bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 628: Molecular Mechanisms of Covid-19 in the Lungs and Heart: Insights from Spatial Transcriptomics
Authors: Veizades Stefan , Solomonidis Emmanouil G , Li Ziwen , Passi Rainha , Berkeley Bronwyn , Stewart Katherine R , Perez-Vicencio Daniel , Barton Anna K , Sellers Stephanie , Mills Nicholas L , Newby David , Dweck Marc R , Dorward David A , Lucas Christopher D , Baker Andrew H , Brittan Mairi
Published: October 2023

Introduction:
Spatial transcriptomics has become a powerful tool for interrogating a disease-specific transcriptome within the context of tissue architecture. In this study, we used spatial transcriptomics to investigate the molecular mechanisms underlying COVID-19 in the lungs and heart.

Methods:
We performed sequencing-based spatial transcriptomics (ST), using 10X Genomics’ Visium, on 6 paired fatal COVID-19 lung and heart tissue samples and 2 control samples per organ. With the gene by spot matrix, we performed dimensional reduction, clustering (Louvain), differential expression (Wilcoxon Rank sum test), and pathway analysis (GSEA). spacexr was used to estimate the cell type composition of ST spots.

Results:
By histology and ST, COVID-19 tissue was defined by a loss in parenchymal cells and increased inflammation (
fig. 1E
). In both the heart and lung, we found a cluster of ST spots specific to fatal COVID-19 (
fig. 1A, B
; P<0.001). Between the two organs, these clusters shared genes and pathways relating to tissue remodeling, B cell action, and complement pathway activation (
fig. 1C, D
). Response to wounding and blood vessel endothelial cell migration pathways were distinct to the heart cluster, also explaining the increased capillary cell weight in the COVID-19 heart compared to the control (
fig. 1D, E
; P=0.0167).

Conclusion:
Our results suggest that there is a shared spatial niche between the heart and lungs in COVID-19 infection and highlight the importance of studying multiple organs in understanding the disease. These findings provide new insights into the molecular basis of COVID-19 and have the potential to inform the development of novel therapies for this disease.

Figure 1
(A)
UMAP colored by (left) clusters and (right) disease.
(B)
Cluster proportions between COVID-19 and control tissue
(C)
Scatter plot of shared differentially expressed genes.
(D)
GSEA pathway score.
(E)
Spatial mapping of clusters and cell-type deconvolution.
Abstract 631: Neointima Abating Yet Endothelium Preserving-An Adventitia-localized Nanoformulation To Inhibit The Epigenetic Writer Dot1L
Authors: Li Jing , Shirasu Takuro , Yodsanit Nisakorn , Huang Yitao , Xie Xiujie , Tang Runze , Wang Qingwei , Zhang Mengxue , Urabe Go , Webb Amy , Wang Yuyuan , Wang Xiuxiu , Xie Ruosen , Wang Bowen , Kent K. Craig , Gong Shaoqin , Guo Lian-wang
Published: October 2023

Open vascular reconstructions such as bypass are common treatments for cardiovascular disease. Unfortunately, neointimal hyperplasia (IH) follows, leading to treatment failure for which there is no approved therapy. Here we combined the strengths of tailoring nanoplatforms for open vascular reconstructions and targeting new epigenetic mechanisms. We produced adhesive nanoparticles (ahNP) that could be pen-brushed and immobilized on the adventitia to sustainably release pinometostat, an inhibitor drug selective to the epigenetic writer DOT1L that catalyzes histone-3 lysine-79 dimethylation (H3K79me2). This treatment not only reduced IH by 66.3% in injured arteries (mimicking open reconstructions) in obese Zucker rats with human-like diseases, but also avoided endothelial impairment-a shortcoming in current IH managements. In mechanistic studies, chromatin immunoprecipitation (ChIP) sequencing revealed co-enrichment of the histone code H3K27ac(acetyl) and its reader BRD4 at the gene of aurora kinase B (AURKB), where H3K79me2 was also enriched as indicated by ChIP-qPCR. Accordingly, DOT1L co-immunoprecipitated with H3K27ac. Furthermore, the known IH driver BRD4 governed the expression of DOT1L which controlled AURKB’s protein level, revealing a BRD4->DOT1L->AURKB axis. Consistently, AURKB-selective inhibition also reduced IH. Thus, this study presents a prototype nanoformulation suited for open vascular reconstructions, and the new insights into epigenetic regulators may aid future translational advances.
Abstract 632: Cell Populations In Pre-access Veins And Arteriovenous Fistulas: A Single Cell Perspective
Authors: Rojas Miguel G , Martinez Laisel , Tabbara Marwan , Vazquez-Padron Roberto I
Published: October 2023

Objective:
A comprehensive picture of the cellular events dictating the transformation of the vein after arteriovenous fistula (AVF) creation has remained elusive so far. This study generated the first granular cellular atlas of pre-access veins and AVF from patients with end-stage kidney disease (ESKD).

Methods:
Veins (n=5) and AVF (n=7) were obtained from a total of 12 ESKD patients undergoing fistula creation in two stages. 41,000 enzymatically dissociated cells were single cell sequenced using the 10x Chromium system. Monocle3 inferred the trajectory leading to neointimal cells and signaling interactome in the AVF.

Results:
Cells populated 16 clusters containing canonical and non-canonical vascular markers after integrating the single cell transcriptomic profiles in Seurat v4. The majority of cells in both veins and AVF were smooth muscle cells (6.6 vs. 9.4%), fibroblasts (12.4 vs. 26.7%), neointimal-like cells (11.5% vs, 17.0, endothelial cells (10.5 vs. 10.5%), macrophages (26.1 vs. 19.5%), lymphocytes (16.4 vs.10.3), and neutrophils (0.7vs. 3.8 %). CENPF+ dividing cells were abundant in the AVF (0.2 to 1.2%). For the first time, we have detected a potential transcriptomic neointimal signature defined by the expression of BGN and PRELP genes that was increased in AVFs, but not absent in the pre-access veins, confirming previous histological findings. Neutrophilic infiltration (S100A8/9+ and CSF3R+) was increased in the AVF. Interestingly, lymphocytes and macrophages were decreased in the AVF with respect to the vein. These changes in cell populations in the AVF wall correlated with up-regulation of GO terms such as ECM deposition, neutrophil activation, and blood vessel remodeling in the AVF. Finally, we established a pseudotime cellular trajectory from contractile SMCs to neointimal-like cells, defined by COL8A1, PRELP, and BGN by Cohen’s I.

Conclusions:
We present a comprehensive single-cell atlas of the AVF transformation and assign identity to the cellular sources and cell trajectories in this remarkable vascular transformation.
Abstract 636: Spinning Disk Confocal Intravital Imaging Revealed Platelet And Neutrophil Dynamics In Microvascular Obstruction Following Ischemia-reperfusion Injury And Their Synergistic Interventions
Authors: Chen Zengrong , Xu Pengfei , Wang Miao
Published: October 2023

Objective:
Microvascular obstruction is associated with poor outcome in patients with acute myocardial infarction, however the underlying cell dynamics is unclear. We report that phospordiesterase-4 inhibition by roflumilast improves microcirculation following myocardial ischemia-reperfusion (I/R) injury. This study aimed to elucidate the cell dynamics in microcirculation following I/R.

Approach and Results:
In eight-week-old C57B6 mice, 30min coronary ischemia followed by reperfusion resulted in impaired microcirculation, as assessed by laser Doppler flow. This I/R response was also reproduced with hind-limb I/R. Micro-vessels and capillaries (with diameter of 10-25 μm and <10 μm, respectively) were non-invasively imaged at the plantar skin area of hind-limb extremity by spinning-disk confocal intravital microscopy. The impaired microcirculatory perfusion following I/R was exemplified with platelet aggregation, platelet-neutrophil aggregate (PNA) formation, neutrophil adhesion, and enhanced vascular permeability. Platelet aggregation and neutrophil adhesion were often spatially different from each other in capillaries. Aspirin treatment suppressed the I/R induced platelet aggregations and PNA formation, whereas roflumilast, without affecting platelet aggregation, attenuated the neutrophil adhesion and PNA formation, and both treatments reduced vascular permeability. Furthermore, treatment with aspirin and roflumilast each ameliorated microvascular obstruction after I/R injury, and combination of the two drugs provided synergistic protection.

Conclusions:
I/R triggers microvascular obstruction attributable to platelet aggregation, neutrophil activation and increased permeability. Pharmacological interventions of platelets and neutrophils synergistically improve microcirculation post I/R injury, implicating their potential clinical application.

Keywords:
Ischemia-reperfusion injury; Microvascular obstruction; Microcirculation; Platelet; Neutrophil; Aspirin; Roflumilast; Spinning disk confocal imaging
Abstract 637: Exercise-stimulated Resolvin Biosynthesis In Adipose Tissue Is Prevented By High Fat Diet-induced Adrenergic Insufficiency
Authors: Hellmann Jason , Pena Calderin Ernesto , Lynch Will , Zheng Jing-Juan , Sansbury Brian , Spite Matthew R , Bhatnagar Aruni , Hill Bradford G
Published: October 2023

Diet-induced adipose tissue (AT) inflammation is associated with obesity and the development of systemic insulin resistance. Our lab has shown that specialized proresolving mediators (SPMs), which are induced by exercise (Exe), restore insulin sensitivity and promote resolution of adipose tissue resolution. In this study, we sought to determine if exercise-enhanced production of SPMs extends to adipose tissue and has dietary dependence. Compared with sedentary controls (Sed), mice exposed to 4 wk of Exe display elevated AT expression of SPM biosynthetic enzyme
Alox15
(Sed 1.12±0.18 vs. Exe 1.68±0.13 relative expression,
n
=5-7), SPM levels (Sed vs. Exe; RvD1 156.7±12.02 vs. 223.0±24.71; 17
R
-RvD1 354.0±35.86 vs. 534.4±98.92; RvD4 31.31±6.93 vs. 123.1±30.69; and 17
R
-RvD3 2.92±1.08 vs. 8.20±1.70 pg/g,
n
=5-7), and proresolving M2 macrophages (F4/80
+
CD301
+
; Sed 17.5±3.2 vs. Exe 40.9±4.5 %F4/80
+
,
n
=4-6). These changes were dependent upon diet as 6-weeks of high fat diet (60% kcal from fat; HFD) feeding abrogated the proresolving effect of exercise when compared with low fat diet fed (10% kcal from fat; LFD) controls—despite performing the same amount of exercise. Interestingly, exercise-induced epinephrine (Epi) production (LFD Sed 147.2±24.4 vs Exe 312.5±60.9 ng/mL) was inhibited by HFD (Sed 121.1±22.7 vs. Exe 140.0±16.8 ng/mL). HFD feeding resulted in diminished expression of the Epi biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT) in adrenal glands (protein expression: LFD 1.0±0.25 vs. HFD 0.36±0.15,
n
=4). Moreover, Sed HFD fed animals treated with Epi (0.1 mg/kg i.p. daily) for 1 wk display increased abundance of proresolving adipose tissue macrophages (F4/80
+
CD301
+
; Sed 17.8±0.6 vs. Exe 25.2±3.1 %F4/80
+
,
n
=6-7). These results suggest that the resolution promoting effect of exercise in adipose tissue is controlled by high fat diet consumption and adrenergic stimulation.
Abstract 638: Ligands Of The Receptor For Advanced Glycation End Products (Rage) Induce Trained Immunity In Mouse Bone Marrow-derived Macrophages (Bmdms)
Authors: Kumar Vikas , Egaña-Gorroño Lander , Gugger Paul F. , Nimma Ramesh , Schmidt Ann Marie
Published: October 2023

Background:
Bone marrow-derived myeloid precursors are vulnerable to “training” and glucose-driven processes affect accelerated atherosclerosis in diabetic mouse models. AGEs accumulate in diabetes and we hypothesized that RAGE ligand CML-AGE contributes to the induction of trained immunity in macrophages, thereby promoting and exacerbating diabetes-associated complications.

Methods:
BMDMs from Wild Type (WT) C57BL/6 mice were treated with CML-AGE (25 μg/ml) and subjected to removal of CML-AGE during a wash-out period of 48 hours (h). BMDMs were then stimulated with LPS (100 ng/ml) or CML-AGE (25 μg/ml) vs. vehicle (medium alone) for 24 h. BMDMs were subjected to qRT-PCR and Seahorse XF assays. Roles for RAGE in these processes were probed using
Age
r
flox/flox
Lysm Cre
+/+
and control Lysm Cre
+/+
mice.

Results:
RNA sequencing of BMDMs of
Ager
flox/flox
Lysm Cre
+/+
vs. control Lysm Cre
+/+
mice (n= 4 per group) revealed altered expression of multiple markers of inflammation and metabolism. Ingenuity Pathway Analysis stratified
Ager
-deleted macrophages into several canonical pathways, including macrophage classical activation and hypercytokinemia signaling pathway.
Ager
deletion in BMDMs upregulated the expression of
C4a, Tnfrsf4, Il17c, Tnfrsf25
, and
Nfkbid
genes and downregulated the expression of
Ly6i, Cd207, Ccl5
, and
Cxcl3
genes. BMDMs from WT mice exposed to transient treatment with CML-AGE vs. vehicle followed by wash-out and subsequent stimulation with LPS or CML-AGE demonstrated significant upregulation
of IL1b, IL6,
and
TNFα
and downregulation of
Il10
genes. CML-AGE training in
Ager
-deleted BMDMs, compared to control
Ager
-expressing BMDMs, did not result in upregulation of these genes, implicating RAGE in the CML-AGE training response in BMDMs. qRT-PCR analysis, Seahorse glycolytic, and Mito stress tests in BMDMs revealed a metabolic switch, with an overreliance on glycolysis in control Lysm Cre
+/+
vs.
Ager
flox/flox
Lysm Cre
+/+
macrophages.

Conclusions:
CML-AGE induction of trained immunity, at least in part via RAGE, highlights novel inflammatory and metabolic alterations that may contribute to the pathophysiological changes observed in diabetic complications.
Abstract 643: Lactobacillus
Plantarum 299v
Supplementation Does Not Acutely Improve Endothelium Dependent Vasodilation
Authors: Wendel Emily , Aljadah Michael , Widlansky Michael E
Published: October 2023

Introduction:
Endothelial dysfunction is characterized by systemic vasoconstriction, thrombosis, and inflammation. Emerging data suggests that modulation of gut microbiota with probiotics, such as
Lactobacillus plantarum 299v
(
Lp299v
), is an effective way of reducing systemic inflammation and subsequent endothelial dysfunction. Our group previously showed daily supplementation with
Lp299v
in men with stable coronary artery disease improved endothelium dependent vasodilation. However, it was unclear if improvement in endothelial function manifests immediately after supplementation, potentially related to the fermentation products consumed with
Lp299v
, or happens as a result of prolonged supplementation. We therefore set out to assess acute impact of
Lp299v
supplementation on endothelial dependent vasodilation.

Hypothesis:
We hypothesized endothelial dependent vasodilation measured at two- and 24-hours would not differ after
Lp299v
supplementation.

Methods:
A total of 26 patients (ages 47-74) with a diagnosis of hypertension, hyperlipidemia, diabetes mellitus, peripheral vascular disease, cerebrovascular disease, or cardiovascular disease were randomized, to receive 20 million CFU of
Lp299v
supplementation contained in 30 mL of the GoodBelly® Straightshot or matching heat-killed placebo. Endothelial function was measured using brachial artery flow mediated dilation (FMD) at baseline, two-, and 24-hours post-intervention.

Results:
Within the placebo and
Lp299v
groups, there were no significant differences in resting diameter, FMD, and baseline peak shear between time points.

Conclusion:
Lp299v
supplementation does not produce a significant acute effect on FMD within the first 24 hours of supplementation. This supports the hypothesis that the beneficial effect of
Lp299v
supplementation is likely a result of long-term supplementation rather than acute effects of concomitant ingestion of fermentation products.
Abstract 647: Cardiac Cysteine And Glycine Rich Protein 3 Induces Renal Myogenesis Leading To CKD
Authors: Funahashi Yoshio , Hebert Jessica , Munhall Adam , Groat Tahnee , Hutchens Michael P
Published: October 2023

Introduction:
Acute cardiac injury with renal injury (acute cardiorenal syndrome) can induce chronic kidney disease (CKD), but mechanisms by which heart injury signals to kidney are unclear. Cardiac LIM protein (CSRP3) is a cardiomyocyte differentiation factor which is released into plasma after cardiac arrest. We assessed the hypothesis that CSRP3 induces myogenesis in the renal proximal tubule, inducing CKD.

Methods:
Two kidney injury models which result in similar acute kidney injury were tested, renal ischemia-reperfusion injury (IRI) or cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Seven weeks later, blood pressure (BP) and glomerular filtration rate (GFR; μg/min/100g) were measured, then mice were sacrificed. Single nucleus RNA sequencing (snRNAseq) was conducted on mouse kidney cells harvested 24h after CA/CPR. Primary human proximal tubular epithelial cells (hPTEC) were cultured from research-designated human kidneys. Recombinant CSRP3 (rCSRP3) was administered to hPTEC followed by RNAseq. Lastly, mice were injected with rCSRP3 after IRI, then sacrificed 7 weeks later.

Results:
CA/CPR (n=8) induced worse CKD than IRI (n=8) despite identical 24h GFR: GFR (CACPR 732.3+31.03 vs IRI 933.3+52.55, p<0.01); fibrosis (V
aSMA
/V
kid
; CA/CPR 1.05±0.086 vs IRI 0.68±0.068, p<0.05); albuminuria (CACPR 0.032±0.005 mg vs IRI 0.132±0.026 mg, p<0.01). Compared with IRI, CA/CPR caused renovascular hypertrophy (thickness index; CACPR 0.75±0.038 vs IRI 0.49±0.011, p<0.01) and hypertension (BP ratio to sham: CACPR 1.22±0.029 vs IRI 0.98±0.069, p<0.01). SnRNAseq demonstrated that CA/CPR upregulated muscle cell differentiation ontologies in proximal tubule cells, driven by regulation of 110/162 myogenesis genes. In hPTEC, CSRP3 induced myogenesis differentiation factor 1 (MyoD1)>30 fold. CSRP3 injection in IRI mice resulted in lower GFR (CSRP3(+) 785.5±34.16 vs CSRP3(-) 954.2±55.69, p<0.05), and more renovascular hypertrophy (CSRP3(+) 0.61±0.011 vs CSRP3(-) 0.51±0.011, p<0.01, n=8/group) compared with IRI without CSRP3.

Conclusions:
CA/CPR caused CKD, renovascular hypertrophy, and hypertension associated with tubular myogenesis. CSRP3 induced myogenesis pathway in hPTEC and in mice. CSRP3 may drive cardiorenal-induced CKD.
Abstract 649: Mechanical Tension Decreases Fibrillin-1 Calcium Binding And Stiffness
Authors: Haller Stephen , Dudley Andrew
Published: October 2023

Introduction:
Aneurysm pathogenesis involves a complex interplay between vascular tissue remolding and hemodynamics. In Marfan syndrome, vascular tissues fail to remodel appropriately, causing aneurysms. The root cause for this is mutations in the extracellular matrix protein fibrillin-1. However, the precise molecular mechanisms through which fibrillin-1 contributes to aneurysm formation remain unclear.

Hypothesis:
We hypothesize that fibrillin-1 calcium binding, which is known to regulate extracellular proteolysis and growth factor sequestration, decreases under mechanical tension.

Methods:
Recombinant fragments of human fibrillin-1 containing the hypothesized force-sensitive calcium binding domains were genetically engineered with unique N- and C-terminal peptide tags (Fig. 1A). The C-terminal tag was used to irreversibly attach the protein to an atomically flat surface (Fig. 1B) The N-terminal tag was then used to reversibly bind a functionalized atomic force microscopy (AFM) probe (Fig. 1C), thereby facilitating force spectroscopy experiments to measure the unfolding dynamics of fibrillin-1 calcium binding under tension (Fig. 1D).

Results:
Force spectroscopy experiments measured a 27% decrease in peak force required to unfold fibrillin-1 calcium binding domains following calcium removal (112 ± 25 pN vs 93 ± 16 pN; p < 0.031; n = 12). This correlates extremely well with the 25% decrease in peak force predicted from our previously published molecular dynamics simulations. Moreover, characteristic force profiles were observed which support force induced calcium unbinding.

Conclusions:
Force-sensitive calcium binding represents a novel mechanism to explore fibrillin-1 as an extracellular tension sensor in Marfan syndrome related aneurysm pathogenesis. Targeting extracellular mechanotransduction could thus provide a new avenue for regenerative medicine research in vascular disease.
Abstract 650: Disrupting Neointimal Formation In Mouse And Human Vascular Tissues Through Targeting Connexin 43
Authors: Sedovy Meghan , King David , Leng Xinyan , Malek Arya , Baillie George , Baker Joseph W , Joseph Mark , Smyth James W , Isakson Brant E , Johnstone Scott R
Published: October 2023

Vascular damage caused by stent placement triggers smooth muscle cell (SMC) de-differentiation, leading to an aggressively proliferating phenotype that thickens the vessel wall and blocks the artery, which contributes to stent failure. We have identified the gap junction protein connexin 43 (Cx43) as a significant contributor to neointima progression. Phosphorylation of Cx43 by mitogen-activated protein kinase (MAPK) promotes its binding to the cell cycle regulator cyclin E and its kinase CDK2, promoting SMC proliferation. Beyond this, we do not know how these proteins regulate SMC proliferation in humans. We tested the hypothesis that disrupting Cx43-cyclin E binding could reduce neointima formation in human blood vessels. We used peptide arrays to identify cyclin E binding sites on Cx43 and to define critical Cx43 MAPK-phosphorylation residues that control their interactions. Based on this, we created a 15 amino acid, stearate-linked, cell-permeable, connexin mimetic peptide termed CycliCx that binds cyclin E. Experiments were controlled using Cx43 mimetic peptides designed against regions not bound by cyclin E. In cultured human coronary artery SMC, the CycliCx peptide reduces Cx43 MAPK-phosphorylation, disrupts Cx43-cyclin E interactions, and prevents PDGF-induced SMC proliferation. Application of the CycliCx peptide to mouse carotid arteries inhibits neointima formation in a 14-day ligation-induced neointima model (n=8, male and female). We developed an ex vivo model of PDGF-induced neointimal formation in excess human saphenous vein tissues recovered after CABG surgery. In the human saphenous vein preparations treated with PDGF, the CycliCx peptide disrupts neointima formation after 14 days (n=9). In conclusion, our data suggest that disrupting Cx43-cyclin E interactions provides protection against pathological SMC proliferation. These data highlight Cx43 as a potential drug target to prevent human neointima development.
Abstract 662: A Novel Approach To Understanding Geometric Remodeling Of Surgically Created Arteriovenous Fistulas
Authors: Varsanik M. Alyssa , Thaxton Carly , Li Willa , Dardik Alan , Pocivavsek Luka
Published: October 2023

Objectives:
Failure of arteriovenous fistulas (AVF) for dialysis access remains a persistent problem. The reason for failure is largely unknown. Computational models of AVF have not been validated in parallel with animal experiments. We propose a computational AVF model that predicts venous geometric remodeling. We hypothesize that remodeling may precede AVF failure.

Methods:
First, we use a validated murine AVF model to define the parameters and boundary conditions for our computational model. Anesthetized mice undergo laparotomy and aortocaval puncture. Ultrasound confirms AVF patency, flow velocities, and vessel geometry throughout the aorta and inferior vena cava (IVC). In vivo CT angiography scans are segmented using 3D software (Simpleware ScanIP) to establish global geometry. We use this geometry to build a simplified geometric model which is then imported into a fluid dynamics simulation (Xflow) and finite element analysis software (Abaqus).

Findings:
Segmentations of in vivo CT scans establish the global geometry of the AVF (Fig 1a). A fluid dynamics model replicates flow through the fistula (Fig 1b). The model depicts the wall pressure being highest in the artery and around the fistula connection. A computational dynamics model predicts deformation and venous remodeling after pressurization of the system (Fig 1c).

Conclusions:
This is the first in vivo-computational model of AVF geometry. Our results indicate elevated wall pressure at the level of the fistula leads to venous deformation as hypothesized. These results can be used to predict how the underlying anastomotic geometry may impact anastomotic stress, stenosis, and injury, and may help define anatomic parameters that could optimize AVF durability. Future experiments involving models using the in vivo geometry itself, higher resolution micro CT images, and histologic staining of the vessel wall will provide insight into the deformation mechanisms around these observed areas of remodeling.
Abstract 667: Endothelial Glucocorticoid Receptor Deficiency Affects Arteriovenous Fistula Maturation
Authors: Zhang Weichang , Gonzalez Luis , Thaxton Carly , Ohashi Yuichi , aoyagi yukihiko , Xie Yangzhouyun , Cai Yujun , Shu Chang , Goodwin Julie , Dardik Alan
Published: October 2023

Objectives:
Arteriovenous fistulae (AVF) are the optimal conduit for hemodialysis access in patients with end-stage chronic kidney disease (CKD); however, AVF have high rates of failure due to aggressive neointimal hyperplasia (NIH) caused by vascular inflammation and endothelial-to-mesenchymal transition (EndMT). Since the endothelial glucocorticoid receptor is a negative regulator of vascular inflammation, we hypothesized that inhibition of the endothelial glucocorticoid receptor would promote EndMT and accelerate NIH.

Methods:
Aortocaval AVF were created in

GR
fl/fl
, Tie-1 Cre +

(GR
EC-KO
) mice and control mice

GR
fl/fl
, Tie-1 Cre -

(

GR
fl/fl

). Three weeks prior to AVF, CKD was created via 5/6 nephrectomy (Nx); ultrasound was used to determine AVF diameter and patency at postoperative days 7, 14, and 21. AVF were harvested at days 3, 7 and 21 and examined with histology, immunofluorescence, and Western blot.

Results:
At postoperative day 21, AVF diameter was significantly decreased in GR
EC-KO
mice, whereas there was increased AVF wall thickness and accumulation of extracellular matrix (ECM) components fibronectin (p<0.005), collagen1 (p<0.005), collagen3 (p<0.005), as well as p-smad2 (p<0.001), compared to the control mice. Endothelial cells in the AVF of GR
EC-KO
mice had significantly increased immunoreactivity of the mesenchymal markers notch3 (p<0.0001), vimentin (p<0.0001) and FSP-1 (p<0.005) compared to control mice, consistent with increased EndMT. There was no difference between GR
EC-KO
and control mice treated with 5/6Nx regarding body weight or aortic diameter.

Conclusion:
Absence of the endothelial glucocorticoid receptor promotes EndMT and wall thickening during venous remodeling in the CKD environment. These data suggest that the endothelial glucocorticoid receptor serves as a physiologic regulator of venous remodeling during AVF maturation.
Abstract 668: Identification And Functional Characterization Of Novel MicroRNAs Targeting Epsins For Angiogenesis And Vascular Remodeling
Authors: Zhu Bo , Lu Yao Wei W , Wang Beibei , Singh Bandana , Chen Hong
Published: October 2023

Objective:
Epsin is evolutionarily conserved protein that involved in clathrin-mediated endocytosis. Our previous studies showed Epsins regulate VEGF signaling, which is critical for angiogenesis, and Epsins can also augment inflammatory signaling in endothelial cells (EC). However, how Epsins are regulated during inflammation mediated angiogenesis is not clear. We hypothesize that post-transcriptional regulation of Epsins by miRNA are critical for inflammation-induced angiogenesis. In this study, we aim to identify novel miRNAs that can bind to Epsin1/2 genes for regulation of inflammation mediated angiogenesis.

Approach and Results:
In vitro, we found Epsin1/2 are significantly induced by TNFa and oxidized LDL in mouse aortic endothelial cells (MAECs). To screen miRNAs that can regulate Epsin1/2 expressions, we performed small RNA-sequencing for the control and treated MAECs samples, and identified differentially expressed miRNAs that are predicted to bind to Epsin1/2. We identified 78 differentially expressed (DE) known miRNA and 464 DE novel miRNAs. Among them, 12 known miRNAs and 69 novel miRNAs were predicted to bind Epsin1/2. We validated 2 known miR (miR-6390 and miR-6539) and 3 novel miRNA candidates (miR105462, miR39474 and miR67284) by in vitro miRNA mimics experiments, and found that they all can robustly suppress Epsin1/2 expressions. We then generated miR105462-flox mouse lines by homologous recombination using CRISPR/Cas9. We cross the miR105462-flox mice with Cdh5-Cre
ERT2
to generate inducible EC-specific deletion by tamoxifen (miR105562-iEKO). Interestingly, neonatally induced endothelial-specific loss of miR105462 lead to reduced microvascular density and fewer tip cells in the retina of postnatal day6 pups. Additionally, higher Epn1 expressions in retinal EC were detected in EC-specific miR105462 deficient mice.

Conclusion:
We identified novel miRNA that can regulate Epsin expression in EC. Using novel genetic models, we identified angiogenic defects in vivo when miR105462 is conditionally deleted in EC, implicating critical function elicit by these novel miRNAs. Future studies will be directed at defining the novel miRNA-dependent molecular mechanism for the angiogenesis.
Abstract 675: Time Restricted Feeding Regulates Cholesterol Efflux And Atherosclerosis In Macrophage-specific Bmal1 Deficient Mice
Authors: Pan Xiaoyue , Hussain M M
Published: October 2023

Background:
Macrophages play a critical role in atherosclerosis. We have previously shown that Clock mutant and global or tissue-specific Bmal1 deficient mice show enhanced atherosclerosis. Yet, the role of time restricted feeding (TRF) in cholesterol transport and atherosclerosis in macrophage Bmal1 deficient mice remains unknown.

Objectives:
To establish a novel regulatory link between TRF, circadian clock, and atherosclerosis.

Methods:
We studied the effect of day or nighttime TRF on macrophage-specific Bmal1 deficient (M-

Bmal1
–/–
Apoe
–/–

) or control mice on cholesterol efflux to clarify the role of Bmal1 in macrophage function and in atherosclerosis.

Results:
We found that daytime-TRF increased atherosclerosis. Biochemical studies showed that macrophages from daytime TRF M-

Bmal1
–/–
Apoe
–/–

mice exhibit increased expression of Lox-1 and Cd36, and take up more oxLDL compared to nighttime TRF mice. Furthermore, daytime TRF mice showed decreased expression of Abca1 and Abcg1, reduced cellular cholesterol efflux and
in vivo
reverse cholesterol transport compared to nighttime TRF mice. Thus, TRF regulates macrophage cholesterol metabolism by regulating uptake of oxidized lipoproteins and cholesterol efflux pathways. Molecular studies showed that TRF regulates HDAC3 interactions with p300 to regulate
Znf202
gene expression. Thus, TRF regulates Bmal1 and Znf202 to regulate uptake of oxidized lipoproteins and cholesterol efflux.

Conclusions:
TRF is an important regulator of cholesterol metabolism in macrophages and affects atherosclerosis. It regulates macrophage function by modulating the expression of different transcription factors. It is likely that nighttime-TRF may prevent atherosclerosis.
Abstract 681: Development Of An In Vitro Model For The Study Of Polarized Surfactant Lipid Secretion In Pulmonary Type 2 Cells
Authors: ward Rafael , Kiang Christopher , Garcia-Arcos Itsaso
Published: October 2023

Introduction:
Alveolar type 2 cells (T2C) synthesize and secrete pulmonary surfactant, a lipoprotein complex essential for respiration. T2C take up precursors through their basal side and secrete surfactant to their apical side. The study of T2C surfactant metabolism in vitro is challenging because traditional culture models do not preserve the polarized lipid secretion and primary T2C dedifferentiate. Prior research in our lab showed that expression of the low-density lipoprotein receptor-related protein 1 (LRP1) is needed for surfactant lipid synthesis in mice. We developed a type 2 cell in vitro model to recapitulate the in vivo findings and conduct mechanistic studies.

Methods:
Human T2C-derived A549 cells were grown in submerged conditions and plated on transwells prior to the experiment. When they reached confluency, media was removed from the upper chamber, exposing the cellular apical side to air (air-liquid interface, ALI), and maintained for 24h. We then collected the cells for RNA extraction and q-PCR analysis. We tested 3 different types of transwell coating: commercially coated inserts (Corning, discontinued), collagen 1 coated inserts, and equimolar mix of collagen 1 plus collagen 3. The inserts were coated with 2 μg/cm
2
collagen for at least 6 hours.

Results:
Cellular adherence did not differ between the 3 conditions and there was no additive effect for coating longer. Media did not leak from the basal to the apical side. Genes involved in surfactant lipid synthesis and secretion
fatty acid synthase
(FASN),
choline cytidyl transferase
(CCTα),
phospholipase A
2
(PLA2) and
ATP Binding Cassette Subfamily A Member 3
(ABCA3), and those involved in cholesterol export
ATP Binding Cassette Subfamily A Member 1
and
G Member 1
(ABCA1 and ABCG1) were highly expressed in all conditions. Decreased gene expression with LRP1 loss was consistent only for collagen 1 plus collagen 3 coated inserts and commercially coated inserts. Our next steps are to compare apically secreted surfactant phospholipid in all conditions.

Conclusion:
This is the first description of an in vitro model to study surfactant metabolism at the ALI. Commercially coated inserts and collagen 1 plus collagen 3-coated inserts are equally valid options.
Abstract 683: Improved Myocardial Blood Flow And Arteriogenesis With Calpain Inhibition May Be Mediated By Vascular Endothelial Growth Factor And Fibroblast Growth Factor Angiogenic Pathways In A Large-animal Model Of Metabolic Syndrome And Chronic Myocardial Ischemia
Authors: Banerjee Debolina , Sabe Sharif A , Harris Dwight , Sabra Mohamed , Hu Jiayu , Sabe Ashraf A , Potz Brittany , Abid Ruhul , Sellke Frank W
Published: October 2023

Objective:
Ischemic heart disease is the leading cause of death worldwide. Under pathological conditions like myocardial ischemia, endothelial calpain proteases are activated by hypoxia, leading to aberrant angiogenesis. Our group has shown calpain inhibition increases myocardial blood flow and vascular density, microvascular vasodilation, and expression of proangiogenic vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1, VEGF-R2) in a porcine model of chronic myocardial ischemia and diet-induced metabolic syndrome. We aim to assess the role of downstream factors in angiogenic pathways.

Methods:
Yorkshire swine were fed a high cholesterol diet for four weeks, and then underwent placement of an ameroid constrictor to the left circumflex artery. After three weeks, pigs received either daily calpain inhibitor MDL28170 (0.25mg/kg, oral) for five weeks (n = 8) or no drug (n = 7), followed by tissue harvest of ischemic myocardium.

Results:
Treatment was associated with a significant increase in expression of proangiogenic mediators, fibroblast growth factor (FGF) receptor 1 (FGFR1, p <0.001) and matrix metalloprotease-2 (MMP-2, p <0.001). Cathepsins D and L have been implicated in both pro- and antiangiogenic actions. Cathepsin D was significantly increased (p = 0.004), but Cathepsin L was unchanged (p >0.99). Cathepsin L proenzyme, procathepsin L, was significantly decreased (p = 0.04), while there was a strong trend toward decreased procathepsin L:cathepsin L ratio (p = 0.09). Proangiogenic Tie-2 (p = 0.28), and antiangiogenic collagen 18 (p = 0.54) and angiostatin (p = 0.24) were unchanged.

Conclusions:
In metabolic syndrome, calpain inhibition led to increased proangiogenic signaling in chronically ischemic myocardium, suggesting angiogenic VEGF- and FGF-mediated pathways may work in concert with improved microvascular vasodilation to explain the observed increase in vascular density and collateral-dependent coronary perfusion.
Abstract 685: A New Volumetric Quantification Method For Atherosclerosis Using Histological Sections
Authors: Graf David B , Sawada Hisashi , Daugherty Alan , Lu Hong S
Published: October 2023

Background and Objective:
Commonly used quantification methods of atherosclerosis in mouse models include en face analysis of lesions in the aorta and lesion area measurements in the aortic roots using cross-sections. The major shortcoming of these methods is that they quantify surface area, which does not reflect the total volume of atherosclerosis. The purpose of this study is to develop a method to measure atherosclerosis lesion volume.

Methods and Results:
Male LDL receptor -/- mice were fed a Western diet for 12 weeks, and then euthanized to collect aortic roots. Serial cross-sections of mouse aortic roots were obtained through a distance of 1 mm that included both the aortic sinus and a small portion of the ascending aorta. The interval between each two serial sections on a same slide were 100 μm. The cross-sections from one slide were stained with hematoxylin and eosin, then digitized using a robotic slide scanner. Lesion areas were captured using a post-processing stage to output accurate areas of lesions. This algorithm used linear regression combined with integration to give a volumetric approximation. This process acquired area of atherosclerosis in each section without bias, and created a regression line between sections from the area data, then integrated those functions over the distance between sections. Each integral region was aggregated to yield the total volume of atherosclerotic plaques.

Conclusion:
The volumetric approximation using a linear regression combined with integration is a simple and useful method for the quantification of atherosclerosis via histological sectioning.
Abstract 686: Glp-2 Predicts Cardiovascular Outcomes In Patients With Myocardial Infarction And Increases Atherosclerosis In Mice
Authors: Sausen Maximillian , Mertens Robert , Quintana Luis , Rueckbeil Marcia , Idel Paul , Tabaza Nadim , Ganesh Naresh , Just Susanne , Arrivas Maria , Moellmann Julia , Spießhoefer Jens , Biener Moritz B , Giannitsis Evangelos , Katus Hugo , Drucker Daniel J , Marx Nikolaus , Lehrke Michael , Kahles Florian
Published: October 2023

Background:
GLP-1 and GLP-2 (glucagon-like peptide-1/2) are incretin hormones that are co-secreted from intestinal L-cells in response to food. While GLP-1 is known to induce postprandial insulin secretion and to improve cardiovascular outcomes in patients with diabetes, GLP-2 is a local intestinal growth factor enhancing intestinal nutrient absorption. GLP-2 agonists are clinically used for the treatment of patients with short bowel syndrome. The relevance of GLP-2 beyond the gut is not well understood. The aim of this study was to investigate the role of GLP-2 for cardiovascular disease (CVD).

Methods:
Circulating GLP-2 levels were assessed at time of hospital admission in 1929 patients with myocardial infarction. The primary outcome of the study was the first occurrence of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke (3-P-MACE). To induce atherosclerosis, Glp2r
–/–
or WT mice were injected with PCSK9 virus and fed a HCD for 12 weeks.

Results:
Kaplan-Meier survival plots and univariable cox regression analyses found GLP-2 to be associated with adverse outcome (3-P-MACE and all-cause mortality; log. GLP-2 values HR: 2.87; p<.0001). Further adjustment for age, sex, smoking, hypertension, hypercholesterinemia, previous CVD and diabetes mellitus did not affect this association (log. GLP-2 values HR: 2.66; p=0.0055). Receiver operating characteristic curve (ROC) analyses illustrated that GLP-2 is a strong indicator for early events, which proved to be superior to Troponin T and hs-CRP. To asses the functional role of GLP-2 in CVD in an experimental approach we injected

Glp2r
–/–

or WT mice PCSK9 virus and fed these mice a HCD. After 12 weeks

Glp2r
–/–

mice compared to WT littermates presented with a significant reduction in plaque volume and lesion size. While body weight and leukocyte numbers in various organs were unaffected,

Glp2r
–/–

mice had significantly lower cholesterol levels.

Conclusion:
Circulating GLP-2 levels are independently associated with cardiovascular events in patients with myocardial infarction while inactivation of the GLP-2 system reduces atherosclerosis in mice. Future studies are needed to investigate whether the GLP-2 receptor might provide a novel therapeutic target for cardiovascular disease.
Abstract 687: Inhibitory And Metabolic Effects Of Diabetes On Yolk Sac-derived Endothelial-macrophage Progenitors In Postnatal Murine Tissues
Authors: Liyanage Sanuri , Williamson Anna , Salagaras Thalia , Hassanshahi Mohammadhossein , Long Aaron , Tran Dang , Kulesza Sylvia , Primer Khalia , Murphy Andrew , Fernando Sanuja , Tan Joanne , Bonder Claudine , Bursill Christina , Sharma Shiwani , Psaltis Peter
Published: October 2023

Converging evidence indicates developmental overlap whereby some tissue macrophages (MΦs) and endothelial cells (ECs) arise from a common embryonic source, namely yolk sac (YS) erythromyeloid progenitor cells. Although YS-derived MΦs and ECs are widely believed to be maintained after birth by proliferative self-renewal, our group has recently identified YS-derived bipotent endothelial-macrophage (EndoMac) progenitors as an alternative source for their postnatal renewal in tissues. We demonstrated that FACS-isolated and culture-derived progenitors from mouse aorta are Lin-CD45+CD11b-F4/80-Sca-1+c-Kit+ and express fractalkine receptor (CX3CR1) and macrophage colony-stimulating factor (CSF1R). Progenitors from different tissues (aorta, skin, skeletal muscle [SkM]) exhibit important similarities, with subtle tissue-related variations in clonal renewal, angiogenic capacity and gene expression. Finally, with a view to understanding their metabolic regulation, we studied the effects of high glucose and hyperglycaemia on aortic, skin and SkM progenitors using a streptozotocin-induced mouse model of type 1 diabetes and identified glucose-induced reductions in their clonogenicity, differentiation and angiogenic capacity, as well as their DNA integrity, mitochondrial viability and metabolic function. Given that diabetes impairs skin wound healing and ischaemia reperfusion we utilised lineage-mapping techniques, to reveal an attenuation in the rapid burst of proliferative accumulation of YS-derived progenitors in early diabetic skin wounds and ischaemic muscle. This was accompanied by blunting of the expansion of YS-derived MΦs and ECs in later stages of repair. Finally, we showed that progenitors from non-diabetic skin and SkM can engraft, differentiate and promote repair and muscle reperfusion respectively when transplanted into recipient diabetic wounds and ischaemic muscle, whereas these salutary properties are severely diminished for diabetic progenitors. Our findings provide a novel framework to help understand how diabetes impairs wound repair and vascularisation of ischaemic tissue through the lens of YS-derived EndoMac progenitors and the effect of diabetes on their function and reparative potential.
Abstract 689: Loss of Smooth Muscle Cell Nogo-B Protects from Atherosclerosis Plaque Development
Authors: Palos-Jasso Andrea , Shan Dan , Baldini Margaret , Tian Xin , Yu Jun
Published: October 2023

Background:
Atherosclerosis, a major cause of cardiovascular diseases, is a lipid-driven inflammatory disease that occurs as fatty deposits, immune cells, calcium, and waste products accumulate on the walls of arteries over decades and often result in myocardial infarction or stroke. A member of the reticulon family of proteins, Nogo-B localizes in endothelial cells, vascular smooth muscle cells (VSMCs), and macrophages. Global and macrophage-specific Nogo-B deletion exacerbated atherogenesis by increasing lesion area and necrotic core size while decreasing cap thickness and collagen deposition. Our previous studies also suggest Nogo-B attenuates growth factor induced VSMC proliferation and neointimal formation after vascular injury. This study aims to elucidate the role of VSMC-specific Nogo-B in plaque development and stability in mice.

Methods and Results:
A murine atherosclerosis model was generated by specifically deleting Nogo-B in VSMCs of ApoE
-/-
mice with a Cre-recombinase system (Nogo-B
iSMCKO
) and feeding a Western diet (1.25% cholesterol) for 14 weeks. VSMC-Nogo-B loss demonstrates a decreased necrotic core, minimized lesion size, and increased collagen content in aortic root lesions, all of which are indices of a more stable plaque phenotype. Nogo-B deletion in cultured VSMCs stimulated with β-CD cholesterol (20ug/mL) was performed to investigate phenotypic modulation and trans-differentiation
in vitro
. After cholesterol stimulation, expression of smooth muscle contractile genes was maintained in cells deficient in Nogo-B while decreasing in WT cells. Furthermore, VSMCs deficient in Nogo-B had decreased cholesterol uptake in cholesterol-loading experiments. Mechanistically, our results show Nogo-B modulates VSMC plasticity through transcription factor Krüppel-like factor 4.

Conclusions:
VSMC-specific Nogo-B deficiency attenuates atherogenesis and promotes stable plaque phenotypes. VSMC-specific Nogo-B deficiency may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.
Abstract 691: Cardio-renal Effect Of Dapagliflozin Alone Versus Dapagliflozin- Saxagliptin Combination In Type 2 Diabetes (t2dm) Subjects
Authors: Nandula Seshagiri Rao R , Sen Sabyasachi
Published: October 2023

Introduction:
Effect of Dapagliflozin (Dapa) and Dapagliflozin-Saxagliptin combination (Combo) was examined on peripheral blood derived CD34+ HSCs as a cellular CVD biomarker. Both Dapa (a SGLT2 receptor inhibitor) and Saxagliptin (a DPP4 enzyme inhibitor) are T2DM meds, but the combo has not been evaluated for cardio-renal risk.

Hypothesis:
We hypothesized that Dapa will improve the outcomes when compared to placebo and the Combo maybe even more beneficial.

Methods:
15 subjects were enrolled in 16 weeks, double-blind, three-arm, randomized placebo matched trial, with 10mg
Dapa
+ Saxa placebo (n=4), 10 mg Dapa + 5 mg Saxa (n=5)
Combo,
And Dapa placebo + Saxa placebo (n=6),
Placebo
groups. T2DM (30-70 yrs) with HbA1c of 7-10%, were included. CD34+ HSC number, migration, mRNA expression along with biochemistry and urine exosomes were measured. Data were collected at wks 0, 8, and 16. For stats, a mixed model regression analysis was used.

Results:
Significant HbA1c (p=0.0357) reduction was noted in Combo group vs Dapa alone and Placebo. hsCRP levels (P=0.0317) and IL-6, two important inflammatory molecules, were significantly reduced in both Dapa and Combo vs Placebo. Leptin levels decreased significantly in both Dapa alone (p=0.035) and Combo group(p=0.015), vs Placebo, however the Adiponectin levels were higher in Dapa alone group. EPC migration improved significantly in both Dapa alone (p=0.05) and Combo group (p=0.05) vs Placebo. Interestingly, urine exosome-based podocyte specific protein such as podocalyxin was actually higher in Combo vs Control.

Conclusions:
Several parameters showed significant improvement with both Dapa and Combo vs placebo. However, other than glycemic control the Combo didn’t offer any further benefit over Dapa alone in terms of HSC, biochemistry and podocyte markers. Of note, the combination may have deleterious effect on renal podocyte inflammation which needs to be examined in a larger study.