bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 526: Activated Platelets And Osteopontin Contribute To Arterial Stiffness And Are Involved In Abdominal Aortic Aneurysm
Authors: Rhee Yae Hyun , Ibing Wiebke , Deng Alicia , Spin Joshua , Tsao Philip S , Wagenhaeuser Markus
Published: October 2023

Significance:
Chronic inflammation is a consistent theme in the progression of cardiovascular disease including AAA. Chronic inflammation contributes to aortic wall weakening in AAA by disrupting the ECM and promoting aortic stiffness. Various cell types (fibroblast, macrophages, VSMCs, and circulating platelets) and inflammatory signaling cascades are implicated in aortic wall weakening. Osteopontin (OPN) is a well-known pro-inflammatory cytokine, which is secreted by pro-inflammatory cells and has been found upregulated in AAA patients. However, its mechanistical pathway in disease’ progression and which cells participate in OPN signaling are unclear. Interestingly, literature suggests that activated platelets can drive OPN secretion in proximal inflammatory cells. Therefore, the aim of this project was to determine whether a mechanistic link exists among the activated platelets and OPN production in cells associated with AAA that eventually increases local aortic stiffness and promotes AAA progression.

Methods:
Human platelets were isolated from donor blood and activated to obtain activated platelet supernatants (APS). M1 macrophages (M1), adventitial fibroblasts (Fib) and vascular smooth-muscle cells (VSMC) were exposed to APS.Full-length OPN and its fragments, generated by thrombin, plasmin and MMP-2 and -9 cleavage, were incubated with the same cell populations as mentioned above. Stiffness-related gene expression and MMP-2 and -9 activity was investigated utilizing qRT-PCR and gelatin zymography, respectively.

Results:
On exposure to APS, stiffness related gene expression as well as MMPs activity was significantly altered in the vascular cell types. Downstream effects of OPN on aortic stiffness presented changes in OPN treated cells compared to control.

Conclusion:
Our results indicate that activated platelets contribute to aortic stiffness and induction of vascular inflammation by modulating OPN expression, suggesting a potential role in AAA pathobiology.
Abstract 527: Redox Dysregulation Of Vascular Smooth Muscle Sirtuin-1 In Marfan Syndrome
Authors: Seta Francesca , Budbazar Enkhjargal , Sulser Ponce De Leon Sandra , Tsukahara Yuko , Liu Hanxiao , Huangfu Yuhao , Wang Yu , Yang Xiaoqiu , Wan Xueping , Goodman Jena , Seabra Pedro , Han Jingyan
Published: October 2023

Background:
Thoracic aortic aneurysms and dissections (TAAD) are life-threatening aortic conditions and a major cardiovascular complication of Marfan syndrome (MFS). We previously demonstrated that vascular smooth muscle sirtuin-1 (Sirt1), a lysine deacetylase, protects against aortic dissection by suppressing aberrant activation of matrix metalloproteinases (MMP2/9).

Objective:
In this study, we investigated whether Sirt1’s dysregulation contributed to TAAD in MFS.

Approach & Results:
Our results showed that oxidative stress markers (3-nitrotyrosine and 4-hydroxy-2-nonenal) were significantly elevated in aortas of MFS patients, compared to healthy controls. Similarly, reversible oxidative post-translational modifications (rOPTM) of protein cysteines, mainly S-glutathionylation, were dramatically increased in aortas of fibrillin-1 hypomorphic mice (Fbn1
mgR/mgR
), an established model of MFS prone to aortic dissection/rupture. We found that Sirt1 was one of these oxidatively modified proteins in Fbn1
mgR/mgR
aortas and smooth muscle cells (VSMCs). rOPTM of Sirt1 resulted in decreased Sirt1 activity, measured as levels of acetylated p53 and histone 3, and increased MMP2/9 activity, measured by in gel-zymography and in situ, in VSMCs and aortas. Deletion of glutaredoxin-1 (Glrx), a specific de-glutathionylation enzyme, increased rOPTM of Sirt1, rOPTM-mediated inhibition of Sirt1 activity, and MMP2/9 activity in VSMCs. On the contrary, overexpression of Glrx or of an oxidation-resistant Sirt1 mutant with an AAV prevented these changes in VSMCs. Lastly, VSMC-specific deletion of Sirt1 in Fbn1
mgR/mgR
(SMKO- Fbn1
mgR/mgR
) mice significantly worsened TAAD progression leading to aortic rupture in 50% of SMKO-Fbn1
mgR/mgR
mice, compared to 25% of Fbn1
mgR/mgR
mice.

Conclusions:
Our results strongly suggest that preventing rOPTM of Sirt1, thereby preserving its activity, may be a novel therapeutic strategy to prevent TAAD in individuals with MFS, for which currently no targeted therapies are available.
Abstract 530: Explantation Vs Graft Preserving Strategy Following Primary Endovascular Aneurysm Repair
Authors: Sultan Sherif , Acharya Yogesh , Hynes Niamh
Published: October 2023

Background:
Continuous sac expansion could result in rupture, requiring aggressive management with re-intervention to abolish the risk of rupture. Re-intervention could be achieved through salvage of the primary endograft through graft preserving strategy or explantation as necessary. We aim to scrutinise our 20 years of EVAR practice, strategies indications for management and re-interventions following primary endovascular aneurysm repair (EVAR).

Methods:
We performed 1555 aortic interventions over study period, including 910 EVARs. Using conventional and innovative diagnostic modalities with Prone contrASt enHanced computed tomography Angiography (PASHA), 136 endoleaks (ELs) were identified (15 type I, 98 type II; 18 type III; 5 type IV).

Results:
In total, 4.84% (44/910) patients underwent re-intervention post-primary EVAR; 18 EVAR GORE SalvAge Fabric Technique (ARAFAT), 12 double breastings, and 14 explantations
(Figure 1)
. Mean EL detection duration following primary EVAR was 53.3 ± 6.82 months, while re-intervention time was 70.2 ± 6.98 months. Mean sac size before the primary EVAR and re-intervention were 6.00 and 7.51 cm. Polyester (61.40%, 12/18) was the most commonly employed stent-graft material. Factors associated with the need for reintervention and likelihood of chronic fabric fatigue failure (CFFF) included the use of more than three modular stent-graft components (3.42 ± 1.31); with the proximal stent-graft diameter of 31.6 ± 3.80 cm and the use of iliac limbs more than 18 mm. We had one peri-operative mortality following explantation due to sepsis-induced multiorgan failure.

Conclusions:
Increasing complications post-EVAR necessitates developing and studying re-intervention strategies to salvage existing endograft and/or address graft-related complications. Re-intervention could be achieved through salvage of the primary endograft through graft preserving strategy or explantation as necessary.
Abstract 534: Peripheral Artery Disease Increases Platelet Activity and Promotes a Prothrombotic Platelet Phenotype
Authors: De Moura Virginio Vitor , Drenkova Kamelia , Schlamp Florencia , Luttrell-Williams Elliot , Barrett Tessa A , Berger Jeffrey
Published: October 2023

Background:
Peripheral artery disease (PAD), characterized by lower extremity atherosclerosis, increases the risk of cardiovascular morbidity and mortality. Among their many functions, platelets modulate hemostasis, thrombosis, inflammation, and angiogenesis. Previously, we noted the potent pathogenic role of platelets in PAD. However, how vascular dysfunction impacts the platelet phenotype is not fully understood. Using a mouse model of PAD (hindlimb ischemia), we investigated the platelet phenotype and transcriptome.

Methods:
Eighteen C57BL/6J mice (10 weeks) underwent femoral artery ligation (HLI) or sham surgery. Platelet activity, P-selectin, JON/A expression, and platelet-leukocyte aggregation, was assessed at day 0, 3, 7, 10, 14, and 21. At day 21, we performed the FeCl3-induced thrombosis model and assessed time to vessel occlusion. To investigate platelet-relevant mechanisms, we induced HLI or sham surgery in 20 mice and collected platelets for RNA sequencing (day 12).

Results:
Our acute PAD model resulted in >90% drop in blood flow after the procedure with ≈50% recovery at day 21. Following the procedure, platelet activity, defined by PAR4 agonist stimulated P-selectin expression (P=0.001), αIIbβ3 activation (JON/A expression, P=0.002), platelet-neutrophil aggregation (P<0.001) and platelet monocyte aggregation (P=0.047) increased over time in the HLI vs sham group. Mice in the HLI group had faster occlusive carotid thrombosis than the sham group (P=0.026). Comparing the platelet transcriptome in the HLI vs sham group noted 895 differentially expressed transcripts (P<0.05) between groups (516 up-, 79 down-regulated). Pathway analysis between groups demonstrated enrichment of inflammatory pathways, including interleukin-1 mediated signaling (P=0.02), apoptotic process (P=0.02), noncoding RNA processing (P=0.02), and signaling receptor binding (P=0.03).

Conclusion:
In our murine model of acute PAD, we found increased platelet activity, a proinflammatory platelet transcriptome, and a prothrombotic state. These data highlight the feasibility of the HLI model to further investigate platelet-associated mechanisms that underlie PAD.
Abstract 537: Inhibiting Glycolytic Determinants In Hypoxic Endothelial Cells Promotes Angiogenesis In Preclinical Peripheral Artery Disease
Authors: Ganta Vijay C , Kuppuswamy Siva , Wood Madison , Reddy Megna , Thounaojam Menaka
Published: October 2023

PFKFB3 is a critical regulator of Glycolysis, the primary metabolic pathway in endothelial cells (ECs). Recent reports have shown that PFKFB3 regulates the angiogenic potential of ECs. However, in our recent experiments, we paradoxically observed that silencing ischemia-induced PFKFB3 expression in hypoxia serum starved (HSS) ECs significantly enhanced their angiogenic potential on growth factor reduced Matrigel (GFRM) vs. control siRNA (P<0.05). To determine the role of PFKFB3, we inhibited PFKFB3 by electroporating control or PFKFB3 shRNA into C57BL/6 ischemic muscle. Interestingly, inhibiting PFKFB3 showed a numerical increase in perfusion recovery and a significant increase in vascular density (P<0.05) at day 21 post-HLI. These data suggested that inhibiting PFKFB3 allows the activation of other metabolic pathways that can induce perfusion in peripheral artery disease (PAD). To determine the metabolic pathways that are induced upon PFKFB3 inhibition, we performed RT
2
Profiler qPCR arrays consisting of 84 genes that regulate glucose metabolism in PFKFB3 silenced HSS ECs, PFKFB3 inhibited ischemic muscle samples, and respective controls. qPCR arrays showed UDP-glucose pyrophosphorylase 2 (UGP2, the only enzyme that converts glucose 1-phosphate to UDP-glucose for use in anabolic pathways) as a common gene that is induced in both PFKFB3 silenced ECs and PFKFB3 inhibited ischemic muscle. Next, to determine the role of UGP2 in regulating perfusion recovery, we delivered the UGP2 antibody (100μg/100μl, i.m.) at day 0, 3, 7, and 14 in C57BL/6 ischemic muscle. UGP2 inhibition significantly decreased perfusion recovery and vascular density (P<0.05) at day 21 post-HLI vs. IgG indicating that UGP2 plays a causal role in regulating perfusion recovery.
In vitro
, silencing UGP2 in PFKFB3 silenced HSS ECs significantly decreased (P<0.05) the PFKFB3-induced angiogenic potential of HSS ECs on GFRM. Furthermore, supplementing UDP-Glucose (product of UGP2) to D-Glucose in HSS ECs significantly increased (P<0.05) the angiogenic potential of HSS ECs higher than D-Glucose alone on GFRM. These findings indicate that inhibiting ischemia-induced PFKFB3 expression in HSS ECs allows activation of UGP2 that can enhance perfusion recovery in preclinical PAD.
Abstract 539: Mini-swine Model Of Hindlimb Ischemia On A Background Of Metabolic Syndrome Mimics Peripheral Artery Disease And Claudication
Authors: Hakim Ali H , Al-Gahmi Al-Murtadha , Cartwright Sara , zhu zhen , Li Shuai , Schieber Molly , Kim Julian K , Casale George , CARLSON MARK A , Pipinos Iraklis I
Published: October 2023

Background:
There is an ongoing need for an animal model of PAD that is similar to the human patients in its size, anatomy, physiology, comorbidities and suitability for invasive/non-invasive testing and treatment.

Methods:
Three months old Ossabaw swine (N=9) were fed a high fat/fructose/sodium diet for 8 weeks. Right hindlimb ischemia was then induced (N=6, 2 female) using Right Superficial Femoral Artery (RSFA cutdown, right external iliac artery endovascular (R EIA) coil embolization/occlusion and RSFA resection (T0) vs sham (N=3, 1 female). At 4 weeks (T4), all swine underwent R carotid cutdown, 2-D angiography, and bilateral(b/l) gastrocnemius open biopsy. At 8 weeks (T8) swine underwent L carotid cutdown, 2-D angiography, b/l sampling of the gastrocnemius. During all procedures, b/l ankle brachial indices (ABIs), and tissue oximetry on the calf and thigh were measured. All swine underwent weekly measurement of their treadmill walking distance. Gastrocnemius muscle mitochondrial respiration was measured with an Oroboros Oxygraph-2k.

Results:
Blood draws showed hyperlipidemia and hyperglycemia in all swine at T0, T4, and T8 after a 12-hour fasting period. Hindlimb ischemia lowered right-sided ABIs at T4 and T8 by 45 and 39%
(p<0.001)
, and tissue oximetry on R calf (28 and 18%
p<0.01)
and thigh (22 and 18%
P<.01
) with no differences in the sham. Ischemia reduced treadmill times from T0 to T8 from 24 to 9.4 minutes
(p<0.01)
vs sham increase from 23.3 to 32.2
(p<0.09)
. R internal Iliac artery had the largest growth of cross-sectional area at T4 and T8 (111.7 and 115.2%) compared to L EIA (40.6 and 46.1% -
p< 0.02
and
<0.5
) and aorta (14.5 and 29% p
<0.02
and
<0.02
).
R profunda artery angiographic appearance was delayed at T4, which improved at T8 after normalization of the timing ratio from right to left (3.25 vs 2.3
p<0.008
). Mitochondrial oxygen consumption in the ischemic right gastrocnemius was consistently lower ex-vivo (21.96±6.41 vs 15.11±11.1
p<0.03
) with no differences in the sham. We had no fatalities.

Discussion:
We present a porcine model of hindlimb ischemia (iliofemoral artery occlusion) which recapitulates key aspects of the pathophysiology of human PAD/ claudication, and can support the development of new therapies for PAD.
Abstract 540: Satellite Cell Increase Vascular Growth Through Paracrine Signaling Mechanisms
Authors: Yu Tao , Oh Dong Gun , Jean-Baptiste Christopher , Brice Beyonce , Ren Tianjiao , Hansen Laura M
Published: October 2023

Peripheral artery disease is a major health burden that is often underdiagnosed and undertreated. Currently treatment strategies remain limited to medical therapy for cardiovascular risk factors and lifestyle modifications including exercise therapy. Patients that are refractory to these require surgical intervention or amputation. Therefore, the need remains to develop more effective minimally invasive therapy options. More extensive collateral vessel networks have been correlated to better patient outcomes; thus, this study sought to understand and promote vascular growth through the satellite cells. Satellite cells are skeletal muscle progenitor cells, and due to the effectiveness of exercise therapy, we hypothesized that they play a role in improving vascular supply to ischemic tissue. Specifically, we hypothesized that satellite cells produce growth factors that modulate vascular growth through paracrine signaling. To test this hypothesis, we performed in vitro angiogenesis assays with cultured primary satellite cells. Migration assays demonstrated that satellite cells produce chemokines which increased smooth muscle migration (3.5 fold) and endothelial cell migration (2.8 fold) over control conditions (n = 4, p<0.05). We also performed aortic ring assays using satellite cell conditioned media. The proportion of sprout-presenting aortic rings was increased in the conditioned media group (88.24%) compared to control denatured conditioned media (33.33%) and control (58.33%, n=2-6). Finally, we tested the vasculogenic capacity of these cells in a unilateral hind limb ischemia (HLI) model of vascular growth. Cells were delivered into the hypoxic environment at day 2 post surgery. The satellite cell group showed significant higher perfusion ratios compared to the empty capsules and saline groups (n=15, p<0.05) up to 21 days post-surgery. Luciferase imaging demonstrated that cells had a viability of over 25% out to day 14 post HLI. Taken together these results show that satellite cells promote vascular growth both in vitro and in vivo through paracrine signaling. Future work will explore the signaling pathways that produce these changes and further test the efficacy of satellite cells as a cellular therapy in a model of chronic ischemia.
Abstract 543: Leukadherine-1 Improves Neovascularization In A Mouse Model Of Hindlimb Ischemia
Authors: Lassance-Soares Roberta , Falero-Diaz Gustavo , Vazquez-Padron Roberto I , Gupta Vineet
Published: October 2023

Objective:
Critical limb ischemia (CLI) is the end stage of peripheral artery disease characterized by rest pain, gangrene, and amputations. CLI is secondary to severe stenosis/occlusion of arteries and to the failure of compensatory neovascularization; and immune cells play essential role on it. Mac-1 integrin (also known as CD11b/CD18) is a leukocyte surface receptor that contributes to monocytes adhesion and infiltration after vascular injury controlling to excessive inflammation. The goal of this study is to evaluate the pharmacological activation of Mac-1, by a compound called Leukadherin-1 (LA1), in neovascularization after limb ischemia.

Methods:
C57Bl6 mice were divided in three groups: control (vehicle), 1 dose LA1 (2 mg/kg), and 2 dose LA1 (2 doses of 2 mg/kg). Injections of LA1 or vehicle (ip) were given for 14 days starting at hindlimb ischemia surgery day. Blood flow recovery, angigogenesis, and arteriogenesis were measured on these groups 14 days after hindlimb ischemia. Single cell RNA sequencing of ischemic limb was performed on control and 2 dose LA1 groups 7 days after hindlimb ischemia. For proliferation, murine smooth muscle cells (SMCs) were cultured with murine monocytes exposed or not to LA1 and cell counting was performed 24h and 48h after treatment.

Results:
Mice who received LA1 treatment showed an improvement in blood flow recovery and remarkable angiogenesis and arteriogenesis. Interestingly, cell clustering findings from limb ischemic muscle single cell RNA-seq, demonstrated increased percentage of B cells in the ischemic muscle on 2 dose LA1 group compared to control group. Moreover, our in vitro data demonstrated that 48 hours after LA1 exposure SMCs proliferation is increased.

Conclusions:
Our data demonstrate that LA1 treatment improves neovascularization after hindlimb ischemia which might be associated with greater mobilization of B cells to the ischemic tissue and higher proliferation rate of SMCs.
Abstract 546: MicroRNA-1282 As A Cis-Antisense Regulator Of Angiogenesis In Diabetic Critical Limb Ischemia
Authors: Sachan Madhur , Rawal Shruti , Wu Winona , Vaz Camila , McCoy Michael , Cheng Henry , Perez-Cremades Daniel , Goodney Philip P , Creager Mark A , Sebatine Marc S , Bonaca Marc P , Feinberg Mark W
Published: October 2023

Introduction:
Peripheral artery disease (PAD) is an atherosclerotic occlusive disease that leads to reduced blood flow to the lower extremities and endothelial dysfunction. Some patients develop critical limb ischemia (CLI), a severe manifestation of PAD defined as chronic ischemic rest pain with higher risk of amputation. Major risk factors for both PAD and CLI is type 2 diabetes. However, the pathophysiological contribution of microRNAs (miRNAs), in diabetic CLI progression remains poorly elucidated.

Methods:
By overlapping plasma miRNA sequencing in PAD patients that develop CLI with a mouse model, miRNAs were identified in the progression of diabetic limb ischemia. In this study, we describe miR-1282, a previously uninvestigated miRNA that was the highest upregulated miRNA in high-risk diabetic CLI human subjects.

Results:
Preliminary studies uncover a mouse ortholog of human miR-1282, a cis-antisense miRNA, that strongly inhibits the expression of SERF2 (small EDRK-rich factor 2), a protein-coding gene located on the opposite DNA strand in both human and mouse endothelial cells. SERF2 is predicted to affect the formation of aggresomes, the inclusion bodies of misfolded proteins; however, its molecular function is also poorly understood. MiR-1282 expression was decreased by diabetic stimuli such as hypoxia and glucose; and its expression inversely correlated with SERF2 expression in endothelial cells. Our in vitro studies indicate that miR-1282 mimic overexpression or SERF2 siRNA-mediated inhibition reduced a range of endothelial angiogenic assays and inhibits apoptosis. Proteomics by tandem mass-tag mass-spectrometry and transcriptomics by RNA-Seq suggest that miR-1282 likely regulates protein quality control system (SERF2, BAG1, BAG3, and HSPB8) and regulated cell death (CASP3, SLC7A11, and CTH), among others. Intriguingly, intramuscular administration of miR-1282 significantly improved blood flow recovery by laser doppler imaging in the hindlimbs of diabetic db/db mice compared to control injected mice.

Conclusion:
Taken together, the cis-antisense regulatory network of miR-1282-SERF2 axis reveals novel insights in the progression of diabetic CLI.
Abstract 548: Microvessel Oxidative Stress And Damage And Its Relation To Muscle Degeneration And Fibrosis In The Ischemic Limbs Of Patients With Peripheral Artery Disease
Authors: Schieber Molly , Li Shuai , Fuglestad Matthew , Shields Christina , Kim Julian K , Sachdev Ulka , Pipinos Iraklis I , Casale George
Published: October 2023

We have previously documented myofiber oxidative damage and degeneration, interstitial fibrosis, and abnormal microvascular architecture in the calf muscle of patients with Peripheral Artery Disease (PAD). Our objective was to evaluate baseline oxidative stress (Heme oxygenase 1 [HO-1] expression) and oxidative damage (carbonyl adducts) in the microvessels (≤10.5 μm) of patients with PAD in association with myofiber pathology and fibrosis. We hypothesized that microvessel oxidative stress and accumulated oxidative damage in PAD muscle at baseline 1) are greater than in non-PAD control muscle and 2) are associated with myofiber pathology and fibrosis.

Methods:
Oxidative stress was assessed as expression of HO-1, regarded as the most important molecule protective against cellular oxidative stress. Oxidative damage was determined as abundance of cellular carbonyl adducts. The gastrocnemius was biopsied for quantification of HO-1 and carbonyl content in both myofibers and microvessels in claudicating patients with PAD (Fontaine Stage II; n=12), in patients with non-healing ulcers (Fontaine Stage IV (n=13), and in non-PAD controls (n=16; recruited from a vascular clinic by a vascular surgeon).

Results:
Microvessel oxidative damage was greater in Stage IV (1,616,286 gsu*μm
2
) compared to Stage II (1,138,707gsu*μm
2
, p=0.022) and controls (817,384 gsu*μm
2
, p=0.0001), and in Stage II compared to controls (p=0.004). Oxidative stress was greater in Stage IV (273,269 gsu*μm
2
) and Stage II patients (228,936 gsu*μm
2
) compared to Controls (175,471 gsu*μm
2
; p=0.0017 and p=0.0173, respectively). Oxidative damage in microvessels of controls and Stage II patients predicted myofiber oxidative damage (87,457,944 gsu*μm
2
; r=0.688, p=0.0065) and (146,730,298 gsu*μm
2
; r=0.685, p=0.0139), respectively, and interstitial fibrosis (r=-0.753, p=0.0047) in Stage II. Microvessel response to oxidative stress (HO-1) predicted myofiber damage (r=0.614, p=0.007) in controls.

Conclusion:
Microvessel oxidative damage is increased in the ischemic calf muscle of PAD patients, advances with stage, and correlates with myofiber oxidative damage and interstitial fibrosis in Stage II patients. Microvessel pathology may be a central mediator of the myopathy of PAD.
Abstract 555: Technique For Testing Drug-coated Balloons In Deep Veins In Vivo
Authors: Moreno Rocha Oscar Y , Kumar Kiran , Rocco Sabrina , Sharma Sriganesh , Luke Catherine , Durham Laura , Clay Amber , Bamezai Sharika , Myers Daniel D , Wakefield Thomas , Henke Peter , Obi Andrea T
Published: October 2023

Background:
Chronic deep venous occlusions, leading to outflow obstruction in dialysis patients and post thrombotic syndrome (PTS) in DVT patients, are often treated with balloon venoplasty and stenting to restore flow and improve limb outcomes. In stent restenosis and occlusion are primary reasons for late intervention failure. mTOR inhibitors might impact thrombotic and fibrotic pathways on venous endothelial cells (VEC).

Methods:
Immortalized murine VEC (EOMA, ATTC #CRL-2586) were stimulated with sirolimus (1ng) and vehicle (DMSO) performing quantitative real-time PCR (qRT-PCR). Sprague Dawley rats (n=4, 250-400g) were anesthetized (isoflurane). IVC diameter measured by ultrasound. Infrarenal IVC was exposed using a midline laparotomy, side branches ligated, posterior venous branches cauterized, and micro-clips temporarily placed. A 0.014 sharpened guidewire with a DCB back-loaded was inserted retrograde into the infrarenal IVC. After DCB inflation/deflation, the entire system was removed and U-stitch tightened for hemostasis.

Results:
Sirolimus blunted transcription of cellular adhesion molecules (CAMs) and mediators of EndMT. (Fig 1) We achieved total stasis to replicate a vein wall reaction to thrombosis and further EndMT changes, with 75% survival in initial proof-of-concept experiments. (Fig 2)

Conclusion:
IVC retrograde cannulation via U-stitch and sharpened guidewire is a viable animal model for testing venous DCBs. Sirolimus inhibits VEC CAMs critical for thrombus initiation and EndMT mediators involved in PTS remodeling. Future DCB research on adhesive and fibrotic phenotypes is required.
Abstract 586: CCR2+ Macrophages Are Elevated In Individuals With Aortic Aneurysmal And Occlusive Disease
Authors: Hafezi Shahab , Arif Batool , Zaghloul Mohamed , Lin Chieh-Yu , Liu Yongjian , Gropler Robert J , Zayed Mohamed A
Published: October 2023

Background:
Mounting evidence suggests that inflammation plays a key role in arterial pathophysiology. C-C chemokine receptor type 2 (CCR2) is essential for recruitment of macrophages to regions of inflammation. Therefore, we hypothesized that CCR2 and macrophage content will be elevated in human aortic tissue from individuals with aneurysmal or occlusive disease.

Methods:
We conducted a retrospective review of prospectively collected human aortic tissue from our institutional review board-approved vascular biobank to include male, non-diabetic cases with active or recent history of smoking. Diseased aortic tissue was collected from individuals with aortic occlusive disease (AOD), ruptured abdominal aortic aneurysm (rAAA), and non-ruptured AAA. Normal non-diseased abdominal aorta (NAA) was harvested from organ donors. All tissue were fixed in formalin, paraffin-embedded, sectioned, and immune-stained for CCR2 and CD68 (macrophage marker). Two blinded observers evaluated the content of CCR2+ and/or CD68+ cells in three randomly selected 0.1mm2 regions of interest within the arterial intima and media. Cell counts were averaged and analyzed.

Results:
A total of 19 aortic tissue samples were studied; including 5 NAA, 8 AAA (56 to 89 mm diameter), 3 rAAA, and 3 AOD. We observed significantly higher CCR2+ cells in rAAA compared with NAA (p < 0.05) (Fig, A). In addition, patients with AAA demonstrated higher CD68+ and CCR2+ CD68+ cells (p < 0.05) (Fig, B, C). Interestingly, only up to half of CCR2+ cells that were observed in diseased aortic tissue (AAA, rAAA, and AOD) co-localized with CD68+ cells, indicating that CCR2 may also be expressed by other cell types.

Conclusions:
CCR2-expressing macrophages are increased in diseased aortic tissue, and CCR2 cellular content is associated with AAA rupture risk and appears to correlate with disease severity. Therefore, CCR2 represents a potential tissue biomarker for aortic disease molecular imaging and targeted therapy.
Abstract 592: Marfan Syndrome Mice Fed On High Fat Diet Have An Altered Metabolism
Authors: Yap Carmen , Li Siyu , Hoogeland Myrthe , Bakker Iris , Kooijman Sander , De Waard Vivian
Published: October 2023

Introduction:
Marfan syndrome (MFS) is a genetic disorder, caused by different mutations in the fibrillin-1 gene (
FBN1
) and affects the connective tissue. Patients suffer from a wide range of clinical symptoms at the cardiovascular, skeletal and ocular level. However, MFS patients are also often characterized as very slender. Body weight gain in these patients can be a challenge, and the reason for this is still not fully understood.

Objective:
To determine if individuals with MFS have an altered metabolism.

Methods:
9-11 weeks old male Fbn1
C1041G/+
(MFS) mice and wild-type littermates were given either a chow, high cholesterol diet (HCD) or a high fat diet (HFD), for 12 weeks. These mice were then housed individually in a metabolic cage for 1 week before euthanasia was performed.

Results:
HFD resulted in higher body weights compared to chow diet, and the HFD groups became insulin resistant as measured by the intraperitoneal glucose tolerance test. However, body weight gain was not different in this MFS mouse model, as compared to WT mice, regardless of diet. The HFD groups had increased fat weight compared to chow groups. Interestingly, MFS mice had an enhanced heart weight compared to WT mice, independent of the diet. As expected, aortic root diameters were enlarged in MFS compared to WT mice. However, in the ascending aorta there was a significant increase in diameter in the WT HFD as compared to WT chow, indicating that enhanced lipid levels promote aortic growth. Metabolic cage data showed that the HFD groups had a lower respiratory exchange ratio (RER), indicating that HFD groups used fat as a predominant source of fuel and that chow groups used primarily carbohydrates as an energy source. MFS mice had overall lower RER compared to WT mice, revealing that MFS mice use more lipids under any condition, most likely to manage the significantly elevated activity in the MFS mice.

Conclusion:
Taken together, MFS mice seem metabolically different.
Abstract 597: TRAIL-TRAIL-R Ligation Regulates EC-pericyte Crosstalk To Generate Stable Microvessel Networks In Ischemia
Authors: Cartland Sian P , Patil Manisha , Boccanfuso Lauren , Kelland Elaina , Patrick Ralph , Manuneedhi Cholan Pradeep , Su Peter , Alwis Imala , Ganss Ruth , Harvey Richard P , Griffith Thomas S , Powell Joseph , Patel Sanjay , Kavurma Mary M
Published: October 2023

Endothelial cell (EC)-pericyte crosstalk is essential for generating stable capillary networks. Capillary function and development is disrupted in CVD, and processes mediating this are poorly understood. TNF-related apoptosis-inducing ligand (TRAIL) stimulates blood vessel development in pre-clinical models, while circulating levels are suppressed in CVD patients. The contribution of EC-specific TRAIL to angiogenesis in ischemia is unknown. To address this, an EC-specific TRAIL knockout (

Trail
EC-/-

) was generated. Compared to

Trail
EC+/+

,

Trail
EC-/-

mice had ~60% reduction in plasma TRAIL, revealing the endothelium as a significant source of TRAIL in the healthy circulation. Angiogenesis was quantified in the Matrigel plug, aortic sprouting and hindlimb ischemia (HLI) models. EC/pericyte content in plugs were ~50-60% less in

Trail
EC-/-

than

Trail
EC+/+

mice, with a ~50% reduction in mRNA expression of angiogenesis/pericyte markers.

Trail
EC-/-

aortic segments had reduced microvascular sprouts in hypoxia, and ECs lacking TRAIL had an impaired ability to form tubules and recruit pericytes. CD31
+
SMA
+
microvessel numbers (measure of EC-pericyte interaction) were significantly reduced in

Trail
EC-/-

ischemic limbs, associating with decreased expression of pericyte markers and ~50% reduction in blood perfusion. Similar findings were observed in

Trail-receptor
-/-

(

Trail-R
-/-

) mice. In contrast, administration of an agonistic anti-mouse TRAIL-R mAb (MD5-1), restored blood perfusion and increased EC-pericyte content in ischaemic

Trail
EC-/-

limbs. MD5-1 also stimulated aortic sprouts. scRNA-seq data identified 5 EC clusters, 2 of which were markedly altered in

Trail
EC+/+

but not in

Trail
EC-/-

ischemic limbs and multiple EC-pericyte interactions dependent on TRAIL were identified. In humans, TRAIL induces apoptosis through TRAIL-R1 and TRAIL-R2. Importantly, TRAIL and TRAIL-R2 mRNA and cell surface expression was augmented, and TRAIL physically interacted with TRAIL-R2 (but not TRAIL-R1) under hypoxic conditions. These studies provide a novel pathway mediating angiogenesis and vessel stabilisation. TRAIL-R agonists could be used as a potential new therapy to stimulate stable capillary networks in peripheral artery disease.
Abstract 603: Type 2 Cytokines Promote Angiogenesis In Ischemic Muscle Via Endothelial Il-4rα Signaling
Authors: Li Huixian , Wong Wing Tak
Published: October 2023

Peripheral arterial disease (PAD) is one of the leading causes of cardiovascular morbidity and mortality worldwide, yet current trials on therapeutic angiogenesis remain suboptimal. Type 2 immunity is critical for post-ischemic regeneration, but its regulatory role in revascularization is poorly characterized. Here, we show that type 2 cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13), are the key mediators in post-ischemic angiogenesis. IL-4/IL-13-deficent mice exhibited impaired reperfusion and muscle repair in experimental model of PAD. Surprisingly, we found that deletion of IL-4Rα in the endothelial compartment, rather than the myeloid compartment, led to remarkable revascularization impairment. Mechanistically, IL-4/IL-13 promoted endothelial cells proliferation, migration and tube formation via IL-4Rα/STAT6 signaling. Furthermore, attenuated IL-4/IL-13 expression was found to be associated with the angiogenesis deficit in the setting of diabetic PAD, while IL-4/IL-13 treatment rescued this defective regeneration. Our findings reveal the therapeutic potential of type 2 cytokines in treating patients with muscle ischemia.
Abstract 604: Distinct Fibroadipogenic Progenitor Populations And Trajectories Characterize Regenerative And Non-regenerative Responses In The Ischemic Limb
Authors: Xu Yueyuan , Peters Derek , Fei Kaileen , Dai Qunsheng , Garrett Brianna , Otto James , Kontos Christopher D , Diao Yarui , Southerland Kevin
Published: October 2023

Introduction:
Skeletal muscle possesses the capacity for self-repair. This ability is impaired in chronic limb threatening ischemia (CLTI) patients. Myopathy in CLTI patients is associated with adverse clinical outcomes. Skeletal muscle regeneration requires multicellular interactions between muscle progenitor cells and other cell types. Fibroadipogenic progentiors (FAPs) represent a support cell type in the skeletal muscle niche; they modulate inflammatory and myogenic responses via the secretion of a paracrine factors. Hence, FAPs are an attractive therapeutic target for myopathies. We sought to characterize the temporal dynamics and transcriptional heterogeneity of FAPs in failed and successful skeletal muscle regeneration in the ischemic limb.

Methods:
We analyzed the FAP regenerative response in BALB/c (non-regenerative) and C57BL/6 (regenerative) mice undergoing hindlimb ischemia using single-cell RNA sequencing spanning 4 distinct time points. We also performed RNA velocity analysis to determine the cellular dynamics of FAP sub-populations during the repair process.

Results:
We identified 10 FAP populations in the ischemic limb, including specific regenerative and non-regenerative FAP sub-clusters. Gene ontology (GO) enrichment analysis demonstrated distinct transcriptional programs between regenerative and non-regenerative FAPs. Regenerative FAPs were enriched in extracellular matrix and angiogenesis pathways, while non-regenerative FAPs were enriched in chemotactic pathways. RNA velocity analysis demonstrated divergent FAP cellular trajectories.

Conclusions:
Our study maps the dynamic FAP response in the ischemic limb at single-cell resolution. We identify distinct transcriptional programs and cellular trajectories that are associated with failed and successful skeletal muscle repair. This provides a resource to investigate FAP mediated mechanisms of skeletal muscle regeneration in response to limb ischemia.
Abstract 607: Activation Of Yes-associated Protein(yap)/pdz-binding Motif (taz) Signaling By Angiotensin II Contributes To Hypertensive Cardiacand Vascular Remodeling
Authors: Xu Qian , Zhuo Kunping , Zhang Xiaotian , Ren Haolin , Zhang Lu , Liu Limin , Zhou MingSheng
Published: October 2023

Vascular remodeling is pathogenic basis of hypertension and end organ injury, proliferation of vascular smooth muscle cells (VSMCs) is central to vascular remodeling. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key effectors of Hippo pathway and crucial for control of cell proliferation, apoptosis and differentiation. The present study investigated the role of YAP/TAZ in cardiac and vascular remodeling of angiotensin II-induced hypertension. Ang II induced YAP/TAZ activation in the heart and aorta, which were prevented by YAP/TAZ inhibitor verteporfin. Treatment with verteporfin significantly reduced Ang II-induced cardiac and vascular hypertrophy with a mild reduction in systolic blood pressure (SBP), verteporfin attenuated Ang II-induced cardiac and aortic fibrosis with the inhibition of transform growth factor (TGF)b/SMAD fibrotic signaling. Ang II induced Rho A, extracellular signal-regulated kinase 1/2 and YAP/TAZ activation in VSMCs, either Rho kinase inhibitor fasudi, or ERK inhibitor PD98059 suppressed Ang II activation of YAP/TAZ . Verteporfin or fasudi also inhibited Ang II-induce VSMC proliferation and fibrotic TGFβ1/SMAD1/2 pathway. These results demonstrate that Ang II activates YAP/TAZ via Rho kinase/ERK1/2 pathway in VSMCs, which may contribute to Ang II-induced cardiac and vascular remodeling in hypertension. Our results suggest that YAP/TAZ plays a critical role in hypertension and hypertensive end organ injury, targeting YAP/TAZ pathway may be a new strategy for prevention and treatment of hypertension and cardiovascular diseases.
Abstract 613: Association Between Left Ventricular Ejection Fraction And Thrombus Resolution: Based On A Prospective Study In Patients With Left Ventricular Thrombus Treated By Rivaroxaban
Authors: Yang Qing , Liang Yan
Published: October 2023

Background:
Previous studies have demonstrated that poor left ventricular ejection fraction (LVEF) levels are independently associated with left ventricular (LV) thrombus formation. However, the relationship between LVEF and LV thrombus outcomes remains sparse.

Methods:
We conducted a prospective, interventional, single-arm study from Oct 2020 to June 2022 in Fuwai Hospital, China. Patients with a history of LV thrombus < 3 months were included and then assigned to rivaroxaban. Contrast-enhanced echocardiography (CE) was used at the baseline and follow-up visits to measure thrombus and LVEF (Simpson's methods). The primary efficacy outcome was the rate of thrombus resolution at 12 weeks. Restricted cubic splines were applied to depict the relationship between LVEF levels and thrombus resolution. The hazard ratio (HR) and 95% confidence intervals (CIs) were estimated using Cox regression models.

Results:
A total of 64 patients with complete CE results were analyzed for efficacy outcomes. The median LVEF was 24 (20, 31)%. Patients with baseline LVEF ≥ 24% had a less likelihood of thrombus resolution compared to those with LVEF < 24% (adjusted HR 0.40, 95%CI 0.22 - 0.72, P = 0.002). According to cumulative event probability curves derived from Kaplan-Meier estimates at 12 weeks, patients with LVEF < 24% had higher resolution rates than those with LVEF ≥ 24% (P = 0.0015). Furthermore, after adjusting for characteristics such as diagnosis and therapy, patients who improved their LVEF by more than 10% at 12 weeks had a greater rate of resolution (HR 1.05, 95% CI 0.45 - 2.41, P = 0.914).

Conclusions:
Among patients with LV thrombus who were treated with rivaroxaban, we reported a higher thrombus resolution rate in patients with LVEF < 24% than those with LVEF ≥ 24% at baseline, though no significant difference was observed in the relationship between LVEF changes at follow-ups and thrombus resolution. Large randomized controlled studies are required to generalize our findings.
Abstract 620: Hsf-1 Enhances Cardioprotective Potential Of Stem Cells Via Exosome Biogenesis And Their Mirna Cargo Enrichment.
Authors: Guru Sameer A , Saha Progyaparamita , Mishra Rachana , Ge Zhi-Dong , Chen Ling , Tulshyan Antariksh , Simms Lydia , Ciolak Emily , Bailey Jeanette , Krishnan Swetha , Stefanowicz Artur , Mehta Vivek , Bilewska Agata , xuebin Fu , Gunasekran Muthu Kumar , Kaushal Sunjay , Sharma Sudhish
Published: October 2023

Introduction:
Stem cells work via different mechanisms of which paracrine mechanism is able to justify most of the effects. Therefore, identifying the control arms for paracrine cocktail production is necessary to tailor stem cell functions.

Objective:
This study shows how HSF1 determines stem cell potential to repair ischemic heart via affecting production of paracrine factors including exosome and their miRNA cargo.

Methods and Results:
CRISPR-Cas9 gene editing tool was used to knock out HSF1 gene in nMSCs while lentiviral hsRNA constructs for HSF1 were used to overexpress it in aMSCs. We identified that HSF1 knockout resulted in reduced cardiac regeneration abilities in nMSCs while its overexpression in aMSCs had opposite effects as depicted by functional characteristics including LVEF, LVEDV, LVESV and LVFS in our rat ischemic model. Further, we found that exosome number in nMSCs decreased significantly after HSF1 knockout while it went up in aMSCs after HSF1’s overexpression as determined by NanoSite particle tracking analysis. The exosomes from nMSCs (nEXOs), aMSCs (aEXOs) and their genetically modified versions (nKO-EXOs) and aOE-EXOs) respectively) were analysed for miRNA cargo using RT-PCR. nKO-EXOs showed significantly reduced candidate miRNA expression (miR-145, miR-146, 199-3p, 199b and miR-590) than nEXOs while aOE-EXOs had increased candidate miRNA expression compared to aEXOs. Since HSF1 does not bind to RNA molecules, we hypothesised that YBX1, another protein highly expressed by nMSCs than aMSCs and has RNA binding nature, under the influence of HSF1 sorts miRNAs into exosomes. YBX1 expression was found to be controlled by HSF1 as shown by RT-PCR and western results. HSF1 controls YBX1 expression, at transcriptional level, is strongly supported by our finding that YBX1 promoter possesses heat shock elements (HSEs) as HSF1 binding sites. We further went on to see whether YBX1 alteration affects miRNA loading of exosomes and it was demonstrated that absence of YBX1 resulted in accumulation of candidate miRNAs into the cells.

Conclusion:
Together, our data shows that HSF1 plays a critical role in determining the regenerative potential of stem cells. HSF1 does that by affecting exosome biogenesis and miRNA cargo sorting via YBX1 protein.
Abstract 621: A Congenital Right Coronary-Pulmonary Artery Fistula In A 61-Year-Old Man; An Uncommon Cause Of Ischemia
Authors: Kalsoom Sidra , Zaman Muhammad Adnan , Khan Muhammad Shoaib , Sharma Dinesh
Published: October 2023

Introduction:
Coronary pulmonary artery fistulas (CPAF) are abnormal communications between the coronary arteries and major vessels or even cardiac chambers. Most patients with these anomalies remain asymptomatic, however, symptoms and complications may develop with increasing age. We present an interesting case of a 61-year-old man who had a congenital coronary pulmonary fistula presenting with chest pain.

Case Presentation:
A 61-year-old male with a history of chronic kidney disease, hypertension, and pulmonary embolism presented with ongoing atypical chest pain. Initial EKG, troponins, and echocardiography were unremarkable. The cardiac MPI SPECT stress test demonstrated a fixed perfusion defect of the inferior wall. This was followed up with a coronary CT angiography (CTA), which revealed an AV fistula from the proximal right coronary artery to bilateral bronchial and right diaphragmatic vessels. The CTA, however, didn’t reveal any coronary artery disease (CAD). Subsequently, cardiac catheterization was performed that showed a coronary fistula noted from the proximal portion of the RCA extending and connecting into the right pulmonary artery, again reaffirming no CAD. The detailed history and multiple investigations ruled out any cardiogenic causes of chest pain along with acquired causes of CPAF. A shared medical decision was made to pursue only a conservative approach with a focus on noncardiogenic etiologies of chest pain which helped the patient.

Discussion:
The systemic manifestations depend principally on the resistance of the fistulous connection and on the site where the fistula terminates. The majority of CPAF may have no clinical significance, however, up to 19% of cases are associated with an aneurysm, ischemia, infarction, or heart failure. Although the nuclear stress test showed a defect in the inferior wall; however, it was not deemed as the cause, considering the size of the fistula was too small and there was no evidence of a left-to-right shunt as well.

Conclusion:
Shared decision-making is of paramount importance in treating CPAF and a diligent approach can save patients from unwanted procedures like embolization and or surgical interventions. A close follow-up is also advised should the symptoms worsen.
Abstract 622: Automated Platform For Large Scale Structural Simulations Of Atherosclerotic Lesions
Authors: Straughan Ross , Kadry Karim , Edelman Elazer R , Nezami Farhad R
Published: October 2023

Coronary artery disease persists as the world’s leading cause of death and often associated with atherosclerotic plaque disruption in the face of excessive plaque structure stress (PSS). Lesion tolerance of PSS correlates with the material and morphological properties of the arterial constituents. 2D-Finite Element Analysis (FEA) performed on models generated from arterial images (e.g. optical coherence tomography (OCT)) has procured a deeper understanding of lesion biomechanics but not the full complex 3D morphological-mechanical response. OCT frame sparsity and manual burden associated with 3D model generation have continuously plagued 3D simulations and automated large-scale studies. We present a fully automated software platform to determine 3D arterial stress distributions from deep-learning labeled OCT images of complex lesions by interpolating labeled frames. A preliminary study showed that frames with a high PSS (over 150 kPa) had significantly larger lipid arc angles, lumen-plaque eccentricity, lipid area, and calcium area, mirroring clinical observations. Such promising results validate the potential for the platform to be utilized for plethora of applications from unprecedented large scale in-silico studies to enhance clinical metrics, optimization of patient-specific angiography operations, and training data for emerging AI simulations.