalar curvature diverges along the spin quantization axis as

1
/

ρ

2

,

ρ

being the cylindrical, radial coordinate defined with respect to the spin axis. These solutions demonstrate the subtle relationship between the Yang-Mills and gravitational interactions, i.e., gauge/gravity duality.
Early and Late Mortality Predictors in Patients with Acute Aortic Dissection Type B
Authors: Lasica Ratko M. , Perunicic Jovan P. , Popovic Dejana R. , Mrdovic Igor B. , Arena Ross A. , Radovanovic Nebojsa L. , Radosavljevic-Radovanovic Mina R. , Djukanovic Lazar D. , Asanin Milika R.
Published: November 2022

Background/Aim. Despite technological advances in diagnosis and treatment, in-hospital mortality with acute aortic dissection type B is still about 11%. The purpose of this study was to assess the risk factors for early and long-term adverse outcomes in patients with acute aortic dissection type B treated medically or with conventional open surgery. Methods. The present study included 104 consecutive patients with acute aortic dissection type B treated in our Center from January 1st, 1998 to January 1st, 2007. Patient demographic and clinical characteristics as well as in-hospital complications were reviewed. Univariate and multivariate testing was performed to identify the predictors of in-hospital (30-day) and late (within 9 years) mortality. Results. 92 (88.5%) patients were treated medically, while 12 (11.5%) patients with complicated acute aortic dissection type B were treated by open surgical repair. In-hospital complications occurred in 35.7% patients, the most often being acute renal failure (28%), hypotension/shock (24%), mesenteric ischemia (12%), and limb ischemia (8%). The in-hospital mortality rate was 15.7% and the 9-year mortality rate was 51.9%. Independent predictors of early mortality in patients with acute aortic dissection type B were uncontrolled hypertension (HR-20.69) and a dissecting aorta diameter >4.75 cm (HR-6.30). Independent predictors of late mortality were relapsing pain (HR-7.93), uncontrolled hypertension (HR-7.25), and a pathologic difference in arterial blood pressure (>20 mmHg) (HR-5.33). Conclusion. Knowledge of key risk factors may help with a better choice of treatment and mortality reduction in acute aortic dissection type B patients.
PADI1 and Its Co-Expressed Gene Signature Unveil Colorectal Cancer Prognosis and Immunotherapy Efficacy
Authors: Zhang Yi-ran , Zhang Lei , Li Feng , He Jia-shuai , Xuan Jin-feng , Chen Cong-cong , Gong Chao , Pan Yun-long
Published: November 2022

Peptidyl arginine deiminase 1 (PADI1) catalyzes protein citrullination and has a role in regulating immune responses. The tumor immune microenvironment has been reported to be important in colorectal cancer (CRC), which was correlated with the ability of CRC patients to benefit from immunotherapy. However, there is a lack of molecular markers for matching CRC immunotherapy. Previously, single-gene risk models have only considered the effect of individual genes on intrinsic tumor properties, ignoring the role of genes and their co-expressed genes as a whole. In this study, we analyzed the differential expression of PADI1 in colorectal cancer (CRC). We found that PADI1 was highly expressed in CRC. Subgroup survival analysis revealed a prognostic survival difference for PADI1 in CRC patients aged less than 65 years, male, T stage, N0, M0, and stage I-II (

p
<
0.05

). In addition, we analyzed the functions and signaling pathways associated with PADI1 in CRC and found that it was highly enriched in several immune-related functions and pathways. Then, a set of PADI1 co-expressed genes (PCGs) risk-prognosis scores was developed with PADI1 as the core, which could accurately predict the prognosis of CRC (

p
<
0.05

). PCGs risk score can be an independent prognostic factor for CRC. A new set of Norman plot models were developed for clinical characteristics with age, sex, and TNM stage, which can accurately predict CRC 1, 3, and 5 years survival, and calibration curves and decision curve analysis (DCA) validated the accuracy of the models. The risk score assessed the immune microenvironment of CRC and found that the immune score was higher in the low-risk group, and CD4+ T cells, helper T cells, and eosinophils were more infiltrated in the low-risk group (

p
<
0.05

). Immunotherapy efficacy was better in the low-risk group (

p
<
0.05

). The underlying mechanism may be that the high-risk group of PCGs was enriched in some pathways that promote immune escape and immune dysfunction. In conclusion, PCGs may better predict CRC prognosis and immunotherapeutic response.
Approach for Multiattribute Decision‐Making with an Interval Grey Number Based on Bonferroni Mean
Authors: Niu Qiuyun , Guo Jinhai
Published: November 2022

Aiming at the multiattribute decision‐making problem in the form of interval grey number, considering the interrelationship between the scheme attributes, this study defines the grey number weighted geometric Bonferroni mean (GNWGBM) operator for aggregating information. The feasibility of the proposed method is verified through a comparison of the example analysis and methods.
Crop raiding and livestock predation by wildlife in Khaptad National Park, Nepal
Authors: Ashish Bashyal  , Sharma Shyam , Koirala Narayan , Nischal Shrestha  , Nischit Aryal  , Bhupendra Prasad Yadav  , Shrestha Sandeep
Published: November 2022

Crop raiding and livestock predation are major conservation problems throughout most protected areas in Nepal, including the Khaptad National Park (KNP). However, no information exists on the extent of crop raiding, livestock predation, and animal attacks among villages surrounding KNP. We conducted a survey of 304 households in 30 villages in four districts (Bajhang, Bajura, Doti, and Achham) in the buffer zone of KNP between 24 May and 20 June 2019, using the snowball sampling technique. All households experienced numerous major incidents of crop raiding between April 2017 and May 2019. Major wildlife involved were Wild Boar Sus scrofa, Himalayan Black Bear Ursus thibetanus, Rhesus Macaque Macaca mulatta, Barking Deer Muntiacus vaginalis, Common Leopard Panthera pardus, Golden Jackal Canis aureus, and Porcupine Hystrix spp. Of the 304 households, all had their crops raided over the past two years, 55.5% (n = 169) faced livestock predation, and 2% (n = 6) attacks resulting in death or injury. Over 40% of households reported taking mitigation measures to minimize crop raiding. Common measures such as night guarding, noise making, use of scarecrows, watch dogs, and fencing were practiced. More than half of respondents had negative opinions towards wildlife but they still believed that wildlife should be conserved. There was no or negligible correlation between general opinion of respondents towards wildlife and wildlife conservation with their education, sex, or involvement in natural resources management group. We established baseline information on crop raiding and livestock predation in villages surrounding KNP. Gathered information will be transmitted to relevant authorities to design and implement measures to mitigate such conflicts.
Diversity patterns and seasonality of hawkmoths (Lepidoptera: Sphingidae) from northern Western Ghats of Maharashtra, India
Authors: Shere-Kharwar Aditi Sunil , Magdum Sujata M. , Khedkar G.D. , Supriya Singh Gupta
Published: November 2022

As most of the biodiversity studies report the abundance and enlist the species, there is severe data deficiency in understanding the diversity patterns. The present study was designed to carry out periodic diversity assessments to understand the trends in diversity patterns of hawk moths. The study was carried out in the northern Western Ghats in Nashik district.  Seven sampling stations were identified and periodic visits to these places were carried out over the span of five years (2011–2015). A total of 463 moths were recorded belonging to 18 species, represented by 10 genera. A new record from Western Ghat, Theretra sumatrensis (Joicey & Kaye 1917) is reported for the first time along with its DNA barcode. Six diversity indices (four alpha diversity indices and two beta diversity indices) were employed to understand the diversity dynamics. Whittaker’s plot was generated using the rank abundance suggesting high species evenness for all sampling stations. Maximum diversity was observed during Monsoon. Wani was the most diverse sampling station throughout the study period (Shannon’s Index = 2.7132±0.060; Simpson’s Index = 0.9273±0.006; Brillouin’s Index = 2.252±0.089; Fisher’s alpha = 10.9472±1.685). Beta diversity was assessed with the help of Dice’s coefficient and Jaccard’s similarity index. Hence, we recommend rigorous periodic diversity assessments to generate adequate information about diversity that expedites conservational strategies’ pace.
An annotated checklist of odonates of Amboli-Chaukul-Parpoli region showing new records for the Maharashtra State, India with updated state checklist
Authors: Dattaprasad Sawant  , Hemant Ogale  , Rakesh Mahadev Deulkar
Published: November 2022

Amboli region, consisting Amboli, Chaukul, Nene, and Parpoli villages is one of the biodiversity rich areas in northern Western Ghats. We opportunistically surveyed odonates from the region and prepared an annotated checklist of 93 species belonging to 12 families. We report 15 Western Ghats endemic species and six new records for the State of Maharashtra. We further present an updated checklist of Odonata of Maharashtra state with a total of 144 species.
The new addition of Blue Pimpernel of Primulaceae to the state flora of Assam, India
Authors: Kalita Sushmita , Das Barnali , Nath Namita
Published: November 2022

Lysimachia arvensis var. caerulea (L.) Turland & Bergmeier, a variety belonging to the family Primulaceae is reported as new to the state flora of Assam, northeastern India.  The authors encountered the specimen during field survey at Kamrup Rural District of Assam in 2020.  This manuscript describes taxonomy and morphology details along with pictorial illustration of the specimen.
Population trends of Mugger Crocodile and human-crocodile interactions along the Savitri River at Mahad, Maharashtra, India
Authors: Utkarsha Manish Chavan  , Borkar Manoj Ramakant
Published: November 2022

In this paper, we report monitoring of a resident population of Mugger Crocodile Crocodylus palustris (Lesson, 1831)  along a stretch of 3.5 km of the river Savitri on the outskirts of Mahad town of Raigad District in Maharashtra, on monthly basis from 2014 to 2021.  This river is increasingly becoming a sink of anthropogenic wastes emerging from adjacent settlements impacting its habitat value, and puts the reptile side by side with humans and human-wastes that could be a cause of rising incidents of crocodile mortality in the recent times here, as also reported from elsewhere.  Savitri River has been a fishing ground for local indigenous communities, who also use the river bank for washing clothes and utensils, and for swimming.  Such proximity between people and crocodiles creates a potential for negative interaction.  This long term study monitored the Mugger population trends for the last eight years at four transect stretches along the river.  Counts are suggestive of a healthy viable population of Mugger in this river currently, but a future conflict situation cannot be ruled out.  Being generalist feeders, Muggers can sustain themselves on fish, and scavenge on dumped carrion and other anthropogenic organic wastes.  With the exception of a few sporadic incidents of aggression by the Muggers at this location, no human casualties have been reported thus far, however, this does not rule out fatal reciprocal interactions in future and hence a few practical mitigation measures have been suggested.
Paresis as a limiting factor in the reproductive efficiency of a nesting colony of Lepidochelys olivacea (Eschscholtz, 1829) in La Escobilla beach, Oaxaca, Mexico
Authors: Alejandra Buenrostro-Silva  , Jesús García-Grajales  , Petra Sánchez-Nava  , María de Lourdes Ruíz-Gómez
Published: November 2022

Rear flippers are crucial in the nesting process of Olive Ridley Turtles Lepidochelys olivacea, so any impact on them could constitute a limiting factor in reproductive efficiency. Muscle weakness of the rear legs has been observed in some nesting females on La Escobilla beach in Oaxaca state, Mexico; however, this disorder has not been sufficiently researched. The aim of this study was to identify and describe this problem in a nesting colony of L. olivacea in La Escobilla. We obtained the biochemical profiles of eight females with clinical signs of muscle weakness of the rear legs, that could not build the incubation chamber for their nest. In order to compare their blood characteristics, we selected eight seemingly healthy turtles that successfully built their nests, laid eggs through oviposition and covered the nest. We found no significant differences in most of the blood parameters, except for Creatinine-Kinase (CK). Female turtles with muscle weakness presented significantly higher concentrations of CK (t = 2.1448, d.f. = 2, P <0.0001) when compared to the healthy turtles. CK is an appropriate enzyme for identifying the integrity of the muscle cell and is a muscle damage indicator. Our hypothesis is that the paresis observed in the rear legs of the female turtles in La Escobilla could be a chronic debilitation caused by a gradual exposure to biotoxins such as saxitoxins.
Foraging activity and breeding system of Avicennia officinalis L. (Avicenniaceae) in Kerala, India
Authors: K. Vinaya  , C.F. Binoy
Published: November 2022

Field studies were carried out to assess the foraging activity and the breeding system of Avicennia officinalis L. in Chettuwa, Thrissur, India during the two flowering seasons of 2018–2019.  A. officinalis, also known as the Indian mangrove is a common mangrove occupying the river banks of the Kerala coast of southern India.  The plant blooms massively during the onset of the first summer showers.  The flowering to fruiting period lasted from April to July. Bagging experiments revealed that A. officinalis preferred a mixed breeding system though they had a low fruit set recorded in self-pollination experiments.  A total of 15 species of foraging insects belonging to three orders, Hymenoptera, Diptera, and Lepidoptera, were observed.  The three most abundant insect foragers were Apis florea Fabr., Campsomeriella collaris Fabr., and Chrysomya megacephala Fabr.  Among these, A. florea showed a significantly high visitation rate followed by C. collaris and then C. megacephala.  C. collaris, however, had the highest and most significant handling time for Avicennia officinalis than A. florea.  The peak foraging activity was recorded from 1000h to 1100h and from 1500h to 1700h.  These findings emphasize the importance of insect flower visitors in the breeding of A. officinalis, highlighting the need to maintain the plant-pollinator relationships for the protection of mangrove ecosystems.
Abstract 105: Proprotein Convertase Subtilisin/kexin Type 6 (Pcsk6) Plays A Role In Angiogenesis Regulation
Authors: Chemaly Melody , Dong Xinran , Suur Bianca E , Jin Hong , Malarstig Anders , Matic Ljubica
Published: November 2022

Background:
PCSK6 cleaves and activates growth factors involved in cell differentiation and
Pcsk6-/-
mice exhibit 25% embryonic lethality, while surviving offspring have blindness and cyclopia. We have shown that PCSK6 localizes to vascular smooth muscle cells of intra-plaque neovessels. We therefore hypothesize that the processes of angiogenesis, atherosclerosis and plaque neovascularisation share common molecular regulators and that PCSK6 plays a role in these processes.

Methods:
Association between SNPs near the
PCSK6
gene and levels of 90 plasma proteins was tested using data from 30,931 individuals in the SCALLOP consortium. Associations of angiogenic factors with PCSK6 (transcriptomic and proteomic) and patient symptomatology were explored in a biobank of plaque endarterectomies (BiKE). Morpholino technology was used for
Pcsk6
knockdown in zebrafish, coupled with vascular phenotype studies.
Pcsk6-/-
mice were used to study the expression of vasculogenesis related genes and vascular morphology at baseline (12 weeks).

Results:
Rs7178801 and rs11639051 in the
PCSK6
gene were found to relate to plasma PDGFB and VEGFD levels. TGFB2, VEGFA, VEGFC, PDGFA and PLAUR associated with patients symptomatology in BiKE and correlated with PCSK6 levels. Gross phenotypic and histological examination of zebrafish embryos with ablated PCSK6 showed improper peripheral vascular patterning of intersegmental vessels along with cerebral and myocardial haemorrhage hemorrhage. Expression of angiogenic factors;
Vegfa, Vegfb, Angpt1
and
Tgfb2
were altered in the heart, liver and adipose tissue in
Pcsk6-/-
mice. Vessel density, length and branch numbers were reduced in the
Pcsk6
-/- compared to the controls in the superficial but more significantly in the deep peripheral vessels. The smooth muscle cell coverage of the retinal vasculature was also reduced in the
Pcsk6 -/-
mice vs controls and
Pdgfa
was significantly downregulated compared to controls in mouse retinas.

Conclusions:
PCSK6 associates with levels of angiogenic factors on the genetic, transcriptomic and proteomic level. Lack of
Pcsk6
in mice and zebrafish led to vascular patterning defects. Further planned studies will examine PCSK6 in pathological neovascularization.
Abstract 107: A Human Arterial Cell Atlas
Authors: Cheng Paul , Pedroza Albert J , Sharma Disha , Weldy Chad , Ryan Yana , Nguyen Trieu , sundaram laksshman , Dalal Alex , Shad Rohan , Kim Hyun-Jung , Shi Huitong , Kundu Ramendra K , Kundaje Anshul , Kim Juyong B , Fischbein Michael P , Wirka Robert , Quertermous Thomas
Published: November 2022

Background:
Human vascular diseases are the worldwide leading causes of morbidity and mortality. Nearly all human vascular diseases have arterial segment-specific tropisms despite identical exposures to genetic and environmental risk factors. Understanding the cellular and transcriptomic determinants of arterial identities may hold the key to identifying novel pathophysiology and potential therapies.

Methods:
To specifically determine arterial site-specific differences independent of inter-individual variation, we have generated a human arterial cellular atlas by simultaneously collecting and analyzing up to 8 arterial sites from multiple healthy transplant donors. We performed single cell transcriptomic analysis on arterial segments to determine the differences in cellular composition and transcriptomic programs. We subsequently integrated human genetic data with cell-type specific transcriptomic differences across vascular beds to identify probable causal cells and causal genes associated with human vascular phenotypes.

Results/Conclusions:
Single cell transcriptomic analysis of > 150,000 cells sequenced at > 50,000 reads per cell revealed that the dominant cellular drivers of transcriptomic differences between distinct arterial segments, i.e. determinants of arterial identity, are fibroblasts and smooth muscle cells, not endothelial cells or macrophages. Adult vascular cells from different segments clustered not by anatomical proximity but by embryonic origin. Differentially regulated genes in fibroblast across different vascular beds were particularly enriched for vascular disease associated genetic signals, suggesting a prominent role for these cells in human disease. While the majority of endothelial cells were transcriptionally similar across vascular beds, a rare, previously undescribed, cluster of endothelial cells were identified who expressed segment-specific transcriptomic signatures. Differentially expressed genes in these cells were enriched for vascular disease signals, suggesting a possible role of these rare cells in human disease.
Abstract 113: Novel Tissue Factor Inhibition For Thromboprophylaxis In COVID-19: Primary Results Of The ASPEN-COVID-19 Phase 2b Randomized Trial
Authors: Hess Connie N , Nehler Mark R , Hsia Judith A , Ruf Wolfram , Morrow David A , Nicolau Jose , Berwanger Otavio , Szarek Michael , Capell Warren , Johri Shilpa , Pursley Michael , Gupta Ryan , Marshall Debra , Graybill Christopher , Carroll Ian , Keuer Thomas , Bristow Michael , Bonaca Marc P
Published: November 2022

Background:
Tissue factor contributes to thrombo-inflammation in viral infection and may be a target in COVID-19. Recombinant nematode anticoagulant protein c2 (rNAPc2), a tissue factor inhibitor with anticoagulant, anti-inflammatory, and antiviral properties, has not been studied in COVID-19.

Methods:
In Phase 2b of ASPEN-COVID-19 (NCT04655586), an open-label blinded endpoint study, COVID-19 inpatients with elevated D-dimer are randomized 1:1:2 to higher or lower dose rNAPc2 given subcutaneously up to 5 days or local standard of care heparin. Primary efficacy and safety endpoints are D-dimer change (to Day 8 or discharge if earlier) and ISTH major bleeding. Recovery time is an exploratory outcome.

Results:
160 patients were randomized at 24 sites in 3 countries. The average age was 56 years, 43% were female, 19% were Hispanic, and 21% were black. Baseline comorbidities included hypertension (51%), diabetes (35%), and smoking (35%); 11% were critically ill. On average, randomization occurred 10 days after COVID-19 symptom onset, and qualifying D-dimers were elevated 3.7-fold above the upper limit of normal. Median recovery time was 6 days (aggregate distribution shown in Figure). Enrollment has completed, database lock is anticipated Q1 2022, and final results will be ready for presentation at Vascular Discovery.

Conclusion:
ASPEN tests the hypothesis that compared to heparin, rNAPc2 reduces D-dimer in COVID-19. Results may help to provide a novel therapy for thromboprophylaxis in virus-related coagulopathies.
Abstract 115: N-terminus Of ApoB Mediates Endothelial Cell Chylomicron Uptake
Authors: Cabodevilla Ainara G , Ananganti Narasimha , Abumrad Nada , Aleman Jose O , Hussain Mahmood M , Goldberg Ira J
Published: November 2022

The development of atherosclerosis is triggered by the endothelial crossing and subendothelial retention of apolipoprotein B (ApoB)-containing LDL and chylomicron remnants. LDL has been reported to cross the endothelial cell (EC) barrier via two receptors: scavenger receptor-B1 (SR-B1) and activin receptor-like kinase 1 (ALK1). We have shown that SR-B1 also mediates EC uptake of nascent chylomicrons, leading to the accumulation of lipid droplets (LD) within the aortic endothelium and skin macrophages of hyperchylomicronemic LpL-deficient mice. In vitro, chylomicron-exposed ECs induced LD accumulation in co-cultured macrophages in the absence of any other lipid source. Surprisingly, conditioned media from chylomicron-exposed ECs primarily contained phospholipids and diglycerides, rather than triglycerides and fatty acids. Electron microscopy analysis revealed the presence of small extracellular vesicles (sEVs), and filtration of the media to remove sEVs prevented LD accumulation in macrophages. To determine the molecular interaction of chylomicrons and SR-B1 we performed a series of competition experiments. At physiologic concentrations found in postprandial blood, chylomicrons blocked LDL uptake into cultured ECs. Moreover, chylomicron uptake was blocked by co-incubation with ApoB18, a lipid-free peptide comprising the 18% N-terminus of ApoB. To assess this in vivo, we used an adeno-associated virus to overexpress ApoB18 in LpL-deficient mice. ApoB18-expressing mice exhibited a ≈ 50% reduction in aortic EC LD content, suggesting that the peptide effectively inhibits chylomicron uptake in vivo. Further, ApoB18 blocked LDL uptake by ECs in culture. These results suggest that a) the N-terminus of ApoB harbors a binding sequence for SR-B1 and b) endothelial uptake of both chylomicrons and LDL can be blocked by competition with non-lipoprotein ApoB peptides.
Abstract 120: Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation By Preserving Cardiac Nanostructure
Authors: Mezache Louisa , Soltisz Andrew , Johnstone Scott R , Isakson Brant , Veeraraghavan Rengasayee
Published: November 2022

Atrial fibrillation (AF) patients experience inflammation and vascular dysfunction and have elevated levels of cytokines that promote vascular leak and edema, such as vascular endothelial growth factor (VEGF). We previously identified edema-induced disruption of sodium channel (Na
V
1.5) -rich intercalated disk (ID) nanodomains as a novel arrhythmia mechanism. Therefore, we hypothesized that: (i) VEGF-induced vascular leak acutely slows action potential propagation in the atria and increases arrhythmia risk by disrupting ID nanodomains, and (ii) protection of the vascular barrier can prevent vascular leak-induced atrial arrhythmias. Electron microscopy revealed ID nanodomain swelling, near both gap junctions (GJ) and mechanical junctions (MJ) following VEGF treatment (60 minutes) in mouse hearts. Super-resolution STORM and STED microscopy both revealed Na
V
1.5 enrichment at GJ and MJ relative to other ID sites in control hearts. VEGF reduced Na
V
1.5 enrichment at both sites, consistent with Na
V
1.5 translocation from ID nanodomains. VEGF increased distance from GJs to 90% of Na
V
1.5 signal (3.17μm vs. 0.47 μm in vehicle controls), measured by a distance transformation-based analysis of 3D confocal images of IDs. VEGF slowed atrial conduction (optical mapping) and increased atrial arrhythmia incidence (ECG) relative to vehicle controls in both
ex vivo
(80 vs 0%) and
in vivo
(70 vs 20%) studies. Overall,
in vivo
arrhythmia burden was higher in VEGF-treated mice (7.5±11 vs. 0±6s/hr in vehicle controls). Preserving the vascular barrier by blocking endothelial Panx1 channels (PxIL2P peptide) decreased VEGF-induced
in vivo
arrhythmia burden (0 ± 6.09 s/hr with 1.6 μM PxIL2P). Concurrently, distance from GJs to 90% of Na
V
1.5 was restored to control levels (0.57μm) in these hearts. In summary, these results highlight inflammation-induced vascular leak as a novel AF mechanism, and suggest vascular barrier protection as an anti-arrhythmic strategy.
Abstract 122: Rabeprazole Enhances Vascular Repair And Resolution Of Sepsis-induced Lung Injury Through HIF1/FoxM1 Signaling
Authors: Evans Colin E , Peng Yi , Dai Zhiyu , Zhang Xianming , Zhao Youyang
Published: November 2022

Objective:
There are currently no effective treatments for sepsis-induced inflammatory lung injury, including acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The repositioning of existing drugs is one possible strategy for the treatment of ALI/ARDS. We previously showed that vascular repair and resolution of ALI is dependent upon hypoxia-inducible factor 1 alpha (HIF1a) and forkhead box M1 (FoxM1) in endothelial cells. The aim of this study was to identify a candidate agonist of HIF1a/FoxM1 signaling to treat ALI/ARDS.

Approach and Results:
First, we adopted a high throughput screening approach of a library of 1200 FDA-approved drugs by using hypoxia response element (HRE)-driven luciferase reporter assays, cell toxicity assays, and molecular analyses of human lung endothelial cells, to identify candidate HIF1 agonists
in vitro
. One of the top candidate drugs, Rabeprazole (also known as Aciphex), gave rise to dose-dependent increases in HRE luciferase activity without concurrent increases in cell death. Next, we performed dose-response studies of Rabeprazole in a mouse model of sepsis-induced ALI. By treating wild type mice orally with Rabeprazole on 2 consecutive days after endotoxic sepsis challenge, we were able to identify a dose of Rabeprazole that is well tolerated and enhanced vascular repair and resolution of ALI. In timeline studies of murine inflammatory lung injury, we found that Rabeprazole treatment resulted in reductions in lung vascular leakage, edema, and inflammatory cytokine expression during the repair phrase following sepsis-induced ALI. Finally, we used conditional knockout mice to show that Rabeprazole increases vascular repair and resolution of ALI via HIF1a/FoxM1 signaling.

Conclusions:
Rabeprazole is a HIF1a agonist that improves vascular repair and resolution of sepsis-induced inflammatory lung injury. Rabeprazole represents a promising candidate for repurposing to treat ALI/ARDS.
Abstract 123: Genetic Deficiency Of The Ectoenzyme CD73 Increases Neutrophil Extracellular Trap Formation In Patients With ACDC
Authors: Becicka Wyatt , Garrad Evan , Berreby Gil , Wang Jintao , Nghiem Khanh , Ramos-Benitez Marcos , Cowling Bretagne , Moitra Jaideep , Huffstutler Rebecca , Carney Katherine , Ferrante Elisa , Cudrici Cornelia , Brofferio Alessandra , Tourdot Benjamin , Jacobson Kenneth , Knight Jason , Kanthi Yogendra , Boehm Manfred
Published: November 2022

Background/Purpose:
CD73 is a ecto-5′-nucleotidase located on the plasma membrane that generates adenosine from AMP in the extracellular space, and functions as a checkpoint against immune and vascular activation. CD73 inhibitors that eliminate this immune checkpoint blockade are being studied in phase 2/3 cancer clinical trials. We recently identified that lack of CD73 promotes vascular thromboinflammation and neutrophil extracellular trap (NET) formation in mice. The role of CD73 in human NETosis, however, remains to be studied. Arterial calcification due to deficiency of CD73 (ACDC) is a rare, autosomal recessive condition (~20 patients globally) that results in occlusive peripheral vascular disease. We hypothesized that the congenital absence of CD73 and its function as an immune checkpoint would heighten innate immune activation in patients with ACDC.

Methods:
To test this hypothesis, six patients with molecularly confirmed ACDC were recruited to the NIH for mechanistic studies. Using purified neutrophils from ACDC patients and healthy controls, spontaneous and LPS-induced NET formation was quantified by both Sytox extracellular DNA (eDNA) labeling and NET-associated MPO activity.

Results:
Absence of surface CD73 was confirmed by flow cytometry. Relative to healthy controls, four of six (4/6) patients with ACDC demonstrated increased ex vivo spontaneous NET formation, but not LPS-triggered NETosis. The four ACDC patients with increased spontaneous NETosis had a mean 1.7-fold increase in Sytox-stained eDNA and 2-fold increase in MPO activity compared with contemporary healthy controls.

Conclusion:
These data reveal marked, spontaneous NETosis in patients deficient in the ectoenzyme CD73. Comparison with LPS-stimulated neutrophils demonstrates near maximal NETosis at baseline in patients with ACDC, consistent with a model where CD73 tonically suppresses neutrophil activation to maintain vascular and immune homeostasis. Ongoing studies are exploring the mechanism of spontaneous NETosis, thrombin formation, and fibrinolysis in patients with ACDC. Taken together, these data will have relevance to patients with ACDC, and inform the thromboinflammatory risk of cancer patients receiving CD73 inhibitors.
Abstract 125: Reversible Epigenome Editing Of PCSK9 As A Therapeutic Strategy
Authors: Wang Xiao , Whittaker Madelynn N , Jindal Ishaan , Cortez-Alvarado Saúl V , Qu Ping , Musunuru Kiran
Published: November 2022

Among the best-established causal risk factors for cardiovascular disease is the blood concentration of low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 (
PCSK9
), an antagonist to the LDL receptor, has emerged as a promising therapeutic target for the prevention of coronary heart disease. Current PCSK9 inhibitors are administered via subcutaneous injection, and their effects are short-lived. An alternative “one-and-done” strategy using genome editing to disrupt
PCSK9
at the DNA level has been demonstrated in preclinical animal models, including non-human primates. However, concerns about the permanence and irreversibility of the genomic changes have been raised and might limit the acceptance of the therapies. Recently, a set of CRISPR-based epigenome editing tools, CRISPRoff and CRISPRon, were reported to regulate gene expression via site-directed methylation and demethylation of gene promoters, respectively. We hypothesized that these epigenome editing tools could durably and reversibly induce methylation changes in the
PCSK9
promoter and thereby modulate its expression. We first screened CRISPRoff guide RNAs (gRNAs) targeting the
PCSK9
promoter, individually and in dual combinations, for their ability to reduce
PCSK9
expression in the human HuH-7 hepatoma cell line. We then performed long-term experiments with the lead candidate gRNAs. We found that these CRISPRoff gRNAs induced profound increases in methylation at CpG dinucleotides in the
PCSK9
promoter, with up to 80% decreases in
PCSK9
expression. These methylation increases and gene expression decreases have endured through >56 cell divisions so far, with only mild attenuation over time. Using the same gRNAs with CRISPRon in cells previously treated with CRISPRoff, we observed moderate decreases in methylation and increases in
PCSK9
expression. Having established these effects
in vitro
, we are similarly assessing the durability and reversibility of epigenome editing using the lead gRNAs in a
PCSK9
-humanized mouse model. Overall, this work provides a proof of concept of precise gene regulation via methylation and demethylation and suggests a potential new therapeutic approach for protection against coronary heart disease.
Abstract 144: Nuclear Smooth Muscle α-actin Is Critical For Smooth Muscle Cell Differentiation And To Prevent Cerebrovascular Disease
Authors: Kwartler Callie , Pedroza Albert J , Kaw Anita , Ma Shuangtao , Duan Xue-Yan , Kernell Caroline , Waters Mikayla , Chen Jiyuan , Fischbein Michael P , Milewicz Dianna M
Published: November 2022

Missense pathogenic variants in
ACTA2,
encoding α-smooth muscle actin (SMA), predispose to thoracic aortic aneurysms. A subset of these
ACTA2
pathogenic variants also predispose to childhood onset cerebrovascular disease characterized by occlusion of the distal internal carotid arteries and small vessel disease. In our studies to identify how specific
ACTA2
pathogenic variants predispose to cerebrovascular disease, we confirmed that SMA is localized to the nucleus in wildtype (WT) smooth muscle cells (SMCs), is enriched in the nucleus over β-actin with differentiation of SMCs, associates with the INO80 chromatin remodeling complex, and selectively binds to the promoters of SMC contractile genes. Pluripotent stem cell-derived SMCs from patients with
ACTA2
-associated cerebrovascular disease (
ACTA2
R179C) as well as SMCs explanted from a knockin mouse model (
Acta2
SMC-R179C/+
) confirm that R179 variants disrupt SMA nuclear localization. Both mouse and human cells harboring the R179C pathogenic variant proliferate and migrate more than WT SMCs, are less differentiated than WT SMCs, and have increased expression of pluripotency-associated genes. Although the variant is heterozygous, these cells show a dominant negative impact on nuclear SMA function through dramatically reduced levels of SMA in the nucleus, in the INO80 chromatin remodeling complex, and on the promoters of SMC-specific genes. Finally, single cell RNA sequencing analysis of aortic tissue from an
ACTA2
R179H patient confirms dedifferentiation of SMCs as a key phenotype associated with this mutation. Taken together, we have identified a novel role for α-SMA in driving differentiation of SMCs, and our data supports that defects in this nuclear role drive cellular phenotypes consistent with the cerebrovascular disease seen in patients with
ACTA2
R179 pathogenic variants.
Abstract 151: Macrophage Specific Ablation Of
Bmal1
Regulate Cholesterol Metabolism And Atherosclerosis
Authors: Pan Xiaoyue , Mota Samantha , O’Hare John , Murphy Jolee , Mowdawalla Cyrus , Hussain M. Mahmood
Published: November 2022

Macrophages play a critical role in atherosclerosis. We have previously shown that Clock mutant and global or liver-specific Bmal1 deficient mice show enhanced atherosclerosis. Yet, the role of macrophage Bmal1 in cholesterol transport and atherosclerosis remains unknown. Here, we used global and macrophage-specific ablation of Bmal1 to clarify the role of Bmal1 in macrophage function in atherosclerosis. We found that macrophage-specific Bmal1 deficient mice show increased atherosclerosis. Biochemical studies showed that Bmal1 deficient macrophages exhibit increased expression of Lox-1 and Cd36 and take up more oxLDL. Furthermore, they show decreased expression of Abca1 and Abcg1 and reduced cholesterol efflux as well as
in vivo
reverse cholesterol transport. Thus, Bmal1 regulates macrophage cholesterol metabolism by regulating uptake of oxidized lipoproteins and cholesterol efflux pathways. Molecular studies showed that Bmal1 indirectly regulates Cd36 and Abca1/Abcg1 by modulating Nr1d1 and Znf202, respectively. We provide evidence that Bmal1 collaborates with Clock and interacts with Nr1d1 promoter. Furthermore, our studies show that Bmal1 interacts with both Clock and Npas2 to regulate Znf202. Thus, Bmal1 regulates two different transcription factors to regulate uptake of oxidized lipoproteins and cholesterol efflux. In summary, our data show that Bmal1 is a key regulator of cholesterol metabolism in macrophages and atherosclerosis. It regulates macrophage function by modulating the expression of different transcription factors. It is likely that augmentation of Bmal1 may prevent atherosclerosis.
Abstract 154: Endothelial Dysfunction In Experimental Celiac Disease Is Mediated By Gut-Derived Vascular Inflammation And Oxidative Stress
Authors: Keppeler Karin , Helmstädter Johanna , Kuester Leonie , Strohm Lea , Ubbens Henning , Bayo Jimenez Maria Teresa , Kuntic Marin , Djordjevic Ivana , Finger Stefanie , Oelze Matthias , Schuppan Detlef , Wenzel Philip , Wild Philipp , Muenzel Thomas , Daiber Andreas , Steven Sebastian
Published: November 2022

Objective:
Celiac disease (CeD) is an autoimmune enteropathy triggered by dietary gluten, with a prevalence around 1%. Since many patients show heterogeneous symptoms, it is vastly underdiagnosed and yet without any treatment option besides lifelong gluten restriction. Vascular function in CeD patients compared to healthy controls has been assessed and hypothesized CeD as a cardiovascular risk factor. Herewith we aimed to explore the cardiovascular effects of CeD in a murine model, with special emphasis on endothelial dysfunction, vascular inflammation, and oxidative stress, known triggers for cardiovascular disease.

Methods:
NOD.DQ8 mice were raised on a zein-based, gluten-free diet. During the experiment, mice received gavage with peptic-tryptic digest of gliadin and were switched to a gluten-containing diet (Gliadin group). A control group (Zein group) was continued on gluten-free diet and received accordingly digested zein.

Results:
Blood pressure measurements revealed arterial hypertension in the Gliadin group. Endothelium-dependent relaxation was impaired in the Gliadin group compared to Zein, whereas no change in endothelium-independent was observed, tested by isometric tension recordings. Pro-inflammatory genes, like
Tnfa
and
Nox2
were upregulated in the Gliadin group measured by quantitative rtPCR in aortic and intestinal tissue. Infiltration by intraepithelial T-lymphocytes into the epithelium of the small intestine was shown by IHC. In cardiac tissue, gliadin-treatment increased the oxidative stress parameters 3-nitrotyrosine and 4-hydroxy-2-nonenal shown in dot blots. To investigate the inflammatory link between gut and the cardiovascular system plasma proteomics were performed and revealed elevated levels of IL-17A.

Conclusion:
CeD is a cardiovascular risk factor. However, the mechanism remains unresolved. In a mouse model, we demonstrate that gluten treatment elevates blood pressure and impairs vascular function. Intestinal inflammation conveys to the cardiovascular system, leads to oxidative stress, and thereby impairs endothelial function. A potential mediator might be IL-17A, whose role in other autoimmunity-based cardiovascular risk factors (e.g., psoriasis) has already been demonstrated.
Abstract 195: Transcriptional Regulation Of Transcriptional Regulation Of Triggering Receptor Expressed On Myeloid Cells-1 By Low Shear Stress In A Three-dimensional Co-culture Of Coronary Artery Smooth Muscle Cells, Endothelial Cells, And Monocytes
Authors: Liu Martin , Samant Saurabhi , Panagopoulos Anastasios , Khan Behram , Elsayed Lina , Agrawal Devendra K , Chatzizisis Yiannis S
Published: November 2022

Objectives:
To identify transcriptional factors involved in the expression of Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) in response to low shear stress in an
in-vitro
model of coronary artery atherosclerosis.

Methods:
Human coronary artery smooth muscle cells were mixed with neutralized type I collagen solution and plated on glass coverslips. The human coronary artery endothelial cells (HCAECs) were then plated on top of the collagen gels followed by plating monocytes (THP-1 cells) on top of the HCAECs. After 48 h, the co-culture was treated with or without low shear stress (LSS, 5±3 dyne/cm
2
) using the Flexcell Streamer for 30 min. Protein and mRNA of TREM-1 expression was assessed by immunofluorescence staining and real time RT-PCR. The expression of transcriptional factors involved in the expression of TREM-1 was assessed by ChIP assay.

Results:
LSS significantly stimulated TREM-1 expression as evidenced by IF staining and mRNA expression (fold increase vs control: 43.2 ± 9.7,
Fig 1A
and
1B
). ChIP assay demonstrated that, under control condition (no flow), NF-κB (p65), PU.1 and activation transcription factor 2 (ATF2) bound to TREM-1 promoter region 2, but not to region 1 (
Fig 2A
). In response to LSS for 30 min, however, binding of the NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 2 was significantly enhanced; furthermore, LSS treatment induced binding of NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 1 (
Fig 2B)
.

Conclusions:
Transcriptional factors NF-κB (p65), PU.1 and ATF2 are critically involved in the TREM-1 expression in response to LSS stimulation in an
in-vitro
model of human coronary artery atherosclerosis.