bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 212: MicroRNA-663 is a Novel Regulator of Human Vascular Smooth Muscle Cell Phenotypic Switch in vitro and Neointimal Formation in vivo
Authors: Li Pan , Yi Bing , Qin Qing , Chen Ming , You Xiaohua , Zhao Xianxian , Qin Yongwen , Sun Jianxin
Published: July 2021

Background

Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Recently, microRNAs (miRNAs) emerge as critical regulators for vascular smooth muscle cell (VSMC) function. Our initial study identified miR-663 as one of the most sharply downregulated miRNAs in human proliferative aortic smooth muscle cells.

Hypothesis

MiR-663 is implicated in human VSMC phenotypic switch and the development of neointima formation.

Methods and Results

By using quantitative real-time PCR (qRT-PCR), we found that microRNA-663 (miR-663) was significantly downregulated in cultured human aortic VSMCs upon platelet-derived growth factor (PDGF) treatment, whereas its expression was markedly increased during VSMC differentiation as induced by either retinoid acid or SMC differentiation medium, a condition which induces SMC differentiation and inhibits cell proliferation. Furthermore, we demonstrated that overexpression of miR-663 significantly increased the expression of VSMC differentiation marker genes, such as SM22α, SM α-action, calponin, and SM myosin heavy chain, suggesting that miR-663 is a novel modulator implicated in human VSMC phenotypic switch. Moreover, miR-663 potently inhibited PDGF induced VSMC proliferation and migration. Mechanistically, we identified JunB as a downstream target of miR-663 in human VSMCs. Indeed, overexpression of miR-663 markedly inhibited the expression of the transcription factor JunB as well as its downstream molecules including matrix metallopeptidase-9 (MMP-9) and myosin light chain-9 (Myl9), thus inhibiting VSMC proliferation and migration. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by approximately 50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression.

Conclusion

These results indicate that miR-663 is a novel modulator implicated in human VSMC phenotypic switch through targeting JunB expression and suggest that specific modulation of miR-663 in human VSMCs may represent a novel and attractive approach for the treatment of vascular proliferative diseases.
Abstract 214: Protein Restriction Attenuates Intimal Hyperplasia and Alters Blood Lipid Profiles
Authors: Mauro Christine R , Tao Ming , Yu Peng , Liu Chengwei , Mitchell James , Zheng Hanqiao , Kristal Bruce S , Bird Susan S , Ozaki C. Keith
Published: July 2021

Introduction

Dietary restriction (DR: reduced enteral intake without malnutrition) diminishes the acute phase response to surgical stress in preclinical models. We hypothesized that short-term preoperative protein-free DR would attenuate the vascular response to injury (intimal hyperplasia: IH) while perturbing systemic circulating lipids.

Methods

After 2 weeks of 60% fat kcal diet, 8 week old B6D2F1/J mice had ad lib access to a complete 10% fat kcal diet (n=10) or a reduced calorie, protein-free diet (DR group, n=10) 1 week before induction of IH, then an ad lib complete diet. IH was induced via a validated model placing a nylon suture tie around the distal carotid artery and external 35g needle mandrel (outer diameter=0.14mm). Subsequent removal of the mandrel created a focal area of stenosis (~78% lumen diameter/~85% flow reduction). 4 weeks later, tissues were harvested for morphology and immunohistochemistry for CD45. Separately, serum was collected from mice fed a complete or DR diet for 1 week absent any surgical stress (n=20/group) for mass spectrometry-based lipidomics.

Results

DR mice showed less intimal area (p = 0.032) vs controls with statistically equivalent intimal leukocyte infiltration. DR mice also had significantly larger internal elastic lamina length (p = 0.003), a remodeling measure. DR serum exhibited significant decreases in certain classes of circulating lipids, including a collapse of multiple triglyceride types.

Conclusions

One week of protein-free DR dramatically decreased circulating lipids and attenuated arterial IH. Preoperative dietary manipulations may offer a practical means of extending durability of vascular interventions.
Abstract 215: Targeting MT1-MMP via Polymeric Transfection is More Efficient to Inhibit the Cellular Processes of Intimal Hyperplasia
Authors: Mountain Deidra J , Arnold Joshua D , Kirkpatrick Stacy S , Stevens Scott L , Goldman Mitchell H , Freeman Michael B , Grandas Oscar H
Published: July 2021

OBJECTIVE

Polymeric transfection could be a valuable therapeutic tool as it may be less toxic and more predictable than traditional methods, which are often poorly translated to in vivo models. Matrix metalloproteinases (MMP), in particular MT1-MMP, play a role in intimal hyperplasia (IH) and their targeted inhibition should be examined in vivo for the prevention of IH. Here we established conditions for polymeric transfection in vascular cells and ex vivo tissue and assayed its efficiency in targeting MMP-modulated cellular processes of IH.

METHODS

Vascular smooth muscle cells (HASMC) and rodent carotid artery segments were transfected with poly(β-amino ester) polymers (Stem) or equivalent DharmaFECT2 (Dharm) conjugated to GAPDH, negative control (NC) or MT1-MMP siRNA.

RESULTS

Bioconjugate translocation was observed at 0.65μl polymer: 100pmol siRNA/ml (Fig 1A). Gene silencing was polymer concentration dependent in vitro, and the most efficient bioconjugate (1.95μl polymer: 100pmol siRNA/ml) resulted in significant silencing ex vivo (Fig 1B). Using these empirically derived conditions, polymeric silencing of MT1 resulted in increased gene knockdown (Fig 2A) and inhibition of HASMC migration (Fig 2B) and proliferation (Fig 2C) compared to Dharm.

CONCLUSIONS

HASMC and arterial tissue were successfully transfected, and silencing of MT1 via polymeric transfection was more effective in gene knockdown and in inhibiting the cellular processes of IH development in vitro when compared with traditional techniques. Future studies using targeted polymeric gene therapy to inhibit MT1 in animal models may modify vascular response to injury and prevent vascular disease.
Abstract 221: Integral Role of Akt-Serum Response Factor-Myocardin Signaling in Myofibroblast Differentiation with Implications in Pulmonary Arterial Hypertension
Authors: Abdalla Maha , Goc Anna , Segar Lakshman , Somanath Payaningal R
Published: July 2021

Objectives

Myofibroblast (MF) differentiation is marked by the
de novo
expression of smooth muscle alpha-actin (αSMA) stress fibers, a function critical for tissue repair both physiologically and pathophysiologically. However, the role of Akt in regulating MFs with implications in pulmonary arterial hypertension (PAH) and fibrosis, and the effect of pharmacological inhibition of Akt pathway on PAH and fibrosis remains poorly defined.

Methods

We used NIH 3T3 fibroblasts and primary human lung fibroblasts (HLFs) in the current study. Cells were treated with TGFβ, the profibrotic cytokine and the role of Akt, SRF and myocardin in αSMA expression, MF differentiation and collagen gel contraction was studied with the use of various constructs encoding expression of specific genes.

Results

Here, we show that Akt mediates MF differentiation as evident by a 5-fold increase in αSMA expression and assembly in mouse embryonic fibroblasts (NIH 3T3) over-expressing active Akt1 (myr-Akt). As for the molecular mechanisms governing MFs, we found that αSMA expression was mediated by Akt, independent of mTOR; noteworthy, fibronectin expression, an ECM protein, is Akt-mTOR dependent. Inhibiting Akt activation utilizing triciribine blunted αSMA and its transcriptional activators, myocardin and SRF, inhibiting MF differentiation in NIH 3T3, myr-Akt-NIH 3T3, and human lung fibroblasts. Furthermore, blocking Akt reversed MF differentiation as evident by a blunted gel contraction primed with TGFβ.

Conclusions

In conclusion, our study clearly demonstrates the crucial role of Akt in mediating TGFβ-induced MF differentiation. We show a previously uncharacterized link between Akt and TGFβ-induced αSMA synthesis through myocardin and SRF, independent of mTOR, in MFs. These data constitute evidence of novel signaling cascade and targeting Akt pathway might be beneficial for the treatment of PAH and fibrosis.
Abstract 223: Is Oxidative Stress Important in AAA pathogenesis?
Authors: Urbonavicius Sigitas , Urbonaviciene Grazina , Sandermann Jes , Lindholt Jes S
Published: July 2021

OBJECTIVE

Active investigations continue to identify markers other than size that would predict a risk of abdominal aortic aneurysms (AAA) rupture. Circulating biomarkers could also indicate optimal intervals between the surveillance intervals. Finally, the identification of biomarkers also may identify potential pathogenic pathways, and thus may open possibilities for pharmacological inhibition of growth. In the search of novel biomarkers of AAA progression, serum and wall material proteins were analyzed by a differential proteomic approach.

METHODS AND RESULTS

The same layers of AAA wall from ruptured (rAAA) and non-ruptured AAA were incubated, and the proteins released were analyzed by 2-dimensional difference in-gel electrophoresis. Proteins from serum were analyzed and correlated with AAA annual expansion rate. Several differentially expressed proteins involved in main AAA pathological mechanisms were identified by mass spectrometry. Among the 8 proteins identified in AAA wall, peroxiredoxin-2 (PRX-2) was more permanent, which was further validated by Western blot and immunohistochemistry. We demonstrated increased PRX-2 levels in rAAA patient wall material compared with AAA subjects and also positive correlation in serum among PRX-2 and AAA diameter and annual AAA expansion rate.

CONCLUSIONS

Eight proteins associated with AAA pathogenesis have been identified in AAA wall by a proteomic approach. Protein profiles identified in the wall from the rAAA and the serum would appear to be a convenient monitoring tool that has the ability to be both sensitive and specific for AAAs. Among them, PRX-2 serum levels are increased in AAA patients and correlate with AAA size and growth rate, suggesting the potential use of PRX-2 as a biomarker for AAA evolution. Characterizing such diagnostic protein profiles, combined with known risk factors as well as emerging genetic data, would contribute to the long-term objective of applying effective non-surgical therapies to the treatment of small AAA disease. Larger studies are required to confirm the potential and clinical role of the identified proteins.
Abstract 226: Thyroid Hormone Stimulated Arteriolar Growth Along With Myocyte Hypertrophy Following Myocardial Infarction In Rats
Authors: Chen Yuefeng , Guo Songchuan , Carrey Zev , Alaie Dariush , Petrillo Richard , Gerdes Anthony M
Published: July 2021

Background

Thyroid hormones (THs) have been shown to improve LV function and remodeling in animals post-MI and in the human setting, as well as induce capillary and arteriolar growth in the hypertrophic

heart. Whether THs can improve arteriolar growth in the infarcted heart is unknown.

Methods

MI was produced by ligation of the LAD in female SD rats. Rats were divided into the following groups: (1) Sham MI, (2) MI, and (3) MI+T4 treatment (T4 pellet 3.3mg, 60 days release, implanted subcutaneously immediately following MI). Each group was further divided into two subgroups (n=9 per subgroup) for myocytes isolation and whole heart tissues collection eight weeks after surgery. Isolated myocyte shape and arteriolar density in the non-infarcted area were measured at terminal study.

Results

T4 treatment improved LV ±dp/dt, normalized TAU, and increased myocyte cross-sectional area without further increasing myocyte length in MI rats. T4 treatment tended to increase the total length of smaller arterioles (5 to 15 μm) proportional to LV weight increase.

Conclusions

These results suggest that long-term T4 treatment after MI has beneficial effects on myocyte and arteriolar remodeling in the non-infarcted area.
Abstract 270: Acute Reductions in Wall Strain Precede Formation of Intimal Hyperplasia
Authors: Favreau John T , Liu Chengwei , Yu Peng , Mauro Christine , Tao Ming , Gaudette Glenn R , Ozaki C. K
Published: July 2021

Intimal hyperplasia (IH) remains the major culprit in revascularization failures. We aimed to unravel relationships between acute changes in circumferential arterial wall strain and genesis of IH.

Methods

To induce IH, we employed a validated model using a 9-0 nylon suture tie around the distal mouse common carotid artery (n=10) and an external 35-gauge needle mandrel (OD=0.14mm), with subsequent removal of the mandrel to create a distal common carotid focal stenosis (~78% lumen diameter/~85% flow reduction). Wall strains were measured in three, 1 mm wide regions along the vessel proximal to the focal stenosis at pre-op day 1 and at post-op day 4 (before detectable IH) using Vevo 2100 ultrasonography with VevoVasc software. At post-op day 28, arteries were perfusion fixed and IH was assessed in the same regions as those where strain was analyzed. Strain and morphology were also assessed in the contralateral control artery.

Results

Decreased wall strain was noted in all regions proximal to the focal stenosis from 0.26 ± 0.01 to 0.11 ± 0.02 (p<0.001) with no change in the control artery from pre-op to post-op day 4 (p=0.45). Based on a strain level histogram, vessels were divided into groups with strain ≤0.1 and >0.1. All segments (n = 13) with wall strain ≤0.1 at post-op day 4 had significant IH at day 28. In regions with strains >0.1 at day 4, only 30% had IH at day 28. The average pre-op strains were identical in >0.1 and ≤0.1 strain groups (0.27 ± 0.09 and 0.27 ± 0.08). Mean intimal thickness in vessels with strain ≤0.1 was 32 ± 20 μm, significantly greater than 8.0 ± 16 μm in the group with strain >0.1 (p<0.01). To further understand the mechanisms underlying changes in strain, systolic and diastolic lumen areas were assessed. Although systolic lumen areas in both >0.1 and ≤0.1 groups remained unchanged from pre-op to post-op day 4 (p=0.46), diastolic area was significantly increased in regions with post-op day 4 strain ≤0.1 (p=0.04) but remained unchanged in mice with post-op day 4 strain >0.1 (p=0.67).

Conclusions

Acute reduction in wall strain precedes the formation of IH in this murine model and this change is primarily caused by an increase in diastolic lumen area. Manipulations of wall strain offer a strategy to prevent and attenuate occlusive IH lesions after revascularizations.
Abstract 273: New Reconstruction Technique for MRA to “Fly Through” the Lumen of the Cerebral Vasculature
Authors: Kasam Mallikarjunarao , Ketha Siva S , Konduru Soumya
Published: July 2021

Methods

Patient exams were performed under an IRB-approved protocol. Two 3D SWIRLS [3] single phase contrast-enhanced angiographies with 1mm3 spatial resolution were used for this work. Clinical exams were acquired on 3.0 T scanners (GE, DVMR 20.1IB, Milwaukee, WI).

Data analysis

The angiogram was post-processed using tree analysis and virtual endoscopy modules of Analyze 11.0 software (Analyze 11.0; Biomedical Imaging Resource, Mayo Clinic, Rochester, MN) to fly through the artery. A statistical parameter called Brightness Area Product (BAP) was defined as the sum of the intensities above the sample minimum intensity/threshold set by the user.

Results and Discussions

Figures (I-IV) show the methodology to fly through the part of anterior carotid artery (ACA) and all statistics of part of the artery (Fig. IV) with angles from the “Root” to its different branches of A1 to G2 (Fig. III). Figure II shows the snap shot of the fly through/internal cross sectional structure of ACA of branch “E2”, at the location marked as “red dot”. Table (IV) represents different statistics including length, angle from the root, area of cross section and the BAP of the branches mentioned in Fig. III. The computation time for this method is < 5 minutes compared to the other reconstruction techniques (>~30 minutes). We proposed a simple and fast automated post-processing method to fly through the artery using Analyze 11.0 software. This technique can be extended to any arteries to monitor internal vasculature and to estimate the stenosis using the statistical parameter BAP, which generates a master profile/database of arteries.

References

1 Shu Y, Bernstein MA, ISMRM 2009 p764.
Abstract 279: Role of Caveolin-1 in Abdominal Aortic Aneurysm induced by Angiotensin II
Authors: Takayanagi Takehiko , Crawford Kevin , Kobayashi Tomonori , Rizzo Victor , Eguchi Satoru
Published: July 2021

Abdominal aortic aneurysm (AAA) is a significant cause of mortality for adults aged >60 years. Accumulating evidence suggests that activation of the AT1 receptor by angiotensin II (AngII) in AAA formation. While several downstream signals and target proteins have been identified in this pathway, there is a huge void in our knowledge regarding the AngII-sensitive proximal events primarily responsible for AAA formation. We recently reported that caveolae membrane microdomains in vascular smooth muscle cells (VSMC) mediate a metalloprotease ADAM17-dependent EGF receptor (EGFR) transactivation which linked to vascular remodeling induced by AngII. Given that ADAM17 expression is one of the key features in AAA, we have tested our hypothesis that caveolin-1 (Cav1), a major structural protein of caveolae, in the vasculature plays a critical role for development of AAA via its regulation on ADAM17. 8 week old male Cav1-/- mice and the control C57Bl/6 wild-type (WT) mice were co-infused with AngII and BAPN, a lysyl oxidase inhibitor, to induce AAA. We found that Cav1-/- mice did not develop AAA compared to C57Bl/6 mice in spite of hypertension assessed by telemetry in both groups. This finding suggests that the AngII signaling essential for vascular contraction remains in place in Cav1-/- mice. We found an increased expression of ADAM17 and auto-phosphorylation of EGFR in WT abdominal aortae with aneurysms that were markedly attenuated in Cav1-/- mice infused with AngII+BAPN. Furthermore, Cav1-/- mice with the infusion showed less oxidative stress and ER stress than their WT counterparts as assessed by nitrotyrosine staining and KDEL/p-eIF2a staining, respectively. In conclusion, Cav1 and presumably vascular caveolae micro-domain appear to play a critical role in the formation of AAA in mice via regulation of the ADAM17/EGFR signaling axis and subsequent induction of ER/oxidative stress.
Abstract 282: Pattern of Development of Abdominal Aortic Aneurysms Induced by Angiotensin In Hypercholesterolemic Mice
Authors: Prins Petra A , Hill Michael , Airey David , Nwosu Sam , Perati Prudhvidhar R , Kon Valentina , Fazio Sergio , Sampson Uchechukwu
Published: July 2021

Background

Understanding the pattern of development of abdominal aortic aneurysms (AAA) may help optimize translational research strategies. Thus we sought mechanistic insight on the pattern of AAA evolution in apolipoprotein E -/- mice.

Methods

Fifty-nine mice underwent a 4-week pump-mediated infusion of saline (controls; n=13) or 1500 ng/kg/min of AngII (n=46), and AAA development was tracked via in vivo serial ultrasound imaging. We adjusted for hemodynamic covariates in the mixed logistic regression model for AAA occurrence in relation to time.

Results

The incidence of intact AAA in AngII infused mice was 59% (27/46) of which 55.6% (15/27) were small AAA and 44.4% were large AAA (1.2-1.5 vs. >1.5x baseline diameter, respectively). The incidence of rupture was 22% (10/46). All large aneurysms and most ruptures occurred within the first week of AngII exposure. Subsequent aneurysms were small and evolved in the second week of AngII infusion. The overall effect of time was statistically significant (p<0.001) (Figure). When compared to day 7 of the study period, there was no decrease in the log odds of AAA occurrence by day 14 (-0.234, p = 0.65). However, compared to 21 and 28 days into AngII infusion, the log odds of an AAA occurrence decreased by -9.07 (p< 0.001) and -2.35 (p= 0.04), respectively (Figure).

Conclusions

These are the first formal estimates of the odds of AAA occurrence vis-à-vis time in the context of a consistent pattern of AAA development in this mouse model of AngII-induced AAA. They occur in the first (large AAA) and second (small AAA) weeks of AngII infusion, and the odds of occurrence diminish thereafter. These findings provide guidance for experimental strategies.
Abstract 283: Platelet Inhibition Increases Angiotensin II-induced Abdominal Aortic Aneurysm Incidence and Rupture in Mice
Authors: Owens A. Phillip , Boulaftali Yacine , Bergmeier Wolfgang , Luyendyk James P , Mackman Nigel
Published: July 2021

Objective

Platelets play a central role in both hemostasis and thrombosis. The coagulation protease thrombin activates platelets by cleavage of protease-activated receptors (PAR1 and PAR4 in humans, and PAR3 and PAR4 in mice). Circulating thrombin is increased in patients with abdominal aortic aneurysms (AAAs). We recently demonstrated that PAR4 deficiency in mice increased the incidence of aneurysm (P = 0.001) and rupture-induced death (P = 0.003) in an angiotensin II (AngII) infusion model of AAA. Furthermore, platelet depletion significantly increased rupture in this model (P = 0.048). The purpose of this study was to examine clinically used anti-platelet drugs in this mouse model of AAA.

Methods and Results

Male
Ldlr
-/-
mice (8-12 weeks in age) were fed a fat and cholesterol-enriched diet (21% milk fat, 0.2% cholesterol). Groups of mice received either aspirin (30 mg/L via drinking water [ASA]), or diet supplemented with the direct thrombin inhibitor dabigatran etexilate (10 g/kg chow [DE]) or the P2Y
12
inhibitor clopidogrel (50 mg/kg/day [Plavix]) 1 week prior to and throughout AngII (1,000 ng/kg/min) infusion for 28 days. Drug bioavailability was confirmed with all treatments. Medial diameters in the suprarenal aortic region were increased significantly from baseline to day 28 in all groups infused with AngII, as measured by in vivo ultrasound. Medial diameters were not different in any of the treatment groups compared with placebo controls. However, DE (87% vs. 47%) and Plavix (82% vs. 40%) significantly increased the incidence of AAA versus placebo groups (P < 0.05). ASA also increased the incidence of AAA (93% vs. 70% P = NS). Importantly, all treatments had a significant increase in aortic rupture-induced death versus placebo groups (P < 0.05; DE [67% vs. 7%]; Plavix [41% vs. 0%]; and ASA [64% vs. 10%]). None of the treatments affected total plasma cholesterol, lipoprotein-cholesterol distributions, or AngII-induced increases in systolic blood pressure.

Conclusion

This study indicates that the presence of functional platelets reduces the formation and rupture of AAA in this mouse model. This suggests that inhibition of platelet function may be detrimental to patients with existing AAAs, a conclusion that will be addressed in future mouse studies.
Abstract 29: Efficient Differentiation and Polarization of Macrophages Differentiated From Human Peripheral Blood Mononuclear Cells-derived iPS Cells
Authors: Zhang Hanrui , Shah Rhia , Hinkle Christine , Yang Wenli , VanDorn Daniel , Chou Stella , Reilly Muredach P.
Published: July 2021

Macrophages fulfill homeostatic functions beyond defense. Monocytes/macrophages derived from human induced pluripotent stem cells (hiPSCs) are useful tools to study vascular diseases and potential sources of cell-based therapies. The objective of this study is to generate PBMC-hiPSC derived monocytes, macrophages, as well as M1 and M2 type differentiated macrophages and to examine multiple macrophage characteristics of relevance to cardio-metabolic disease. We briefly cultured human adult PBMCs from healthy volunteers to enrich for the erythroid progenitors. These progenitors were used to generate hiPSCs using lentivirus-mediated transduction of Oct3/4, Sox2, Klf4 and c-Myc. A stepwise protocol was used for the differentiation of PBMC-hiPSCs to monocytes/macrophages. The protocol involves primitive streak and mesoderm induction (stage 1), hematopoietic specification (stage 2), hematopoietic cell maturation and myeloid expansion (stage 3), monocytes collection and differentiation to macrophages (stage 4). Differentiation cultures were set up in six-well plate format, from 20 embryoid bodies per well, and produced up to 30 million macrophages. hiPSC-derived macrophages (hiPSC-MΦ) exhibited spindle morphology and incorporated acetylated low-density lipoprotein. Immunocytochemical analysis of hiPSC-MΦ revealed positive staining for CD68, CD11b, and CCL2. FACS analysis indicates >90% expression of macrophage lineage markers, including CD45, CD14, CD16, CD115, CD68, CX3CR1, CD11b, and CD18. Migration of hiPSC-MΦ in response to M-CSF was confirmed by Boyden Chamber assay. hiPSC-MΦ were polarized to M1 (classical activation stimulated by TLR ligands LPS and IFN-gamma) and M2 (alternative activation stimulated by IL-4) types. M2 macrophages were characterized by more efficient phagocytic activity of zymosan particles and greater migration capacities compared with M1 macrophages. The results demonstrated the feasibility of differentiation/polarization of macrophages from PBMC-hiPSCs. This study advanced our understanding of the suitability of subject-specific hiPSC for deriving myeloid/monocyte/macrophages for study of monocyte-macrophage functions of direct relevance to cardio-metabolic disease.
Abstract 294: Expression of Matrix Metalloproteases during the Differentiation of Porcine Adopse-Drived Mesenchymal Stem Cells ro Endothelial Cells
Authors: Almalki Sami G , Rao Velidi , Pankajakshan Divya , Agrawal Devendra K
Published: July 2021

Rationale

Adipose-derived mesenchymal stem cells (ADMSCs) are multipotent cells that have the potential to differentiate into different cell linages, and represent promising tools in various clinical applications. However, the molecular mechanisms that control the ability of ADMSCs to remodel 3-dimensional extracellular matrix (ECM) barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of ADMSCs to endothelial cells (ECs)
in
vitro
.

Methods

MSCs were isolated from porcine abdominal adipose tissue, and characterized by positive staining for MSC markers, CD44, CD73, CD90, and negative staining for CD11b, CD34 and CD45. The plasticity of MSCs was detected by bi-lineage differentiation to osteocytes, and adipocytes. The mRNA transcripts for different MMPs and TIMPs and protein expression of EC markers were analyzed by RT-PCR and immunostaining. The enzyme activity and protein expression were also analyzed by gelatin zymography, ELISA, and Western blot.

Results

The differentiation of ADMSCs to ECs was confirmed by the positive staining and mRNA expression of the endothelial markers. The mRNA transcripts for MMP-2 and membrane type 1 MMP (MT1-MMP) was significantly increased by 2.5 and 2.0 fold, respectively, during the differentiation of MSCs into ECs. Western blot and ELISA showed an elevated MT1-MMP and MMP-2 expression. The enzyme activity of MMP-2 was also observed by gelatin zymography.

Conclusion

We demonstrated that porcine ADMSCs have the ability to differentiate into ECs, and this process involves the up-regulation of MMP-2 and MT1-MMP. The increase in the expression of MMP-2 and MT1-MMP may, at least partially, facilitate the change in morphology of MSCs by degrading the ECM barriers. These findings may provide a potential mechanism for the role of MMP2 and MT1-MMP in the differentiation of ADMSCs into ECs.
Abstract 296: The RNA-Binding Protein Quaking Maintains Endothelial Barrier Function Through Targeting Vascular Endothelial Cadherin mRNA
Authors: van Gils Janine M , de Bruin Ruben G , Schmidt Iris , van der Veer Eric P , Roeten Marko K , Rabelink Ton J , van Zonneveld Anton Jan
Published: July 2021

Endothelial cells (ECs) form a vital barrier between the blood and the artery wall, and play a major role in the onset of atherosclerosis. Endothelial monolayer integrity is determined largely by EC-cell interactions, where vascular endothelial cadherin (VE-cadherin) is the central adhesive component at these endothelial adherens junctions. While many of the receptors and signaling proteins involved in regulating these adherens junctions have been identified, surprisingly little is known regarding their regulation at the post[[Unable to Display Character: ‐]]transcriptional level. The RNA-binding protein Qauking (QKI), originally known for its function in the nervous system, has been demonstrated to be essential for blood vessel formation. We find that QKI is highly expressed in quiescent ECs, in vitro and in vivo. In contrast, human umbilical vein ECs displayed reduced levels of QKI in response to the inflammatory stimuli TNF-α as well as in cells lacking cell-cell contacts, suggesting that QKI may act to enhance barrier function. To test this, we specifically abrogated QKI expression in ECs and measured their capacity to form a high-resistance monolayer with Electrical Cell-substrate Impedance Sensing. Silencing of QKI did not affect EC adhesion or spreading, but markedly affected the capacity to form a high resistance endothelial monolayer. Consistent with these data, and the fact that VE-cadherin mRNA contains a putative QKI-response element, the targeted reduction in QKI was accompanied by a significant reduction in VE-cadherin expression at cell junctions. Importantly, we identified a direct role for QKI in regulating VE-cadherin mRNA biology, as RNA immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the VE-cadherin mRNA and regulate transcript stability, respectively. In conclusion, we show that the modulation of QKI expression levels affects endothelial monolayer integrity by functioning as a critical regulator of the VE-cadherin mRNA. These studies provide novel insight into a role for post-transcriptional regulation in the maintenance of endothelial barrier function, and may have wide ranging implications for the preservation of vascular integrity in disease.
Abstract 303: CD44 Activation Protects Human Sclerotic-derived Valvular Interstitial Cells from Calcification
Authors: Poggio Paolo , Branchetti Emanuela , Sainger Rachana , Grau Juan , Lai Eric , Ferrari Giavanni
Published: July 2021

Introduction.

The activation of Valvular Interstitial Cells (VIC) towards an osteoblast-like phenotype is a cellular hallmark of pathological progression towards Aortic Stenosis (AS). In recent years several clinical trials have failed to halt or revert the progression of this prevalent disease. The ability to prevent end-stage AS requires the understanding of the molecular events associated with the early phase of valve degeneration, a condition known as Aortic Sclerosis (ASc). In the last few years the transmembrane receptor CD44 has been studied as a putative molecule for cardiovascular drug therapy. We reported that the functional interaction between CD44 and one of its ligand, Osteopontin (OPN), protects vascular smooth muscle cells from calcification. More recently, we demonstrated that sclerotic tissues show increased expression of Bone Morphogenetic Protein 4 (BMP4) and BMP4 directly stimulates osteoblast-like transdifferentiation and calcification of VICs. Therefore we hypothesized a direct role of CD44 activation in protecting human Aortic Sclerosis-derived VICs from calcification.

Methods.

Human VICs from Control, ASc, and AS (n=5 each group) were isolated. Histological, cellular and molecular analysis, and in situ Proximity Ligation Assay were used to investigate the role of CD44 and OPN in VIC calcification. BMP4 treatments were used to promote VIC activation. Osteoblast-like transdifferentiation was analyzed using Alkaline Phosphatase (ALP) expression.

Results.

CD44 and OPN, as well as their functional binding, were increased in sclerotic and stenotic tissues compared to healthy controls in vitro and ex vivo. CD44-OPN binding prevented in vitro calcification induced by inorganic phosphate on human ASc-derived VICs. A neutralizing antibody against CD44, under BMP4 treatments, promoted calcium deposition along with increased expression of OPN and ALP.

Conclusion.

Our results generate an important insight into the molecular mechanism of VIC calcification. We proved that CD44-OPN direct interaction inhibits calcification of Aortic Sclerosis-derived VICs, suggesting that CD44 activation could have a protective role against VIC osteoblast-like transdifferentiation and calcification in the early stage of the disease.
Abstract 307: Deficiency of Lim-only Protein Fhl2 Accelerates Neointima Formation After Carotid Artery Ligation
Authors: Kurakula Kondababu , de Waard Vivian , van Tiel Claudia M , de Vries Carlie J
Published: July 2021

Rationale

The LIM-only protein FHL2, also known as DRAL or SLIM3, has a regulatory function in many physiological processes and is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells, but not in macrophages. FHL2 regulates SMC phenotype, but its function in vascular injury is unknown.

Objective

To assess the role of FHL2 in SMC-rich lesion formation after vascular injury and to elucidate the underlying mechanism.

Methods & Results

Cultured aortic SMCs from FHL2-KO mice showed increased migration and increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. In agreement with this, overexpression of FHL2 in SMCs reduced CyclinD1 expression. In addition, FHL2-KO SMCs showed enhanced expression of pro-inflammatory cytokines in a NFkB dependent manner. Consistent with these findings, NFkB activity is higher in FHL2-KO SMCs. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice developed accelerated lesion formation compared with wild-type (WT)-mice. Furthermore, FHL2-KO mice displayed high number of macrophages in lesions and enhanced expression of RANTES and stromal derived factor-1α (SDF-1α) in ligated carotid arteries. SDF-1α expression was also increased in plasma of FHL2-KO mice. Finally, FHL2-KO mice showed enhanced Ki67 expression in lesions compared with wild-type (WT)-mice.

Conclusion

FHL2 deficiency in mice results in an exacerbated neointima formation by enhanced proliferation and migration of SMCs, possibly regulated via ERK1/2 and NFkB pathway.
Abstract 326: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix Stores and Induces Vascular Permeability in vivo
Authors: Hendel Alon , Granville David J
Published: July 2021

Introduction

The formation of unstable and leaky neovessels underlies the pathogenesis of a large number of chronic inflammatory diseases. Granzyme B (GZMB) is a serine protease that is expressed and released by a variety of immune cells and accumulates in the extracellular matrix (ECM) during chronic inflammation where it cleaves a number of ECM proteins, including fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM by binding FN in both normal and diseased tissue. We hypothesize that GZMB cleavage of FN will release VEGF from its extracellular stores and promote vascular permeability as a mechanism that contributes to neovessel leakage during chronic inflammation.

Methods

GZMB-mediated VEGF release from either FN coated wells or endogenously produce endothelial cell (EC) matrix was measured by VEGF ELISA. VEGF-release supernatants were used to treat EC and VEGF receptor 2 (VEGFR2) activation was evaluated by immunoblotting for phosphorylated VEGFR2. Evan’s blue was injected intravenously to CD1 mice followed by ear injection of either mouse GZMB, saline control, GZMB + neutralizing mouse VEGF antibody or GZMB+ IgG control (n=5 for each experimental group). Vascular leakage was evaluated by Evan’s blue dye extraction.

Results

GZMB effectively releases VEGF from both FN and from EC matrix, while inhibition of GZMB prevented VEGF release. GZMB-mediated VEGF release resulted in significant activation of VEGFR2 in EC monolayer signified by increased VEGFR2 phosphorylation. GZMB ear injection resulted in a significant increase in vascular permeability in vivo. Importantly, co-injection of GZMB and neutralizing mouse VEGF antibody significantly reduced vascular leakage compared to co-injection of GZMB and matching IgG control.

Conclusions and Impact

GZMB increases VEGF bioavailability by releasing it from the ECM leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.
Abstract 328: Viral Anti-Atherogenic Proteins Alter Monocyte and Neutrophil Invasion in Mice and Bag3 Gene Expression
Authors: Davids Jennifer , Saikaly Sami , Lucas Alexandra
Published: July 2021

Aim

Atherosclerosis is characterized by chronic inflammation and cell death (apoptosis). Serine protease inhibitors, or serpins, regulate inflammatory, thrombotic and apoptotic pathways. Poxviruses encode cross-class serpins that prevent host cell apoptosis. Serp-2, from Myxoma, reduces plaque, inflammation and apoptosis in animal models; CrmA, from Vaccinia, does not. Both serpins target Caspase 1 and Granzyme B, but the reasons for the differing effects in vivo are unknown. In prior research three Myxoma viral proteins, including Serp-2, reduced monocyte invasion, plaque growth, and aneurysm formation in ApoE-/- mice after angioplasty. These same proteins alter expression of a shared cohort of 48 apoptosis-related genes in human monocytes treated with camptothecin. This study assesses the effects of Serp-2 and CrmA on apoptotic gene expression in a mouse peritoneal inflammation model.

Methods

Mouse strains deficient for Granzyme B (GzmB, N=13) and Caspase 1 (Casp1, N=15) were compared to their respective background mice (C57Bl/6 and Nod, N=15 each) 18 hours after treatment with PMA and either Serp-2 or CrmA. RNA was isolated and analyzed by RT-PCR and normalized to GAPDH, then to the PMA-only treated control.

Results

Compared to human monocytes, mouse peritoneal exudates from knockout mice displayed differential alteration of BCL2-associated athanogene 3 (BAG3) in response to treatment with Serp-2 or CrmA. Serp-2 treatment reduced expression levels compared to CrmA treatment in GzmB (p=0.0267) and C57Bl/6 mice (p=0.0280). Casp1-/- mice treated with Serp-2 downregulate BAG3, expressing 9.7-fold less than Nod mice (p=0.0006), but CrmA had no significant effect in either strain. An associated differential migration of Ly6Chi and Ly6Ghi cells was also discovered in knockout mice with serpin treatments.

Discussion

One candidate gene found in human monocytes, BAG3, has reduced gene expression after Serp-2 treatment in mouse peritoneal exudates but increased by CrmA. BAG3 is known to alter cell migration and apoptosis, important to atherosclerotic progression. BAG3 is regulated by 3 myxomaviral proteins in human and mouse cells, underscoring a potential role as a lynchpin in viral protein anti-inflammatory and anti-apoptotic pathways.
Abstract 331: Caeolin-1 Deficiency Increases LDL Cholesterol Uptake but Attenuates Inflammation of Endothelial Cells
Authors: Aryal Binod , Lin Chin-Sheng , Salerno Alessandro , Canfran Duque Alberto , Aranda Gomez Juan Francisco , Fernandez-Hernando Carlos
Published: July 2021

Caveolin-1 (Cav-1) is the major structural protein essential to the formation of the caveolae in endothelial cells. Cav-1 is thought to play an important role in the regulation of cellular cholesterol homeostasis, a process that needs to be properly controlled to limit and prevent cholesterol accumulation and eventually atherosclerosis. Genetic ablation of Cav-1 on an apoE knockout background inhibits the progression of atherosclerosis, while re-expression of Cav-1 in the endothelium promotes lesion expansion by affecting several processes including endothelial cell proliferation, migration, and nitric oxide production in vitro and increased expression of vascular cell adhesion molecule-1. Specifically, in vivo study shows that loss of Cav-1 reduces LDL infiltration into the artery wall and inhibits progression of atherosclerosis. However, surprisingly, we found that knock down of Cav-1 in human endothelial cells increases the binding and uptake of diI-LDL in vitro suggesting that Cav-1 decreases transcytosis of LDL. Moreover, Caveolin-1 has also been implicated in the regulation of inflammation in endothelial cells. Here we show that Cav-1 affects the induction of cell adhesion molecules in endothelial cells by inflammatory mediators including TLR2 ligand Pam3cys, IL-1B, and TNF. Specifically, knock-down of Cav-1 in human endothelial cells decreases the induction of ICAM-1, VCAM-1 and SELE in both mRNA and protein levels. Furthermore, we also show that silencing of Cav-1 attenuates the major signaling pathways that are involved in the induction of cell adhesion molecules including MAPK, AKT, JNK-AP1 and NF-κB pathways.
Abstract 35: Macrophage Depletion Limits Experimental Aneurysm Progression
Authors: Xu Baohui , Xuan Haojun , Tanaka Hiroki , Glover Keith J , Nagai Taku , Fujimura Naoki , Iida Yasunori , Wang Wei , Furusho Yuko , Miyata Masaaki , Michie Sara A , Matsuyama Takami , Dalman Ronald L
Published: July 2021

Macrophage activity mediates aortic aneurysm pathogenesis. Aortic mural macrophage density, and activation status, may alter progression of existing abdominal aortic aneurysms (AAAs). We examined the consequences of macrophage depletion on aneurysm progression using the porcine pancreatic elastase (PPE) AAA model. Following PPE infusion, aortic diameter enlarged progressively through 14 days as measured via serial transabdominal ultrasound examinations in control mice. Total macrophage depletion via diphtheria toxin (DT) injection, initiated prior to PPE infusion in CD11b-diphtheria toxin receptor (DTR) transgenic mice, abrogated aortic enlargement. Initiation 4 days following PPE infusion promoted AAA regression. In separate experiments, depletion of activated macrophages, those characterized by folate-receptor β (FRβ) expression, was accomplished via FRβ-directed immunotoxin treatment in C57BL/6 mice. FRβ immunotoxin limited AAA progression in this construct. Both DT injection in DTR-transgenic mice, and FRβ immunotoxin treatment in C57BL/6 mice, preserved medial smooth muscle cellularity and elastin content, and reduced mural inflammation and neovessel formation on histopathologic analysis. In conclusion, modulation of mural macrophage cellularity may represent an effective method of non-surgical AAA disease management.
Abstract 350: Influence of HIV-1 Infection and Antiretroviral Therapy on the Coagulation System
Authors: Fasing Kevin , Weil Brian R , Diehl Kyle J , Greiner Jared J , Stauffer Brian L , Connick Liz , DeSouza Christopher A
Published: July 2021

HIV-1-infected individuals have a two- to four-fold greater incidence of cardiovascular disease and atherothrombotic events compared with the general population. The mechanisms responsible for this heightened cardiovascular risk are not fully understood. We have previously reported that the capacity of the endothelium to release tissue-type plasminogen activator (t-PA), the primary activator of fibrinolysis and a key endogenous defense mechanism against intravascular fibrin deposition and thrombosis, is impaired in HIV-1-seropositive adults. Whether diminished fibrinolytic activity is coupled with a hypercoagulative state in this population is unknown. Elevations in specific coagulation markers such as tissue factor and Factor VII are associated with increased thrombotic risk. The experimental aim of this study was to determine the influence of HIV-1 infection and antiretroviral therapy (ART) on markers of coagulation. To address this aim we studied 33 men: 16 HIV-1-seronegative (age: 41±3 yr); 7 HIV-1-seropositive treatment-naïve (34±2 yr); and 10 HIV-1-seropositive receiving ART (42±3 yr; efavirenz-based regimen). All subjects were non-obese, normotensive and free of overt cardiometabolic disease. Circulating concentrations of tissue factor, Factor VII, Factor VIII and Factor X were determined by immunoassay. There were no significant differences in plasma concentrations of tissue factor (32+3 vs 40+3 pg/mL), Factor VII (103+9 vs 100+5 %), Factor VIII (111+13 vs 117+8 %) and Factor X (89+5 vs 91+2 %) between HIV-1-seropositive treatment-naïve and healthy men. Moreover, there was no influence of ART on these circulating markers. Plasma tissue factor (41+5 pg/mL), Factor VII (107+6 %), Factor VIII (103+7 %) and Factor X (90+3%) were similar in the HIV-1-seropositive receiving ART compared with HIV-1-seropositive treatment-naïve and seronegative groups. These data suggest that neither HIV-1 infection per se nor ART are associated with unfavorable changes in specific coagulation markers. Thus, changes in the coagulation system that have been linked to increased thrombotic burden are not apparent in HIV-1-seropositive adults.
Abstract 354: Prevalence and Predictors of Non Alcoholic Fatty Liver Disease Among Normal Weight Non Diabetic Individuals
Authors: Sinha Pragya , Oni Ebenezer T , Jamal Omar , Blaha Michael J , Agatston Arthur A , Blumenthal Roger S , Nasir Khurram , Santos Raul
Published: July 2021

BACKGROUND

Nonalcoholic fatty liver disease(NAFLD) is generally considered a close manifestation of obesity and metabolic abnormalities. In the past, autopsy studies have also demonstrated NAFLD in 15% of non-obese subjects. Increasingly, NAFLD is considered an early manifestation of CAD. However, there is limited data on the prevalence and predictors of NAFLD in normal weight (NW) individuals.

METHODS

We evaluated 6,385 healthy non diabetic Brazilian subjects (43±10 years, 79% males) without clinical coronary heart disease between November 2008 and July 2010. NAFLD was diagnosed by ultrasound.

RESULTS

Overall 39% (n=2494) individuals (41±9 years, 62% males) had normal weight (BMI<25 kg/m
2
& normal waist circumference). The prevalence of NAFLD in this group was 11% compared to 43% in the overweight (BMI 25-29.9) and 79% in the obese (BMI≥30), respectively (p<0.0001). As shown in the table below, increasing age, male gender and metabolic risk factors (MRF) remained significant predictors of NAFLD in NW individuals in univariate and multivariate analyses. As compared to those with no known MRF, the adjusted odds ratio for presence of NAFLD with increasing risk factors (1,2 and 3) were 1.56 (1.13-2.14), 3.93 (2.75-5.75) and 3.81 (1.92-7.59) respectively.

CONCLUSION

Approximately 1/10th of normal weight non-diabetic individuals had NAFLD in our cohort. High triglyceride and low HDL were independent predictors of NAFLD. Increasing metabolic risk factor burden can be considered for further testing to identify those at risk for NAFLD in this group.