bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 316: Hydroxytyrosol, An Olive Oil Phenol, Reduces
Ex Vivo
Thrombin-induced Platelet Activation
Authors: Zhang Ruina , Moscona Alberto , Myndzar Khrystyna , Luttrell-Williams Elliot , Vanegas Sally , Calderon Karry , Berger Jeffrey S , Heffron Sean
Published: November 2022

Background and Aims:
Olive oil consumption is associated with lower cardiovascular disease (CVD) risk. We recently demonstrated that frequent olive oil consumption was inversely associated with thrombin-induced platelet activation
ex vivo
. While various olive oil phenolic compounds, notably hydroxytyrosol, have been shown to reduce human platelet aggregation
ex vivo
, little is known about how olive oil affects platelet activation, a key mediator of atherothrombosis. Therefore, we sought to investigate whether hydroxytyrosol exposure affects platelet activation.

Methods and Results:
Ten healthy, non-smoking men and women (25.0±1.6 years) with normal body mass index (BMI 22.2±3.3 kg/m
2
) were recruited for
ex vivo
platelet studies. Individuals on antiplatelet or anticoagulation medications or medications known to influence lipid levels were excluded. Subjects attended a single visit at NYU Langone where platelet-rich plasma (PRP) was isolated and incubated with three different concentrations of hydroxytyrosol or ethanol vehicle for 60 minutes at room temperature. PRP was then incubated with bovine thrombin for 5 minutes followed by anti-CD62P for 15 minutes in the dark. Platelet activation was determined by platelet surface expression of P-selectin, assessed via flow cytometry.
E
x vivo
exposure to hydroxytyrosol inhibited thrombin-induced platelet P-selectin expression in a dose-dependent manner (Figure).

Conclusions:
We demonstrate that hydroxytyrosol exposure reduces thrombin-induced platelet P-selectin expression in a dose-response fashion. The dose response seen in this
ex vivo
platelet activation study is consistent with our clinical observation that more frequent olive oil intake was associated with reduced
ex vivo
thrombin-induced P-selectin expression. This laboratory study suggests a putative mechanism underlying our clinical observation as well as the established inverse association of olive oil consumption with CVD.
Abstract 318: Fxr1 Regulates Vascular Contractility And Decreases Blood Pressure
Authors: St Paul Amanda , Preston Kyle , Corbett Cali , Leigh Tani , Kelemen Sheri E , Eguchi Satoru , Autieri Michael
Published: November 2022

Introduction:
Hypertension is a major risk factor for cardiovascular disease and stroke. Optimally treated hypertensive patients have a 50% greater cardiovascular risk than untreated normotensive subjects presenting a need for additional targets. Vascular smooth muscle cells (VSMC) are the fulcrum of vascular disease, particularly hypertension. VSMC play a critical role in vascular contractility and the regulation of blood pressure. Fragile X-related protein (FXR1) is a muscle-enhanced RNA binding protein and we previously found siRNA knock down of FXR1 increases inflammatory mRNA stability. Overexpression of FXR1 decreases inflammatory mRNA stability in VSMC. Little is known concerning FXR1 protein binding partners and its role in vascular disease. The specific aim of this study is to test the hypothesis that FXR1 regulates vascular contractility by RNA stability and protein interactions.

Results:
Gene ontology of RNA immuno-precipitation sequencing analysis in human VSMC identified that FXR1 binds to mRNA that participate in VSMC contractility and regulation of blood pressure- related pathways. FXR1 depletion in human VSMC decreases mRNA abundance of contractile machinery such as RhoA, Cortactin, ARP2 and Dynactin. While considered an RNA stability protein, mass-spectrometry identified that FXR1 interacts with proteins related to contractile processes such as cell migration, adhesion and stress fiber formation. Additionally, siRNA knock down of FXR1 decreased VSMC adhesion, migration and collagen gel contraction. FXR1 depletion decreases CDC42 activation in VSMC. CDC42 is a small GTPase involved in cell morphology, adhesion and migration. In order to establish an
in vivo
role of FXR1 in vascular disease we generated a novel, VSMC-specific FXR1 conditional knock out mouse (FXR1
VSMC/VSMC
). Preliminary data indicates that these mice show decreased diastolic (P < 0.05) blood pressure at baseline compared to controls.

Conclusion:
These data are the first to suggest FXR1 regulates blood pressure and vascular contractility potentially by two mechanisms: mRNA stability and functional activity by protein-protein interactions. These findings suggest FXR1 as target for therapeutic intervention to regulate blood pressure.
Abstract 319: Electronic Cigarettes Activate The Heat Shock Response In Vascular Smooth Muscle In Atherosclerosis
Authors: Damiani Isabella , Easwaran Meena , Qin Tracy Guyu , Cheng Paul , Kim Hyun-Jung , Martinez Joshua , DiRenzo Elizabeth , Quertermous Thomas , Kim Juyong Brian
Published: November 2022

Introduction:
Electronic cigarettes pose a serious emerging risk for cardiovascular disease. Despite a significant association between exposure to e-cigarettes (e-cigs) and burden of coronary artery disease, the pathogenic mechanism is not well understood. The environment sensing transcription factor aryl-hydrocarbon receptor (Ahr) pathway has been implicated in tobacco-induced atherosclerosis but its role in e-cigarette exposure is not known.

Methods:
SMC-lineage tracing mice (WT, n=3) and SMC-specific Ahr knockout (KO, n=3) mice on a ApoE null hyperlipidemic background were put on high fat diet for 12 weeks and exposed to pod-based e-cigs (Juul) daily for 2 weeks in a whole body chamber (inExpose, Scireq) and compared to WT mice exposed to air (n=3). The aortic sinus was dissected and digested, FACS was performed to sort for live single cells, then single-cell RNA-Seq and ATAC-seq were performed on the 10X Genomics platform. Analyses were completed with Seurat and Signac tools.

Results:
There were total of 20102 cells included in the scRNA-Seq analysis (WT Control 5282, WT exposed to e-cig (WT-Ecig) 9425, and KO exposed to
Juul
(KO-Ecig) 5395). We found increased
Cyp1b1
expression in the modulated SMC population in the WT-Ecig group, suggesting activation of the Ahr pathway. The proportion of modulated SMC to quiescent SMC remained comparable following E-cig exposure in the WT mice. We identified a new cluster of SMC lineage cells that were enriched for markers of heat shock response, including
Hspb1, Hsp90aa1
, and
Cryab
. This subpopulation of SMC was nearly absent in the SMC-specific Ahr KO mice exposed to E-cigs. The scATAC-seq profiles showed enrichment for motifs of CCAAT/enhancer-binding proteins, and pathways of cytokine production, ECM organization, and cell migration in the clusters of heat shock response-activated SMC, as well as significant enrichment for Hsf2 motifs in open chromatin regions following E-cig exposure.

Conclusion:
Using single-cell genomics of atherosclerotic plaque from mice exposed to e-cigs, we identified the heat shock response pathway to be uniquely activated in the modulated SMC-lineage population, and that this process may be mediated by Ahr.
Abstract 335: Macrophage Interleukin-1 Beta Dependent Angiogenesis During Wound Healing
Authors: Sharma Sheila , Mantsounga Chris S , Neverson Jade , Lee Cadence , Carley Rachel T , Butler Celia , Choudhary Gaurav , Morrison Alan R
Published: November 2022

Angiogenesis during wound healing is thought to require an inflammation suppressed state. However, there is growing evidence that early infiltrating inflammatory macrophages may play an important role in setting the stage for angiogenesis. Here we sought to understand the relationship between the potent inflammatory cytokine, IL-1β, and vascular endothelial growth factor-A (VEFG-A) in the context of two experimental models of angiogenesis-dependent healing: skin punch biopsy and hind limb ischemia. We established a macrophage-specific
IL-1
β
-deletion model (
mIL-1
β
) to study the relationship between IL-1β and VEGF-A in the context of these models. Using a punch biopsy model, wounds were quantified, showing remarked expansion of the wounds in
mIL-1
β
-deleted mice on day 2 of the healing process, resulting in slower wound closure. Analysis from
mIL-1
β
-deleted mice confirmed decreased macrophage expression of IL-1β and VEGF-A early at day two post-wounding by punch biopsy. In our second approach, we used femoral artery ligation to demonstrate hind limb ischemia. We hypothesized that
mIL-1
β
-deleted mice would exhibit reduced blood flow recovery due to impaired angiogenesis. Analysis from
mIL-1
β
-deleted mice indicates reduced IL-1β and VEGF-A expression. In establishing a pathway of IL-1β-dependent macrophage VEGF-A upregulation in the acute injury state, we examined transcriptional factors STAT3 and NF-κB. Constitutively active STAT3 and IKK-2 (NF-κB activator) appear to increase VEGF-A expression, while inhibition of STAT3 and NF-κB exhibits a dose-dependent reduction of VEGF-A expression. In summary, two models of angiogenesis-dependent healing from injury indicate that macrophage VEGF-A expression is dependent on IL-1β expression during early inflammation. Strategies to alleviate impaired wound healing where VEGF-A levels may not be sufficient could require upregulation of the downstream effectors of IL-1β to restore macrophage VEGF-A expression levels required for adequate angiogenesis.
Abstract 336: Comparing Of
In-vivo
Carnosine Levels In Blood, Urine, And Muscle
Authors: Amraotkar Alok R , Hoetker David , Negahdar Mohammad J , Ng Chin K , Lorkiewicz Pawel , Owolabi Ugochukwu , Baba Shahid P , Bhatnagar Aruni , O Toole Timothy
Published: November 2022

Background:
Dietary supplementation of carnosine, an endogenously produced antioxidant, can be potentially useful in decreasing oxidative stress in the vasculature. However, a safe and accurate measurement of in-vivo Carnosine levels has not been identified.

Methods:
We enrolled 28 healthy male (50%) and female participants. In-vivo levels of carnosine were measured in three distinct tissues in all 28 participants - urine, blood red blood cells (RBC), and muscle via proton magnetic resonance spectroscopy (1H-MRS). Generalized Linear Model with gamma-distribution and log-link was adjusted for age, sex, race, and diet. Coefficient of variance (CoV) was calculated for 1H-MRS repeat measurements: single location with multiple measurements and multiple locations with single measurements.

Results:
Univariate regression analysis showed a significant positive association between RBC and urinary carnosine levels (β=0.02; 95% CI: 0.004-0.03; P=0.01). The adjusted model also showed significant positive association between RBC and urinary carnosine levels (β=0.02; 95% CI: 0.002, 0.03; P=0.03). There was no association between 1H-MRS and RBC carnosine or urinary carnosine or carnosine conjugates in urine. The median CoV for multiple readings at single location were 8% and 1% for C2 and C4 peaks respectively, however 12% and 13% for C2 and C4 peaks respectively for single reading at multiple locations.

Conclusions:
Even though urine, blood (RBC), and muscle represent in-vivo tissues of the human body, the levels of carnosine pools in these different compartments are not in equilibrium with each other. 1H-MRS is highly sensitive to location of measurement. In-vivo levels of carnosine in urine and blood depot are in synchrony and should be used for large scale clinical and research studies like the NEAT clinical trial (NCT03314987), which studies the use of carnosine dietary supplementation to mitigate harmful cardiovascular effects of PM2.5 air pollution.
Abstract 345: Natural Vascular Scaffolding Suppresses Experimental Abdominal Aortic Aneurysms
Authors: Xu Baohui , Ikezoe Toru , Guo Jia , Shoji Takahiro , Warner Kevin S , Kauser Katalin , Dalman Ronald L
Published: November 2022

Objective:
Proteolytic destruction of aortic extracellular matrix (ECM) is central to abdominal aortic aneurysm (AAA) pathogenesis. Matrix metalloproteinase inhibition with doxycycline failed to limit AAA progression. However, photochemical modification of collagen and elastin fibers may provide an alternative approach to ECM stabilization. We investigated the effectiveness of this treatment in limiting experimental AAA progression.

Methods:
AAAs were created in 8-10 weeks old male C57BL/6J mice via intra-aortic elastase infusion. Natural vascular scaffolding (NVS) (2 mg/ml, Alucent Biomedical) or vehicle solution was applied to the abluminal aortic wall immediately following elastase infusion and aortotomy closure and exposed to laser light activation. AAA progression was assessed via serial ultrasound aortic diameter measurements and histopathologic analysis at sacrifice.

Results:
Ultrasonography confirmed progressive aortic enlargement and AAA formation in all vehicle-treated mice within 14 days following elastase infusion. NVS treatment substantially attenuated AAA development and progression with reduced medial elastin degradation and smooth muscle cell depletion, as well as mural neovessel development. No difference was seen in aortic CD4 or CD8 T accumulation between the two treatment groups.

Conclusion:
Photochemical linking of extracellular matrix proteins attenuated experimental AAA progression, suggesting a potential translational application for this approach in clinical disease management.
Abstract 348: Angiotensin 1-7 Exerts Protective Effects In Thoracic Aortic Aneurysm By Attenuating Smooth Muscle Cell Phenotypic Switching
Authors: Jadli Anshul S , Ballasy Noura , Gomes Karina , Mackay Cameron , Meechem Megan , Wijesuriya Tishani Methsala , Belke Darrell D , Fedak Paul , Patel Vaibhav B
Published: November 2022

Background:
Thoracic aortic aneurysm (TAA) involves extracellular matrix (ECM) remodeling of the aortic wall, leading to reduced biomechanical support with risk of aortic dissection and rupture. Elusive pathophysiology of initiation and progression of TAA has hindered efforts to develop pharmacological therapeutic interventions to delay or prevent progressive aortic dilatation. Activation of the renin-angiotensin system, and resultant Angiotensin (Ang) II synthesis, is critically involved in the onset and progression of TAA. The current study investigated the effects of Angiotensin (Ang) 1-7 on a murine model of TAA.

Methods & Results:
8-10 weeks old apolipoprotein-deficient mice (ApoEKO) were infused with Ang II (1.44 mg/kg/day) and treated with Ang 1-7 (0.576 mg/kg/day). Echocardiography and histological analyses revealed increased thoracic aortic dilatation, ECM remodeling, perivascular fibrosis, and inflammation in Ang II-treated mice. Infusion of Ang 1-7 led to suppression of Ang II-induced dilatation, remodeling, and inflammation in the thoracic aorta. The immunofluorescence imaging showed reduced α-SMA fluorescence (contractile marker) in vascular smooth muscle cells (VSMCs) of the aortic media indicating phenotypic switching. In response to Ang II, the thoracic VSMCs from ApoEKO mice exhibited phenotypic switching indicated by reduced contractile (
Acta2, Cnn1, Myh11
) and increased synthetic (
Il-6, Mmp2, Mmp9, Col1a1, Col3a1
) gene expression. Ki67 staining and flow cytometry analysis showed VSMCs hyperproliferation with Ang II treatment. Ang 1-7 preserved the contractile phenotype of VSMCs and attenuated hyperproliferation. The mitochondrial structure was assessed using MitoTracker Red staining which showed Ang II-induced excessive mitochondrial fission. DHE and MitoSOX Red staining were used to assess the reactive oxygen species (ROS). Ang II-treated thoracic VSMCs demonstrated elevated cellular and mitochondrial ROS generation. Ang 1-7 mitigated Ang II-induced mitochondrial fission and oxidative stress.

Conclusion:
Ang 1-7 prevented Ang II-induced vascular remodeling and the development of TAA. Enhancing Ang 1-7 actions may provide a novel therapeutic strategy to prevent or delay the progression of TAA.
Abstract 350: Disruption Of Proprotein Convertase Subtilisin/Kexin 9 Suppresses Inflammation And Attenuates Abdominal Aortic Aneurysm Formation
Authors: Peng Zekun , Chen Hong , Wang Miao
Published: November 2022

Objective:
Abdominal aortic aneurysm (AAA) is a degenerative inflammatory disease, with its rupture highly lethal. There have been no effective pharmacological medications. Proprotein convertase subtilisin/kexin 9 (PCSK9), which is known to modulate cholesterol metabolism, has recently been associated with AAA in human genome-wide association studies. However, its role in AAA pathogenesis is unknown.

Approach and Results:
Histology staining of mouse aortic aneurysmal tissue showed PCSK9 expression and its colocalization with macrophages. To investigate the role of PCSK9 in AAA formation, mice deficient in PCSK9 were crossed with

ApoE
SA/SA

mice, which are hypercholesterolemic and inducible to develop angiotensin II-mediated hypertension upon doxycycline (Dox) administration. Deletion of PCSK9 reduced both incidence and severity of AAA and increased survival rate, without changing plasma cholesterol levels. This was associated with decreased aortic expression and activity of matrix metalloproteinase 9 (MMP9), less elastin breakdown, and reduced aortic expansion and aneurysm rupture. Additionally, PCSK9 deficiency suppressed systemic inflammatory response and macrophage infiltration into AAA lesions. In isolated macrophages stimulated with LPS, the upregulated expression of pro-inflammatory cytokines (
Tnfa, Il1b, Il6
) was dramatically diminished in PCSK9-deficient macrophages. Furthermore, administration of PCSK9-specific siRNA in mice resulted in less macrophage accumulation in the aneurysmal aortic wall, decreased MMP9-mediated elastin degradation, and substantially constrained AAA formation.

Conclusions:
Endogenous PCSK9 promotes AAA formation, consistent with its novel action in mediating systemic and local inflammation, particularly, the macrophage-dependent MMP9 activity. Pharmacological inhibition of PCSK9 might be a promising strategy to curtail AAA.

Key Words:
aneurysm, PCSK9, elastin, inflammation, macrophages
Abstract 354: Beiging Of Perivascular Adipose Tissue Regulates Its Inflammation And Vascular Remodeling
Authors: Adachi Yusuke , Ueda Kazutaka , Ito Kaoru , Nomura Seitaro , Takimoto Eiki , Komuro Issei
Published: November 2022

Background:
Inflammation plays a primordial role in atherosclerosis. Perivascular adipose tissue (PVAT) that surrounds the vasculature has been reported to undergo phenotypic changes in response to vascular inflammation. However, regulatory roles of PVAT in vascular inflammation and subsequent pathological remodeling remain unclear.

Methods and Results:
Endovascular injury was generated by wire insertion into the mice femoral artery. Here, we showed that vascular injury induced the beiging (brown adipose tissue-like phenotype change) of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism. In a mouse model of endovascular injury, macrophages accumulated in PVAT, causing beiging phenotype change. Inhibition of PVAT beiging by adipose tissue-specific genetic ablation of PRDM16, a key regulator to beiging, exacerbated inflammation and vascular remodeling following injury. Local knockdown of
Prdm16
in perivascular tissues of wild-type mice using small interfering RNA similarly exacerbated inflammation and vascular remodeling. Conversely, activation of PVAT beiging attenuated inflammation and pathological vascular remodeling. Single-cell RNA sequencing revealed that beige adipocytes abundantly expressed neuregulin 4 (
Nrg4
) which critically regulated alternative macrophage activation. Furthermore,
Nrg4
knockdown abolished the PVAT-mediated anti-inflammatory effects on macrophages. Importantly, significant beiging was observed in the diseased aortic PVAT in patients with acute aortic dissection.

Conclusions:
Endovascular injury induced the beiging of adjacent PVAT with macrophage accumulation, where NRG4 secreted from the beige PVAT facilitated alternative activation of macrophages, leading to the resolution of vascular inflammation. Our study demonstrated the pivotal roles of PVAT in vascular inflammation and remodeling and will open a new avenue for treating atherosclerosis.
Abstract 357: Expression of a Constitutively Active TGF-β Receptor-1 in Mouse Aortic Smooth Muscle Cells Does Not Cause Severe Aortopathy
Authors: Lee Chloe Y , Angelov Stoyan N , Sethuraman Meena , Dichek David A
Published: November 2022

Background:
Altered transforming growth factor beta (TGF-β) signaling in aortic SMC is implicated as a cause of aortic aneurysms and dissections (AAD), with AAD attributed both to increased and decreased TGF-β signaling. Loss of TGF-β signaling in aortic SMC of young mice unequivocally causes AAD. However, it is unclear whether increased aortic SMC TGF-β signaling is sufficient to cause AAD. To answer this question, we induced expression of a conditional constitutively active Type I TGF-β receptor (TBR1-CA) in adult mouse aortic SMC.

Methods:
Experimental mice
(Acta2
-CreER
T2 +/o
TBR1-CA
+/o
) were injected with tamoxifen at 6 weeks of age and compared to tamoxifen-injected controls (
Acta2
-CreER
T2 o/o
TBR1-CA
+/o
,
Acta2
-CreER
T2 +/o
TBR1-CA
o/o
mice, or
Acta2
-CreER
T2 o/o
TBR1-CA
o/o
). TBR1-CA was detected by immunoblotting; ascending aortas were analyzed by sectioning, histology, planimetry, and wire myography. TBR1-CA
+/o
SMC were explanted, cultured, and treated with adenovirus-Cre (Ad-Cre; to induce TBR1-CA expression) or control AdNull. Extracts were analyzed by western blotting.

Results:
Expression of TBR1-CA in SMC of experimental mice was detected by immunoblotting of aortic media extracts 10 days and 4 months after tamoxifen injection. Ad-Cre-treatment of cultured TBR1-CA
+/o
SMC significantly increased phosphorylation of SMAD2 and SMAD3 (2-3-fold; P
<
0.002 for both). Gross aortic hemorrhage was present in 1 of 68 experimental and 0 of 89 control mice (P=0.4). Wire myography (4 weeks after tamoxifen) showed no differences in vasomotor function. There were no penetrating aortic ulcers, intramural hematomas, or Prussian blue staining in any of 42 experimental or 52 control mice harvested either 10 days or 10 weeks after tamoxifen. 10 weeks after tamoxifen, aortic diameters of experimental mice were normal, but medial thickness was mildly increased (45.2 μm ± 6 vs 39 ± 4.7 μm; P= 0.04), and medial area trended higher (0.15 ± 0.03 mm
2
vs 0.13 ± 0.02 mm
2
; P=0.1).

Conclusion:
Expression of a constitutively active type I TGF-β receptor in aortic SMC of postnatal mice appears to cause—at most—a mild and indolent aortopathy. Further studies are ongoing; however, elevated SMC TGF-β signaling mediated by TBR1 may be insufficient to cause severe aortopathy.
Abstract 358: Smooth Muscle Cell Specific Beclin-1 Deficiency Accelerates Ascending And Abdominal Aortic Aneurysms In Male Hypercholesterolemic Mice Infused With Angiotensin Ii
Authors: Javidan Aida , Jiang Weihua , Yang Lihua , Subramanian Venkateswaran
Published: November 2022

Background and Objective:
Ascending and abdominal aortic aneurysms (AAs) are permanent dilations of the ascending and abdominal aorta with a mortality greater than 80% after rupture. Vascular smooth muscle cells (SMCs) are pivotal in maintaining aortic structural integrity and function, and SMC-rich aortic medial stability is highly disrupted in AAs. Autophagy is a self-regulatory process by which cell digest, and recycles their cytoplasmic materials for energy purposes under stress. Recent clinical studies highlighted that Beclin-1, a gene indispensable for autophagy induction, is highly upregulated in AA tissues from patients. However, the functional contribution of SMC-derived Beclin-1 in the development of AAs is unknown. Using the well-established Angiotensin II infusion model of AAs, we examined the role of SMC-Beclin-1 during the development of AAs in hypercholesterolemic mice.

Methods and Results:
Mice with inducible deletion of Beclin-1 in SMCs were produced by breeding male mice hemizygous for Acta2-Cre
ERT2
(Cre+) to female Beclin-1 floxed mice in an LDLR -/- background. At 8 weeks of age, male Beclin-1 x Acta2-Cre
ERT2
(Cre+) and non-Cre littermates (Cre-) mice were injected with tamoxifen (75mg/kg, i.p.) for 5 consecutive days. After 2 weeks, Western blot analyses showed depletion of Beclin-1 protein in the aortic media from Cre+ mice compared to Cre- littermates. To study the role of Beclin-1in SMCs, male Cre+ and Cre- (N=5-6/group) mice were fed a fat-enriched diet (21% fat; 0.15% cholesterol) for 5 weeks. After 1 week of diet feeding, mice were infused subcutaneously with low dose of AngII (500 ng/kg/min) by osmotic mini-pumps for 4 weeks. Interestingly, depletion of Beclin-1 in SMCs significantly accelerated AngII-induced aortic luminal dilation (Cre-: 1.0 ± 0.02 mm, Cre+: 1.5 ± 0.14 mm;
P
< 0.001), and expansion of ex-vivo maximal diameters of abdominal aortas in mice (Cre-: 1.1 ± 0.1 mm, Cre+: 1.52 ± 0.1 mm;
P
< 0.05). In addition, SMC- Beclin-1 depletion significantly increased AngII-induced ascending aortic aneurysms (Cre-: 12.6 ± 0.4 mm
2
, Cre+: 20.9 ± 1.2 mm
2
;
P=0.008
).

Conclusion:
These findings demonstrate that SMC-Beclin-1 plays a critical role in suppressing AngII-induced ascending and abdominal aortic aneurysm formation in mice.
Abstract 360: Epigenomic Changes Govern Smooth Muscle Cell Phenotype Shift In Marfan Syndrome Aortic Root Aneurysm
Authors: Pedroza Albert J , Dalal Alex R , Shad Rohan , Yokoyama Nobu , Nakamura Ken , Cheng Paul , Mitchel Olivia , Hiesinger William , Quertermous Thomas , Fischbein Michael P
Published: November 2022

Background:
Vascular smooth muscle cell (SMC) phenotype modulation produces a mixed proteolytic/collagen synthetic state in Marfan syndrome (MFS) aortic root aneurysm. While the transcriptomic changes associated with this process are established, upstream regulators governing this plasticity are poorly characterized.

Methods/Results:
We performed concurrent single-cell RNA sequencing (scRNAseq) and assay for transposase-accessible chromatin using sequencing (scATACseq) on aortic root aneurysm tissue from adult

Fbn1
C1041G/+

(MFS) mice and littermate controls. MFS/control scRNAseq data analysis identified four SMC subtypes including MFS-specific ‘modulated’ cells (modSMCs) which enriched for extracellular matrix organization and collagen synthesis pathway activation. These SMC clusters were projected onto the MFS/control scATAC dataset via integrative label transfer to study dynamic chromatin accessibility during SMC phenotype modulation. We compared DNA accessibility in modSMCs versus mature SMCs, finding 336 enriched and 29 suppressed peaks, suggesting increased open chromatin during modulation. Using chromVAR, we identified 242 enriched transcription factor motifs overrepresented in modSMCs. Motifs representing central but nonspecific transcription factor families including numerous AP-1 (FOS/JUN/ATF) heterodimers and TEAD family members showed highest enrichment, while enriched TWIST1, HAND2, and SMAD2:SMAD3:SMAD4 complex motifs suggested more specific functions. To functionally validate these findings, we examined
TWIST1
as a potential regulator of SMC modulation
in vitro
via lentiviral overexpression in MFS patient-derived aortic SMCs.In SMCs with forced TWIST1 overexpression, we found heightened promoter region DNA accessibility (via bulk ATACseq) and increased mRNA expression (via RT-PCR) for specific modSMC markers (e.g.,
COL1A1
and
LUM
confirming TWIST1 promotes collagen synthesis.

Conclusions:
Integrated single-cell transcriptomic/epigenomic analysis permits identification of critical upstream regulatory signals promoting disease-specific cell phenotype changes. TWIST1 is a potential driver of SMC modulation and a target for therapeutic agent design in MFS aortic aneurysm.
Abstract 371: The Role Of Scleraxis In Inducing Vascular Stiffness
Authors: alhattab Danah , Moffat Teri , Ledingham Allison , Czubryt Michael P
Published: November 2022

Vascular dysfunction underlies numerous significant diseases including diabetes, atherosclerosis and hypertension. Vascular dysfunction can be a result of altered smooth muscle contraction/relaxation, and impaired endothelial cell function within the vessel wall. Vascular fibrosis involves an increase in the thickness of vessel wall. This contributes to either an increase in extracellular matrix (ECM) synthesis or induced vascular smooth muscle proliferation within the vessel wall, or both, causing stiffer vessels with impaired tone and reduced lumen diameter.Our lab identified the transcription factor scleraxis as a novel master regulator of fibrotic signaling in the myocardium, showing scleraxis is sufficient to induce fibroblast to myofibroblast phenotype conversion, a critical step in the development of fibrosis, and directly up-regulates ECM genes in cardiac fibroblasts. Angiotensin II (AngII) was reported to induce vascular fibrosis via activation of the transcription factor Smad3 in aortic vascular smooth muscle cells. Our lab has shown that Smad3 physically interacts with scleraxis, and critically requires scleraxis to drive TGFβ/Smad fibrotic signaling in cardiac fibroblasts.Our preliminary data has revealed that scleraxis is detectable in the arterial wall, and scleraxis expression is elevated in high pressure versus low pressure regions of vessels. Loss of scleraxis in the aortas of scleraxis knock-out mice reveals a discontinuation and disarrangement in the structure of vascular wall. We thus hypothesize that scleraxis is sufficient and necessary to induce vascular fibrosis.Pressure myography data reveals an increase in vascular stiffness and thickness of 3rd order mesenteric arteries of smooth muscle-specific scleraxis overexpression mice. Also, our data shows that vascular stiffness is significantly increased in AngII-induced scleraxis overexpression mice with a relative increase in telemetry blood pressure measurements and pulse wave velocity. Histological sections suggest a reduction of the translamellar ECM accumulations in AngII-induced scleraxis overexpression aortas. To summarize, scleraxis may contribute to vascular stiffness by inducing vascular smooth muscle proliferation.
Abstract 373: Senescence Drives Changes In Vesicle Secretion And Extracellular Matrix Organisation To Enhance Vascular Amyloidosis
Authors: Whitehead Meredith , Ahmad Sadia , Yusoff Syabira , Shanahan Cathy
Published: November 2022

Background:
Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid fragment of milk fat globule EGF-factor 8 (MFGE8). Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid metabolism and accumulation in the extracellular matrix (ECM). MFGE8 has been identified as EV cargo in several cell types, including vascular smooth muscle cells (VSMCs). To determine the mechanisms of AMA formation with age, we explored the impact of VSMC senescence, EV secretion and ECM remodelling on medin accumulation.

Methods:
Primary human VSMCs were used for
in vitro
experiments, including EV isolation and synthesis of decellularised ECM for Western blotting, immunofluorescence and mass spectrometry analysis.

Results:
Medin was detected in EVs secreted from VSMCs. Small, round medin aggregates colocalised with EV markers in the ECM and medin was shown on the surface of EVs deposited in the ECM. Decreasing VSMC EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age. VSMC senescence increased EV secretion, increased EV medin loading and triggered extracellular deposition of medin in fibrillar form. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition. These changes led to enhanced EV-ECM binding in the senescent ECM and accelerated aggregation of medin into amyloid fibrils. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalised with EVs and medin. Knock-down of HSPG2 decreased medin fibril formation in the senescent ECM.

Conclusions:
Medin is secreted into the ECM by VSMC-derived EVs, which enhance its aggregation. Senescence induces changes in EV secretion and cargo and ECM composition. Together, these changes trigger accumulation of fibrillar medin, contributing to the age-associated development of AMA.
Abstract 374: Smooth Muscle Cell-specific Deletion Of At1a Receptor Attenuates Vascular Fibrosis In Angiotensin Ii-infused Mice
Authors: Okuno Keisuke , Torimoto Keiichi , Sparks Matthew A , Eguchi Satoru
Published: November 2022

Angiotensin II (Ang II) AT1 receptor has been shown to play a critical role in several cardiovascular diseases. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. Deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affect hypertensive fibrosis. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock-In, KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and control (AT1a flox/flox Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks. In control mice, Ang II infusion induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly decreased in AT1a SMKO mice compared with control mice (6.04 versus 4.89, p=0.032). Cardiac hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 μm vs. 56.4 μm, p<0.0001). Control mice with Ang II infusion showed increased area of perivascular collagen III immunostaining in aorta, which was attenuated in AT1a SMKO mice. In conclusion, AT1a receptors expressed in VSMC could mediate Ang II-induced perivascular fibrosis. Our data together with the past manuscript showing a protective effect of fibroblast AT1a silencing suggest the importance of both VSMC and fibroblast AT1a in fibrosis. However, whether our data can be fully explained by the prevention of hypertension remains to be determined.
Abstract 375: Assessment Of Ultrasound-facilitated Endothelial Permeability Using Transwell Culture Methodology
Authors: Islam Afsana F , Peng Tao , Shelat Harnath S , Gao Song , McPherson David D , Geng Yong-Jian , Klegerman Melvin E
Published: November 2022

Background:
We have previously demonstrated the ability of continuous wave and pulsed Doppler ultrasound (US) to increase the permeability of the arterial wall to echogenic liposomes (ELIPs). We have modeled this phenomenon
in vitro
using transwell endothelial cell monolayers. This study uses the latter to explore potential mechanisms of US-enhanced vascular permeability.

Hypothesis:
US facilitates endothelial cell permeability by radiation pressure, rather than cavitation effects, mediated by nitric oxide (NO) generation.

Methods:
Human umbilical vein endothelial cells (HUVEC) confluently grown in 24-well transwell insert plates were treated with TNF-α (20 ng/mL) and incubated overnight at 37°C. After washing with Dulbecco’s PBS (DPBS), cells were incubated with fluorescently (rhodamine) labeled ICAM-1-targeted echogenic liposomes (Ab-ELIP). Treatments included Ab-ELIP, nonspecific IgG ELIP, non-echogenic Ab-ELIP, and Ab-ELIP with 10 μM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of endothelial nitric oxide synthase (eNOS), or 1H-[1 ,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 0.1 and 1.0 μM), an inhibitor of soluble guanylyl cyclase (sGC). Some plates were treated with continuous wave ultrasound (1 MHz, 2 W/cm
2
, 50% duty cycle for 1 minute). After 30 minutes, the insert plates were washed with DPBS, filled with endothelial cell medium and incubated at 37°C for 24 hours; 150 μL of lower well media were transferred to 96-well plates, then measured for fluorescence intensity with a microplate reader.

Results:
US treatment enhanced endothelial cell penetration of Ab-ELIP relative to non-US treatment (18 DOF, p=0.000012). Non-echogenic ELIP was as effective in this regard as echogenic ELIP (p=0.57), supporting the hypothesis that the ultrasound effects noted in these experiments were due to radiation pressure. L-NAME suppressed US-enhanced penetration of Ab-ELIP to non-US levels (p =0.14), indicating that US enhancement of Ab-ELIP penetration is mediated by NO generation. ODQ (0.1 μM) reduced the US penetration enhancement (p=0.0002), suggesting that the effect is at least partially mediated by NO-induced cGMP production.

Conclusion:
Both aspects of the hypothesis were supported by the experimental results.
Abstract 376: Microscale Mechanisms Regulating Fibrin Clearance During Fibrinolysis
Authors: Lynch Spencer R , Laverty Sean M , Bannish Brittany E , Hudson Nathan
Published: November 2022

During fibrinolysis the enzyme plasmin digests fibrin fibers, the primary structural elements in blood clots. Developing a mechanistic understanding of fibrinolysis is important for understanding the pathophysiology of hemostasis, but lysis mechanisms have long been debated. Using time-resolved fluorescence microscopy, we investigated the plasmin-mediated digestion of individual fibrin fibers. We found that digestion occurs all along the fiber length, but digestion rates vary spatially. At some fiber locations, digestion accelerates over time, likely due to a previously unknown tension-dependent mechanism that pulls fibers apart during digestion. The fiber is eventually cleaved into two halves, often with the halves displaying cone-shaped ends. To determine how fibers are digested after the initial cleavage event, fiber ends were tracked, and surprisingly the cone-shaped structure persisted over for over 13 s. To understand our experimental results, we used mathematical modeling to estimate the mean first passage time of plasmin molecules arriving at the fiber surface and the number of digestion sites on a fiber. Modeling results were comparable to the experimental time evolution of digestion sites data. Taken together, these results provide a detailed understanding of fibrinolytic mechanisms, both during and after fiber cleavage, and underscore the need to consider fibrin tension when assessing fibrinolytic approaches.
Abstract 377: Neonatal Hyperoxia Contributes To Early Cardio-renal Fibrosis And Systemic Vascular Stiffness In 1 Year Old Rats
Authors: Benny Merline , Shelton Elaine , Schmidt Augusto , Kulandavelu Shathiyah , Chen Pingping , Tian Runxia , Abitbol Carolyn , DeFreitas Marissa , Wu Shu , Young Karen
Published: November 2022

Background:
There is a knowledge gap in the mechanisms linking neonatal hyperoxia (O
2
) exposure and its contribution to cardio-renal dysfunction in adults born preterm. TGF-β, a pro-fibrotic cytokine is a key player in cardio-renal fibrosis and vascular stiffness. Here, we test the hypothesis that neonatal hyperoxia induces cardio-renal dysfunction and systemic vascular stiffness in adulthood and this is associated with increased TGF-β levels.

Objective:
To determine the association between cardio-renal dysfunction, systemic vascular stiffness and TGF-β levels in adult rats exposed to neonatal hyperoxia.

Methods:
Newborn rats (N=22) randomly assigned to normoxia (RA) or hyperoxia (85% O
2
) from postnatal day 1 to 14 (P1 to P14), were recovered in normoxic conditions until 1 year of life. At 1 year, left ventricular (LV) function was assessed by echocardiography. Aortic and mesenteric arterial stiffness was assessed by wire myography. RNA-seq of the aorta and kidney was done to assess the transcriptional effects of neonatal hyperoxia at 1 year. TGF-β expression was assessed by multiplex cytokine analysis in heart, aorta and kidney. Data was analyzed by Student’s T test.

Results:
At 1 year, there was no evidence of LV dysfunction in rats exposed to neonatal hyperoxia. In contrast, wire myography revealed that rats exposed to neonatal O
2
had significantly increased stiffness of mesenteric artery and aorta. Neonatal hyperoxia exposure differentially regulated genes in 1 year old aorta and kidney. Gene set enrichment analysis showed that genes most induced in the aorta by hyperoxia were “connective tissue development”, “extracellular matrix remodeling” and in the kidney were “actin cytoskeleton”, “epithelial cell differentiation”. As compared to the RA group, rats exposed to neonatal hyperoxia had significantly increased TGF-β1 and TGF-β2 expression in LV and increased TGF-β1 and similar TGF-β2 expression in kidney at 1 year of age.

Conclusion:
Although LV function was normal in hyperoxia-exposed 1 year old rats, these animals had increased systemic vascular stiffness and increased TGF-β expression in LV, aorta and kidney, implying the crucial role of TGF-β signaling in cardio-renal dysfunction in adult rodents exposed to neonatal hyperoxia.
Abstract 384: Regulation Of The Smooth Muscle Cell Phenotypic Switch In Resolution Of Vascular Injury
Authors: Buchmann Giulia K , Pflüger-Müller Beatrice , Spaeth Manuela , John David , Abplanalp Wesley T , Dimmeler Stefanie , Brandes Ralf P
Published: November 2022

Background:
The modulation of vascular smooth muscle cells (VSMC) phenotypes is a highly important process in the formation of neointima and in resolution of inflammation after vascular injury. Underlying genetic mechanisms that control the phenotypic switch of VSMC in disease are not completely understood. On this basis, the phenotypic regulation of VSMC in restenosis development was examined in the mouse carotid artery injury model.

Methods and Results:
Wire-induced injury, an established mouse-model to study restenosis, was applied to induce vascular injury in C57BL/6 mice. Time-resolved single-cell RNA-sequencing (scRNAseq) and massive-analysis-of-cDNA-ends (MACE)-RNAseq of neointima obtained by laser capture microdissection were performed to detect genetic changes in neointima development. Single-cell RNA-sequencing of C57BL/6 mice at different time points after wire-induced injury revealed an expanding, highly proliferative activated VSMC cluster, which exhibited low expression of SMC marker genes. Analysis for differentially regulated genes (DEG) and pseudotime clustering identified a unique marker gene signature, including high expression of TIMP Metallopeptidase Inhibitor 1 (Timp1), which was identified as main ordering gene of the modulation processes′ pseudo temporal states. MACEseq confirmed upregulation of Timp1 selectively in neointima forming VSMCs. Integration with atherosclerosis scRNAseq data indicated differences in gene signatures of activated VSMC populations. Transcription factor analysis of modulated VSMC identified BACH1, a factor involved in reactive oxidative stress response and proliferation of SMCs, as potential regulatory target for re-differentiation of VSMCs after vascular injury.

Conclusion:
ScRNAseq identified a unique cell cluster responsible for neointima formation. Targeting the transcription factors driving the cluster (like BACH1) can be exploited to combat neointima development.
Abstract 388: Role Of The Antiapoptotic C-iap2 On The Generation Of An Endothelial Memory In Schistosomiasis-associated Pulmonary Arterial Hypertension
Authors: Oliveira Suellen Darc , Erewele Ejehi , Castellon Maricela , Schwartz Andrew , Williams David L. , Morrell Nick , Bonini Marcelo , Silva Claudia L. M. , Lutz Sarah E. , Erzurum Serpil , Minshall Richard D
Published: November 2022

Introduction:
Schistosomiasis-associated Pulmonary Arterial Hypertension (Sch-PAH) is a life-threatening complication of chronic
S. mansoni
infection, which can evolve to heart failure and death. During PAH, hyperproliferation of apoptosis-resistant endothelial cells (ECs) has been extensively reported, but therapeutic approaches to reverse this pathological phenotype remain clinically challenging.

Hypothesis:
Thus, our hypothesis is that antigenic molecules from
S. mansoni
eggs amplify the secretion of inhibitor of apoptosis protein 2 (c-IAP2), leading to generation of an abnormal EC memory of survival and the development of PAH.

Methods/Results:
Previously, we showed that depletion of the anti-apoptotic protein Caveolin-1 (Cav-1) via shedding of extracellular vesicles (EVs) leads to the survival of an abnormal EC phenotype through an unclear mechanism. Analysis of survival-associated genes in isolated murine lung ECs from

Flk1
+/GFP
;Cav1
-/-

mice, revealed high expression of the anti-apoptotic genes BIRC3 and BIRC5 (which encodes cIAP2 and survivin, respectively) when compared to control ECs. Interesting, exposure to 1 μg/mL of the major
S. mansoni
egg antigen Sm-p40 induced time-dependent phosphorylation of Cav-1 at residue Tyr14 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p<0.05; n=4), culminating in c-IAP2 expression with no difference on the constitutive expression of c-IAP1. Furthermore, Sm-p40 treatment of HMVEC-L in the presence of pro-inflammatory mediators IL-6, TNF-α, and ATP induced a significant release of EVs containing c-IAP2. Finally, in vivo analysis of lungs from
S. mansoni
-infected mice confirmed a significant decrease in Cav-1 expression unique to the egg-dependent granuloma area, indicating that local inflammatory response due to egg-derived antigenic molecules is essential for reprogramming and survival of an abnormal EC phenotype during Sch-PAH.

Conclusions:
Our data suggest that
S. mansoni
-induced Cav-1 depletion promotes the secretion of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.