bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 516: Calpain-2 Compensation Promotes Angiotensin II-induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice
Authors: Subramanian Venkateswaran , Moorleghen Jessica J , Balakrishnan Anju , Howatt Deborah A , Chishti Athar H , Uchida Haruhito A
Published: March 2022

Background and Objective

We demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development.

Methods and Results

Male LDLr-/- mice that were either calpain-1 +/+ or -/- were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no effect on body weight or blood pressure during AngII infusion. Intimal areas of ascending aorta and maximum external width of the suprarenal abdominal aorta were measured by en face. Calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AA development (P = NS). Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410 along with AngII infusion significantly attenuated AngII-induced ascending (Vehicle - +/+: 15.0 ± 1.0, -/-: 14.9 ± 0.8; BDA: +/+: 11.5 ± 0.6, -/-: 12.1 ± 0.6 mm
2
; P<0.05) and abdominal (Vehicle - +/+: 1.7 ± 0.1, -/-: 1.6 ± 0.1; BDA: +/+: 1.0 ± 0.1, -/-: 1.1 ± 0.1 mm; P<0.05) AA formation in both calpain-1 +/+ and -/- mice as compared to vehicle administered mice. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A (P<0.05), an actin binding cytoskeletal protein, and medial macrophage accumulation in aorta.

Conclusion

Calpain-2 compensates for loss of calpain-1, and promotes AngII-induced ascending and abdominal AAs in calpain-1 deficient mice.
Abstract 519: Mineralocorticoid Receptor Agonists and Salt Induce Aortic Aneurysms in C57BL/6 Mice
Authors: Liu Shu , Xie Zhongwen , Daugherty Alan , Cassis Lisa , Pearson Kevin , Gong Ming , Guo Zhenheng
Published: March 2022

In an independent study in which we used 10-12 month old male C57BL/6 mice to investigate deoxycorticosterone Acetate (DOCA) and salt (1% saline) induced hypertension, we unexpectedly found that a numbers of mice died of aortic aneurysm rupture. Since aortic aneurysm affects 4-8% elderly men and elevated plasma aldosterone (ALDO) concentrations in patients have been linked to a spectrum of cardiovascular diseases, we therefore tested the possibility that activation of the mineralocorticoid receptor (MR) by DOCA and salt may be involved in aortic aneurysm formation. We reported here that that administration of 10-month-old male C57BL/6 mice with DOCA and salt, but neither DOCA nor salt alone, potently induced abdominal aortic aneurysm (AAA, 62%), thoracic aortic aneurysm (TAA, 44%), and aortic rupture (18%). ALDO and salt also induced similar high rates of AAA (58%), TAA (50%), and aortic rupture (42%). Histological and immunohistological examinations of cross-sections of AAA and TAA at the elastin degradation site demonstrated that DOCA and salt or ALDO and salt induced aortic aneurysm resembled human aortic aneurysm with respect to elastin degradation, macrophage infiltration, smooth muscle cell degeneration and apoptosis, and oxidative stress. Moreover, DOCA and salt also induced aortic aneurysm in 10-week-old mice but with lower incidence and less severe than that in 10-month-old mice. Interestingly, aortic aneurysm formation did not correlate with the increase in blood pressure induced by DOCA and salt. Systemic administration of the angiotensin converting enzyme inhibitor, enalapril, or angiotensin type 1 receptor antagonist, losartan, did not affect DOCA and salt-induced aortic aneurysm. In contrast, the MR antagonist spironolactone significantly attenuated DOCA and salt induced aortic aneurysm. In summary, our study describes a novel aortic aneurysm animal model induced by MR agonist and high salt, and reveals a previously unrecognized but potentially significant role of aldosterone in the pathogenesis of aortic aneurysm. These findings imply that MR antagonists may be effective in the treatment of some aortic aneurysms.
Abstract 534: Superoxide Dismutase 1 Modulates STAT3 pathway in Angiotensin II Induced Vascular Remodeling
Authors: Shiraishi Yasunaga , Kujiraoka Takehiko , Niida Tomiharu , Ishigami Norio , Arai-Nakaya Yuko , Hakuno Daihiko , Isoda Kikuo , Adachi Takeshi
Published: March 2022

[Background]

Superoxide dismutase 1 (copper/zinc SOD; SOD-1) generates hydrogen peroxide (H2O2) from superoxide and is predominantly expressed in cytosol of vascular walls. Angiotensin II (Ang-II) induces vascular remodeling is associated with H2O2 generation from NADPH oxidase. We have previously shown that SOD-1 modulates Ang-II induced vascular remodeling. The aim of this study is to seek the signaling mechanisms for SOD-1to modulate vascular remodeling with Ang-II in in vivo.

[Methods and Results]

We employed SOD-1 deficient mice (SOD-1-/-), which were continuously infused with Ang-II (3.2mg/kg/day) for seven days. There was no difference in blood pressure between SOD-1-/- and their wild-type mice (WT). The serum levels of derivatives of reactive oxygen metabolites were similarly increased in both WT and SOD-1-/- with Ang-II, however, an increase in oxidized glutathione levels in whole blood cells were blunted in SOD-1-/-, suggesting a decrease in cytosolic H2O2 generations with SOD-1 deficiency. In histological analysis, Ang-II increased aortic wall thicknesses in WT, however, the responses were also blunted in SOD-1-/- (wild-type: 91.9±3.0μm SOD-1-/-: 68.4±4.5μm, P<0.001, N=10-12). In western blot analysis, Ang-II infusion enhanced the phosphorylation of signal-transducer-and-activator-of-transcription 3 (STAT3) in aortas from WT, which was blunted in SOD-1-/-, while the phosphorylation of ERK and Akt were identical. The interleukin 6 (IL-6) of serums were equally elevated in both SOD-1-/- and WT with Ang-II infusion. In cultured rat vascular smooth muscle cells (VSMCs), Ang II (24 h) slowly increased, and IL-6 (5 min) or hydrogen peroxide (H2O2) (15min) rapidly increased the phosphorylation of STAT3. With copper-chelating agents (N, N-diethyldithiocarbamate; 1mM 4h), the phosphorylation of STAT3 was blunted with all agonists.

[Conclusion]

These results indicated that the activation of STAT3 plays a role in Ang II- induced vascular remodeling, and SOD-1 is involved with the signaling mechanism in in vitro and in vivo.
Abstract 537: Platelet Derived Growth Factor Induced Vascular Smooth Muscle Cell Proliferation is Associated with Enhanced Glut1 Expression/glycosylation and Glucose Uptake
Authors: Poulose Ninu , Pyla Rajkumar , Osman Islam , Ganapathy Vadivel , Segar Lakshman
Published: March 2022

Objective

The role of glucose transporters (GLUTs) in the phenotypic regulation of vascular smooth muscle cells (VSMCs) has not been fully characterized. The present study has examined the effects of PDGF (
vs
insulin) on GLUT isoform expression/localization, GLUT1 glycosylation, glucose uptake and ATP formation during VSMC proliferation.

Methods

Human aortic VSMCs were exposed to PDGF (30 ng/ml) or insulin (1 μM) for different time intervals. GLUT isoform expression was determined by qPCR and immunoblot analysis. GLUT isoform distribution/localization was determined by subcellular fractionation studies and immunocytochemical analysis. Glucose utilization and ATP formation were determined by glucose oxidation assay and luminescent ATP detection assay, respectively. The role of GLUT1 in VSMCs was determined by GLUT1 siRNA knockdown studies.

Results

Exposure of VSMCs to PDGF resulted in significant increases in glucose uptake and cell proliferation, compared with insulin. In addition, PDGF and insulin enhanced the mRNA expression levels of total GLUT isoforms (GLUT1 through 14) to ~4317/10
5
B2M
transcripts and ~8,174 transcripts/10
5
B2M
transcripts, respectively, compared with control (~3259 transcripts/10
5
B2M
transcripts). In particular, PDGF and insulin enhanced GLUT1 mRNA to 1711 ± 38/10
5
B2M
transcripts and 3387 ± 58 transcripts/10
5
B2M
transcripts, respectively. Immunoblot analysis using whole cell homogenates showed a significant increase in GLUT1 protein expression in PDGF or insulin-treated VSMCs. Importantly, subcellular localization studies using PDGF-treated VSMCs revealed enhanced GLUT1 protein expression and its glycosylation in the plasma membrane. Furthermore, PDGF enhanced glucose metabolism as revealed by increased glucose utilization and ATP formation. Down regulation of GLUT1 using target-specific siRNA significantly attenuated PDGF-induced glucose uptake.

Conclusions

Enhanced expression and glycosylation of plasma membrane-localized GLUT1 isoform plays a major role in promoting VSMC proliferation, which may contribute to accelerated atherosclerosis.
Abstract 543: ADAR1-mediated RNA Editing, a Novel Mechanism Controlling Phenotypic Modulation of Vascular Smooth Muscle Cells
Authors: Fei Jia , Wang JiangNing , Cui XiaoBin , Chen ShiYou
Published: March 2022

Vascular smooth muscle (SMC) phenotypic modulation, the transition from a contractile to a proliferative phenotype accompanied by neointima formation following vascular injury, plays a critical role in the development and progression of several proliferative cardiovascular diseases such as atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. A hallmark feature of the phenotypic modulation is the down-regulation of SMC contractile genes. Platelet-derived growth factor-BB (PDGF-BB), a well-known stimulator of SMC phenotypic modulation, down-regulates SMC gene expression via posttranscriptional regulation of the related genes. The post-transcriptional mechanisms involved in SMC phenotypic gene expression, however, remain largely unknown. We found that the down-regulation of SMC contractile genes is caused by abnormal RNA editing of their precursor mRNAs (pre-mRNAs). This abnormal pre-mRNA editing is facilitated by adenosine deaminase acting on RNA (ADAR), which converts adenosines to inosines (A→I editing). PDGF-BB induces ADAR1 while down-regulating the expression of SMC myosin heavy chain (SMMHC) and calponin (CNN). Knockdown of ADAR1 by shRNA restores PDGF-BB-blocked SMMHC and CNN expression, demonstrating that ADAR1 plays an essential role in SMC phenotype modulation.
In vivo
animal studies show that SMMHC and CNN pre-mRNA is accumulated while their mature mRNA is decreased in balloon-injured rat carotid arteries. Moreover, ADAR1 is highly induced in media layer SMCs initially, and neointima SMCs subsequently following the injury. Of importance, knockdown of ADAR1 dramatically inhibits injury-induced neointima formation, demonstrating a critical role of ADAR1 in vascular remodeling
in vivo
. Taken together, our study unraveled a novel molecular mechanism, i.e., pre-mRNA editing, governing SMC phenotypic modulation.
Abstract 545: Response Gene to Complement 32-deficiency Causes Impaired Placental Angiogenesis in Mice
Authors: Cui Xiaobing , Guo Xia , Chen Shiyou
Published: March 2022

Aims

The objectives of this study are to determine the role of response gene to complement 32 (RGC-32) in the placental angiogenesis during pregnancy and explore the underlying mechanisms.

Methods and Results

RGC-32-deficient (RGC32-/-) mice were generated from C57BL/6 embryonic stem cells with deletion of Exon 2 and 3 of RGC-32 gene. Most of the RGC32-/- mice can survive. However, their body sizes were much smaller compared to their wild-type littermates when they were born. By examining the embryo development and placentas at 16.5 days post-coitum, we found that RGC32-/- embryos and fetal placentas were significantly smaller than the wild-type. Further analysis showed that the labyrinth zone of RGC32-/- fetal placenta was smaller with defective angiogenesis. Mechanistically, vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and placental growth factor (PlGF), but not other angiogenic factors, were significantly downregulated in RGC32-/- placentas, suggesting that VEGFR2 and PlGF may mediate RGC-32 function in placenta angiogenesis. Indeed, knockdown of RGC-32 by shRNA inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation while blocking VEGFR2 expression. Knockdown of RGC-32 also inhibited trophoblast proliferation while blocking the expression of PlGF.

Conclusion

Absence of RGC-32 caused intrauterine growth restriction through interrupting the placental angiogenesis, which was due to the decreased expressions of VEGFR2 in endothelial cells and PlGF in trophoblast cells.
Abstract 548: Scavenger Receptor Bi-deficiency-induced Hypercholesterolemia Impairs Lymphocyte Homeostasis
Authors: Zheng Zhong , Ai Junting , Guo Ling , Howatt Deborah D , Daugherty Alan , Bondada Subbarao , Li Xiang-An
Published: March 2022

Objective-

Deficiency in Scavenger receptor BI (SR-BI), an HDL receptor, leads to a specific HDL-c-elevated hypercholesterolemia and impairs lymphocyte homeostasis in mice. However, how SR-BI-deficiency-induced hypercholesterolemia contributes to the adaptive immunity defects remains unknown.

Hypothesis-

SR-BI-deficiency-induced hypercholesterolemia leads to the impaired lymphocyte homeostasis.

Methods and Results-

We utilized two animal models combined with cell culture to test our hypothesis. First, in a high fat/high cholesterol (HF) diet mouse model (n>10 per group), we introduced a 2-fold increase in plasma cholesterol concentration. For HF diet SR-BI
+/+
mice, the increased cholesterol was distributed in VLDL/LDL fraction, but the lymphocyte homeostasis was not affected. In contrast, for HF diet SR-BI
-/-
mice, in addition to VLDL/LDL-c, HDL-c was also further elevated. These mice exhibited marked disruption in lymphocyte homeostasis, as characterized by a 1.6-fold increase in T cell number (2.7-fold vs SR-BI
+/+
group), a 2.1-fold increase in B cell number (4.2-fold vs SR-BI
+/+
group), exacerbated T cell activation and further accelerated B cell proliferation compared with control diet SR-BI
-/-
mice. Then, in a bone marrow transplantation model (n=5~6 per group), we found that HF diet SR-BI
-/-
recipients whether receiving SR-BI
+/+
or SR-BI
-/-
BMCs displayed higher lymphocyte numbers, greater lymphocyte activation and more lymphocyte proliferation than respective HF diet SR-BI
+/+
controls, indicating that non-hematopoietic SR-BI-deficiency environment was the dominant factor leading to the impaired adaptive immunity. Finally, in a series of
in vitro
lymphocyte activation experiments (n=4 per group, repeated twice), we found that HDL from control diet or HF diet SR-BI
+/+
mice exhibited an inhibitory effect on T cell or B cell proliferation under TCR or BCR stimulation, but HDL from HF diet SR-BI
-/-
mice completely lost that function, indicating that the dysfunctional HDL in HF diet SR-BI
-/-
mice was responsible for impaired lymphocyte homeostasis.

Conclusion-

The SR-BI-deficiency-induced hypercholesterolemia is the leading factor for impaired lymphocyte homeostasis in SR-BI
-/-
mice.
Abstract 553: Expression of TNFalfa and CCL18 is Increased as a Result of Cholesterol Accumulation in Macrophages
Authors: Orekhov Alexander N , Nikiforov Nikita G , Melnichenko Alexandra A , Karagodin Vasily P , Sobenin Igor A
Published: March 2022

Innate immunity plays important role in atherogenesis. Immune reaction in artery wall involves both pro-and anti-inflammatory cytokines. We investigated cytokines’ mRNA expression in uninvolved and atherosclerotic human aortic intima. TNF-α and CCL18 were localized in autopsy aortic samples using immunohistochemistry. Expression of cytokine mRNA was measured by PCR or qPCR in the same samples. Primary culture of blood-derived monocytes/macrophages from healthy donors was used to study TNF-α and CCL18 mRNA expression induced by IFN-gamma and IL-4, respectively. Intracellular lipid accumulation was induced by incubation of cultured cells with atherogenic modified LDL isolated from blood of patients with documented coronary atherosclerosis. Immunohistochemistry demonstrated that the number of cytokine-positive cells (both TNF-α and CCL18) were increased in atherosclerotic lesions as compared with unaffected aortic intima. Cytokines were associated with different cell types including macrophages and non-macrophage cells. Both TNF-α and CCL18 were manly occurred in macrophages. Rice of both cytokines’ mRNA expression measured by PCR was observed in atherosclerotic lesion. In lipid-rich atherosclerotic lesion cytokine expression was the most considerable. Correlation between lipid content in aortic intima and cytokine expression was found. To elucidate the mechanism of elevation of lipid-associated cytokine expression, cell culture study was performed. Cultured monocytes/macrophages were incubated with atherogenic modified LDL of atherosclerotic patients and non-atherogenic LDL from healthy donors. In contrast to non-atherogenic LDL patients’ LDL induced intracellular cholesterol accumulation and increased mRNA expression of both TNF-α and CCL18 in cell culture.
Thus, i
ntracellular lipid accumulation caused by atherogenic modified LDL from atherosclerotic patients induces overexpression of TNFa and CCL18. This finding can explain the elevation of TNF-α and CCL18 expression in lipid-rich atherosclerotic lesions of human aorta.
Abstract 558: A Role for Syndecan-1 in the Motility of Alternatively Activated Macrophages
Authors: Haller Carolyn , Angsana Julianty , Chaikof Elliot
Published: March 2022

Background

Syndecan-1 (Sdc-1) is a member of a family of cell surface proteoglycans that has been reported to participate in the regulation of events relevant to tissue repair and chronic injury responses, including cell-substrate interactions, matrix remodeling and cell migration and proliferation. We have previously demonstrated that macrophage Sdc-1 expression is up-regulated and protective in 2 clinically relevant models of chronic inflammation: elastase-induced AAA and Western diet-induced atherosclerosis. In this study, we aim to define the subtype and functional consequence of Sdc-1 expressing macrophages.

Methods

Human and mouse Sdc-1 macrophages were enriched and characterized against defined macrophage subtypes, including classically activated M1(IFN-γ/LPS), M2 (IL-4), and M2 (IL-10). Endpoints included (i) surface expression of MHCII, CD86, PD-L2, and Sdc-1; (ii) iNOS and arginase activity; and (iii) ELISA or qPCR cytokine and chemokine analysis. Motility was examined using boyden chamber chemotaxis and adhesion to ECM was measured using CSFE labeled macrophages.

Results

In vitro characterization of Sdc-1 expressing macrophages, which can be generated through the activation of cAMP/PKA signaling pathway or through exposure to adenosine, indicates that Sdc-1 expression defines an anti-inflammatory population with high intrinsic motility. Sdc-1 macrophages displayed reduced expression of pro-inflammatory cytokines and chemokines and displayed limited potential to mediate monocyte or CD4 T cell chemotaxis. Using Sdc-1
-/-
macrophages, we found that the absence of Sdc-1 is associated with defective actin reorganization, resulting in dampened macrophage migration and enhanced adhesion to extracellular matrix. Analysis with human macrophages confirmed Sdc-1 expression in an alternatively activated, motile population.

Conclusions

Collectively, these results reinforce the physiological significance of macrophage motility as an endogenous modulator of the inflammatory response and further suggest Sdc-1 as a marker of alternatively activated macrophages in chronic inflammation.
Abstract 560: Role of Paraoxonase 2 In Innate Immune Response in an Acute Infection Model
Authors: Devarajan Asokan , Bourquard Noam , Gao Feng , Ganapathy Ekambaram , Verma Jitendra , Reddy Srinivasa T
Published: March 2022

Background

(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a quorum-sensing molecule produced by certain classes of gram-negative microbial pathogens, such as
Pseudomonas aeruginosa
(PAO1), is involved in the regulation of virulence factors and alters the function of the host cells. Previously, We have shown that paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL in murine tracheal epithelial cells. In this study, we examined the physiological role of PON2 in innate host defense using the PA01-induced acute infection mouse model.

Materials and Results

We demonstrates that both intact cells and membrane-enriched protein lysates obtained from PON2-deficient (PON2-def) macrophages reveal a marked impairment in their ability to hydrolyze 3OC12-HSL. PON2 is neither up-regulated nor down-regulated in response to 3OC12-HSL in control macrophages. In an
ex vivo
model, macrophages derived from PON2-def mice show an increase in ER stress-mediated oxidative stress and defective PI3 kinase/AKT activation, leading to reduced phagocytes function compared to control macrophages following 3OC12-HSL treatment. Moreover, p85, a regulatory subunit of PI3-kinase, shows higher tyrosine nitration in the macrophage of PON2-def mice which is associated with phagocytic function. Restoration of PON2 in the macrophages reduces the ER stress mediated oxidative stress and improves the phagocytosis function in response to 3OC12-HSL treatment. Furthermore, following administration of 1.6x10
7
CFU of PAO1, a decrease in bacterial clearance is found in the lungs, and liver of PON2-def mice by 5.7, and 14.8 fold, respectively.

Conclusion

Increased ER stress mediated oxidative stress decreases phagocytosis function in the macrophages of PON2-def mice in response to 3OC12-HSL may explain the decreased bacterial clearance observed in PON2-def mice following PAO1 infection.
Abstract 561: Serpin Suppression of Macrophage and T Helper Cell Response in Temporal Artery Biopsy Xenograft Implants from Patients with Giant Cell Inflammatory Arteritis
Authors: Chen Hao , Zheng Donghang , Ryden Sally , Hazem Sami , Bartee Mee Y , Davids Jennifer , Sobel Eric , Dai Erbin , Liu Liying , Macaulay Colin , Weyands Cornelia , Thoburn Robert , Lucas Alexandra
Published: March 2022

Background

Glucocorticoids are a principal treatment for giant cell arteritis (GCA), with some studies reporting predominate effect on T helper17 (Th17) cell activity in GCA, with lesser effects on other inflammatory cells. Serp-1 is a 55kDa myxomaviral
serpin
that modifies macrophage, Th1, and Th17 responses in aortic transplants in animal models.

Objectives

We have analyzed the effects of a virus-derived anti-inflammatory
ser
ine
p
rotease
in
hibitor (
serpin
) on human temporal artery (TA) biopsies isolated from patients with suspected GCA.

Methods

Using a newly developed “Aortic Window Xenopatch” model, human TA biopsy sections were divided implanted as full thickness grafts into the abdominal aorta of SCID mice in parallel and tested for response to Serp-1, with and without human PBMC infusion (N = 32).

Results

TA sections positive for arteritis (GCA
pos
) displayed significantly increased inflammatory plaque when compared to negative sections (GCA
neg
). Serp-1 reduced plaque in GCA
pos
sections (<0.01) with concommitant reduction in Th1, and Th17 splenocytes on flow cytometry (P < 0.01), with decreased IFNγ and IL-17 gene expression by RT-PCR array analysis. Serp-1 also reduced arterial CD11b
+
cells after PBMC infusion with reduced TNF-α expression in spleen, but not CD3
+
, CCR6
+
, nor CD86
+
. Splenocytes from mice with GCA
pos
grafts had increased interleukin-1beta (Il-1β), IL-17, and CD25 expression, while IL-1β was significantly reduced by Serp-1.Serp-1 also markedly reducedt gene expression for the FII, FXa-PAR2, tPA, uPA in splenocytes (P <0.05).

Conclusions

Treatment with a virus-derived serpin, significantly reduces inflammation and plaque thickness in human GCA
pos
TA xenograft implants with associated reductions in macrophage and Th1 and Th17 responses in mice.
Abstract 563: Axl Regulates Major Histocompatibility Complex Class II (MHC-II) Expression through STAT1 Pathway in Smooth Muscle Cells
Authors: Batchu Sri Nagarjun , Gerloff Janice , Korshunov Vyacheslav
Published: March 2022

Introduction

Major histocompatibility complex class II (MHC-II) is upregulated in vascular smooth muscle cells (VSMCs) in response to injury. Previously we found that Axl, a receptor tyrosine kinase, is important for vascular remodeling via regulation of MHC-II
in vivo
. The aim of this study was to identify Axl-mediated intracellular signaling that is responsible for upregulation of MHC-II in VSMCs.

Methods and Results

Mouse aortic smooth muscle cells (MASMCs) were isolated from Axl wild type (Axl+/+) or knockout (Axl-/-) littermates from our colony. Cells were treated with interferon gamma (IFNγ; 1,000U/ml) or PBS for various times (10min-24hrs). There was no difference in apoptosis as measured by cleaved caspase-3 in Axl-/- vs. Axl+/+ after treatment with IFNγ. Likewise, phosphorylation levels of Akt were similar between the groups. Expression of MHC-II mRNA and protein was significantly lower in Axl-/- vs. Axl+/+ MASMCs. Levels of major histocompatibility class II transactivator (CIITA) mRNA was also significantly reduced in Axl-/- MASMCs. Reduced expression of number of cytokines and chemokines was observed in Axl-/- vs. Axl+/+ MASMCs after treatment with IFNγ. Similar to our
in vivo
results, a complement component 3 (C3) expression was drastically reduced in Axl-/- vs. Axl+/+ MASMCs. We found that STAT1, a major regulator for CIITA-MHC-II and C3 pathways, was not phosphorylated in Axl-/- vs. Axl+/+ MASMCs upon IFNγ treatment.

Conclusions

This is the first study that demonstrates that Axl affects STAT1-signaling in VSMCs. Our findings suggest that Axl contributes to vascular remodeling via enhancement of pro-inflammatory signals in smooth muscle cells.
Abstract 569: M-RNA Expressions in Carotid Atherosclerotic Lesions Related to Plasma Levels of Lipoprotein (a)
Authors: Bergmark Claes
Published: March 2022

Objective

High plasma levels of Lipoprotein (a), (Lp(a)) is an atherosclerotic risk factor. Individual levels of Lp(a) are genetically determined and constant over lifetime. Thus, individuals with high Lp(a) levels are exposed to this risk factor over a long time. In vitro, Lp(a) has many biochemical and signalling effects which could act proatherogenic, but until now, nothing has been possible to confirm in vivo. Plasma levels of Lp(a) correlate with lesion levels of Lp(a).

Methods

A consecutive series of 153 patients operated for Carotid stenosis by Trombendarterectomy (TEA) had plasma samples taken preoperatively. TEA-specimens were analysed for gene expression by Affymetrix. Plasma samples were analysed for levels of Lp(a) with routine antibody-methods. 20000 different m-RNA expression levels were correlated to 10log levels of Lp(a). Patients with plasma levels of Lp(a) above 60mg/dl were compared with those below 20mg/dl

Results

The most up regulated gene in patients with high Lp(a) compared to normal/low Lp(a) is MIAT (myocardial infarction associated transcript), also known as RNCR2 (retinal non-coding RNA) or Gomafu, a long non-coding RNA. MIAT is associated with risk of myocardial infarction. The most down regulated gene is Glypican 4, a member of heparane sulfate proteoglycans. Lp(a) binds to proteoglycans. Gene enrichment analysis revealed Lp(a) association with cholesterol efflux, oxygen binding and proteasome complex, each with a role in atherosclerosis.

m-RNA from the LPA-gene could not be detected, confirming previous assumption that apo (a) is not synthesised in vascular tissue.

Conclusions

High levels of Lp(a) is associated with gene expression in carotid lesions, some suggestive to a proatherosclerotic pathway. The new finding of association with MIAT and the proteasome complex deserves further studies.
Abstract 63: Post-transcriptional Control of the LDLR Pathway by Cholesterol-responsive microRNAs.
Authors: Salerno Alessandro G , Rayner Katey J , Wanschel Amarylis C , Oldebeken Scott R , Scotti Elena , Tontonoz Peter , Moore Kathryn J
Published: March 2022

Despite the success of statins to reduce levels of LDL-cholesterol (LDL-C), cardiovascular disease remains the leading cause of mortality in westernized countries. PCSK9 inhibition is emerging as an exciting new therapy to further lower LDL and holds tremendous promise to reduce the significant residual cardiovascular risk in many individuals. Recent advances from our group and others have shown that microRNAs (miRNAs) have emerged as critical components in the regulation of cholesterol metabolism. In addition to miR-33, which we discovered as a key regulator of metabolic programs including cholesterol and fatty acid homeostasis, we identified two new cholesterol responsive miRNAs, miR-520d-5p (miR-520d) and miR-224. Here we show that miR-520d and miR-224 target the 3’UTR of both PCSK9 and Idol, “accessory” proteins that regulate cell surface expression of the LDL receptor (LDLR). Moreover, miR-520d and miR-224 simultaneously target the 3’UTR of HMGCR, which codes for the limiting enzyme in cholesterol synthesis and is the target of statins. In mouse and human cells, the overexpression of miR-520d inhibits PCSK9, Idol and HMGCR mRNA levels by 30-50% compared to a control miRNA, while overexpression of miR-224 results in an even more potent 75% decrease in PCSK9, Idol and HMGCR mRNA compared to controls. In human hepatocytes (HepG2/Huh7) and mouse peritoneal macrophages, miR-520d and miR-224 overexpression caused a repression of PCSK9, Idol and HMGCR protein levels, and a decrease in PCSK9 protein secretion. Importantly, these miR-224 and miR-520d-induced changes in PCSK9, Idol and HMGCR expression were associated with a concomitant increase in LDLR protein expression, increased cell surface expression of an LDLR-GFP fusion protein and enhanced LDL binding to the cell surface. Notably, these increases in LDLR were above and beyond those achieved with statin treatment. Together, these findings suggest a role of miR-224 and miR-520d in contributing to the post-transcriptional regulation of LDLR expression, and represent the first evidence of miRNA control of the LDLR pathway. This highlights the therapeutic promise of miR-224/miR-520d for reducing LDL cholesterol beyond statin therapy alone.
Abstract 74: Crucial Role of ROCK2 in Vascular Smooth Muscle Cells in Hypoxia-Induced Pulmonary Hypertension in Mice
Authors: Shimizu Toru , Fukumoto Yoshihiro , Tanaka Shin-ichi , Satoh Kimio , Ikeda Shohei , Shimokawa Hiroaki
Published: March 2022

Background

We have previously demonstrated that Rho-kinase (ROCK) pathway in vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of cardiovascular disease including pulmonary arterial hypertension (PAH). Rho-kinase has 2 isoforms; ROCK1 for inflammatory cells and ROCK2 for VSMC. We thus examined the specific role of ROCK2 in VSMC in the pathogenesis of PAH.

Methods and Results

In patients with PAH as compared with controls, the immunoreactivity of ROCK2 and phosphorylated ERK1/2 co-expressed with αSMA in pulmonary arterial media were markedly increased. Thus, we developed VSMC-specific ROCK2-deficient (ROCK2
+/-
) and VSMC-specific ROCK2-overexpressing (ROCK2-Tg) mice. The extent of hypoxia-induced pulmonary hypertension (10% O
2
for 4 weeks), as evaluated by pulmonary vascular remodeling, right ventricular (RV) systolic pressure and RV hypertrophy, was significantly reduced in ROCK2
+/-
mice and was enhanced in ROCK2-Tg mice compared with the littermates (n=10 each, all P<0.05). The expression of ROCK activity and phosphorylated ERK1/2 in lung tissues analyzed by Western blotting and the numbers of Ki67
+
proliferating VSMC and CD45
+
inflammatory cells in pulmonary arterioles revealed by immunostaining were significantly reduced in the ROCK2
+/-
mice and were increased in the ROCK2-Tg mice compared with the littermates (n=4 each, all P<0.05). In cultured murine aortic VSMC, both scratch assay and Boyden chamber assay showed that migration activity was significantly reduced in the ROCK2
+/-
VSMC and was increased in the ROCK2-Tg VSMC compared with littermates (n=5 each, both P<0.01). In addition, in primary pulmonary arterial VSMC (PASMC) cultured from patients with PAH, siRNA knockdown (-81%) of ROCK2 expression (ROCK2-siRNA) significantly suppressed the expression of ROCK activity and PDGF-induced phosphorylated ERK1/2 (n=4 each, both P<0.05). Compared with control-siRNA, PASMC transfected with ROCK2-siRNA exhibited significant reduction in both migration activity by scratch assay and proliferation activity by growth curve (n=3 each, both P<0.01).

Conclusions

ROCK2 in VSMC plays a crucial role in the pathogenesis of PH through enhanced VSMC migration and proliferation in mice in vivo.
Abstract 78: Inhibition of Mir-33 Overcomes the Deleterious Effects of Diabetes on Atherosclerosis Regression.
Authors: Distel Emilie , Chung Kellie , Murphy Andrew J , Barrett Tessa , Parathath Sajesh , Essau Christine C , Moore Kathryn J , Fisher Edward A
Published: March 2022

Objective

Diabetes increases the risk of cardiovascular morbidity and mortality which remains elevated with conventional therapies, such as statins. We have shown that diabetes also impairs plaque regression following LDL reduction in mice, as evidenced by a higher macrophage (CD68+ cells) plaque content and a higher inflammatory state of these cells compared to non-diabetic mice. Type-2 diabetics have dyslipidemia characterized by elevated triglyceride levels and low HDL, the latter is thought to contribute to their risk, based on epidemiologic studies. In non-diabetic atherosclerotic mice, inhibition of miR-33, an intronic micro-RNA located within the SREBF2 gene, increases the plasma level of apoAI and HDL-cholesterol (HDL-C) and promotes plaque regression. We hypothesize that elevation of HDL through the inhibition of miR-33 will overcome the deleterious effect of diabetes on macrophage content and inflammatory phenotype in plaques in a regression evironment.

Methods and Results

Diabetic (STZ-injected) and non-diabetic Reversa (LdLr-/-Apob100/100Mttpfl/flMx1-Cre+/+) mice, a model in which diet-induced hypercholesterolemia can be quickly and efficiently reversed, were fed a western diet for 16 weeks and then treated with anti-miR-33 oligonucleotide or control anti-miR for 4 weeks. Treatment with anti-miR-33 increased HDL-cholesterol in both diabetic (17%) and non-diabetic mice (30%). Anti-miR-33 treated diabetic mice showed an improvement in plaque regression as evidenced by a 26% reduction in CD68 content compared to diabetic mice receiving control anti-miR, and an enrichment in anti-inflammatory M2 macrophages (assessed by MR 1 and YM1). Monocyte tracking with latex beads suggests that the reduction of macrophage content is due to a decrease in monocyte recruitment to the plaque, rather than an increase in macrophage egress. This reduction in recruitment correlated with a decrease in the monocytosis associated with diabetes in the anti-miR-33 treated diabetic mice.

Conclusion

These findings suggest that miR-33 inhibition is able to overcome the deleterious effects of diabetes on atherosclerosis regression, by decreasing monocytosis, monocyte recruitment and improving the inflammatory status of plaque macrophages.
Abstract 9: Apelin Protects Against Arterial Thrombosis in Mice with Diet-induced Obesity
Authors: Jin Hong , Wilson Katina M , Chauhan Anil K , Tsao Philip S , Lentz Steven R
Published: March 2022

Background and Hypothesis

The prevalence of obesity and related thrombotic diseases is increasing steadily, but the underlying mechanisms of thrombosis are not well elucidated. Apelin is a peptide adipokine that is upregulated in obesity and has protective effects on cardiovascular function. We tested the hypothesis that apelin protects from thrombosis in a mouse model of diet-induced obesity.

Methods

To induce obesity, male apelin null (
Apln -/y
) mice and wild-type (WT) littermates were fed a high fat (HF) diet with 60% of Kcal from fat (TD.06414) for 12 weeks. Susceptibility to thrombosis of the carotid artery was assessed using a photochemical injury (rose bengal) method. Expression of plasminogen activator inhibitor-1 (PAI-1) was measured by ELISA and real-time PCR.

Results

Body weight and fasting glucose were higher in mice fed the HF diet than in mice fed a control diet (P<0.05) but did not differ between
Apln -/y
and WT mice (P>0.05). The time to stable occlusion of the carotid artery was significantly accelerated in
Apln -/y
mice compared with WT mice (24.8±1.5 vs 43.9±6.8 min, P=0.04) fed the HF diet, but not a control diet. Because apelin is known to regulate the expression of the antifibrinolytic serpin PAI-1, we investigated the role of PAI-1 in the antithrombotic mechanism of apelin. Plasma levels of total PAI-1 were elevated 3-fold in WT mice fed the HF diet (P<0.05 vs. control diet). An even larger 5-fold elevation of plasma PAI-1 was seen in
Apln -/y
mice fed the HF diet (P<0.05). Levels of PAI-1 mRNA in lung also were significantly elevated in
Apln -/y
mice (P<0.01 vs. WT mice). To directly determine the role of PAI-1 in the prothrombotic phenotype of
Apln -/y
mice, a PAI-1-blocking antibody was administered prior to inducing thrombosis of the carotid artery. The PAI-1 antibody nearly completely reversed the shortened time to stable occlusion (P=0.03) in obese
Apln -/y
mice.

Conclusions

Endogenous apelin protects from arterial thrombosis in obesity, in part by dampening obesity-induced upregulation of PAI-1. These findings may have pharmacological implications for the prevention of thrombotic complications of obesity and metabolic syndrome.
Abstract 10: Improving Postprandial Lipoprotein Apolipoprotein B-48 Metabolism in Statin-Treated Patients With Diabetes
Authors: Pang Jing , Chan Dick , Hamilton Sandy , Tenneti Vijay , Watts Gerald , Barrett Hugh
Published: March 2022

Background:
Type 2 diabetics often have elevated plasma TG and apolipoprotein B-48 (apoB-48) concentrations, particularly during the postprandial (PP) period. Evidence suggests that apoB-48 plays a central role in the development of atherosclerosis. Statins are frontline therapy to reduce CVD risk, however, residual risk still remains, suggesting that additional interventions are required to further reduce CVD risk.

Aim:
To compare PP apoB-48 kinetics in optimally statin-treated diabetic men with a group of normolipidemic healthy controls, and to investigate the effect of niacin on apoB-48 kinetics in these diabetic men.

Methods:
Twelve type 2 diabetic men and fourteen age-matched non-diabetic controls were recruited. Diabetics required a statin-treated LDL-C < 2.5 mmol/L to enter the trial: they were randomized to rosuvastatin or rosuvastatin plus niacin (titrated from 1 to 2 g daily) for 12 weeks and then crossed to the alternate therapy (3 week washout). PP metabolic studies were performed at the end of each treatment period, and on a single occasion in control subjects. D3-leucine tracer was administered as subjects consumed a high-fat liquid meal. Blood samples were collected over 24 h. ApoB-48 tracer/tracee ratios were measured using GCMS. Kinetic parameters, including fractional catabolic rate (FCR) and production rate (PR), were derived using a compartmental model.

Results:
Fasting plasma TG (+112%, p<0.01), but not apoB-48 was elevated in statin-treated diabetics compared with controls. Incremental AUC TG and apoB-48 (+194% and +79%, respectively, p<0.01) were higher in diabetics. PP apoB-48 PR was higher in diabetics (+96%, p<0.01) and FCR lower (-45%, p<0.05), compared with controls. Niacin significantly lowered TG, plasma C, LDL-C and apoB (all p<0.005). Fasting apoB-48 concentration was lowered with niacin (-33%, p=0.03). ApoB-48 FCR was not altered with niacin, but PP apoB-48 PR was lowered to control levels (-38%, p=0.02) on niacin.

Conclusion:
ApoB-48 PR and FCR are abnormal in statin-treated diabetics. Niacin reduces apoB-48 concentration by lowering apoB-48 PR. This effect may be beneficial for reducing atherogenic PP lipoproteins and may provide CVD risk benefit to patients with type 2 diabetes.
Abstract 100: Smooth Muscle Cell-Specific Deficiency of ACE Attenuates AngI-Induced Ascending Aortic Dilation in Male LDL Receptor -/- Mice
Authors: Chen Xiaofeng , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Cassis Lisa A , Daugherty Alan , Lu Hong
Published: March 2022

Introduction and Objectives:
Angiotensin II (AngII) infusion into hypercholesterolemic mice induces atherosclerosis and luminal dilations in both ascending and abdominal aortic regions. Angiotensin-converting enzyme (ACE) is the major enzyme converting AngI to AngII. We demonstrated previously that AngI induced atherosclerosis and aortic aneurysms through an ACE-dependent mechanism. ACE is abundant in smooth muscle cells (SMCs), the major cell type of the aortic wall. The purpose of this study was to determine whether SMC-derived ACE contributes to AngI-induced aortic pathologies.

Methods and Results:
ACE floxed mice were bred into an LDL receptor -/- background. Subsequently, female mice were bred to male Cre transgenic mice with a SM22 promoter to generate wild type (smACE+/+) and SMC-specific ACE deficient (smACE-/-) mice. SMC-specific deficiency of ACE did not affect serum ACE activity, but ablated ACE protein abundance and activity in the aortic media. smACE+/+ (male n=24 male and female n=7) or smACE-/- mice (male n=33 and female n=7) were infused with AngI (1 μg/kg/min) for 4 weeks through osmotic minipumps. Mice were fed a western diet for 1 week prior to pump implantation and during 4 weeks of AngI infusion. Atherosclerosis was quantified on intimal surfaces of aortas by an en face technique. Maximal ex vivo diameters of suprarenal aortas and intimal area of ascending aortas were measured to compare abdominal and ascending aortic dilation, respectively. SMC-specific deficiency of ACE had no effect on percent atherosclerotic lesion area in aortic arches. There was no significant difference of abdominal aortic dilation between smACE+/+ and -/- mice. In contrast, ACE deficiency in SMCs significantly attenuated dilation of ascending aortas in male (smACE+/+ versus -/- mice: 12.2 ± 0.2 versus 10.7 ± 0.4 mm2, P=0.04) but not in female mice (smACE +/+ versus -/- 8.2 ± 0.3 versus 8.7 ± 0.5 mm
2
; P=0.4).

Conclusion:
SMC-specific deficiency of ACE attenuates AngI-induced ascending aortic dilation in male LDL receptor -/- mice, but had no effect on abdominal aortic aneurysms and atherosclerosis.
Abstract 101: LRP1 Deficiency in Vascular Smooth Muscle Cells Augments Development of Angiotensin II-Induced Ascending Aortic and Superior Mesenteric Artery Aneurysms
Authors: Davis Frank , Haggerty Christopher M , Rateri Debra L , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Fornwalt Brandon K , Strickland Dudley , Daugherty Alan
Published: March 2022

Introduction and Objective:
LDL receptor related protein 1 (LRP1) is an endocytic receptor highly expressed in vascular smooth muscle cells that maintains the integrity of the vessel wall. In addition, a recent genome-wide association study implicated LRP1 as a susceptibility locus for human aortic aneurysms. Chronic angiotensin II (AngII) infusion in mice leads to formation of ascending and abdominal aortic aneurysms (AAs). The purpose of this study was to define whether deficiency of LRP1 in vascular smooth muscle cells alters development of either AngII-induced ascending or abdominal AAs.

Methods and Results:
Eight week old male mice with a smooth muscle cell specific-deficiency in LRP1 (smLRP1-/-) were generated by crossing sm22 promoter-driven
Cre
transgenic mice with
lrp1
flox/flox mice. Littermates not expressing Cre were used as controls (smLRP1+/+). Mice were randomized into 4 groups: smLRP1+/+ saline infusion, smLRP1+/+ AngII infusion (1,000 ng/kg/min), smLRP1-/- saline infusion, and smLRP1-/- AngII infusion for 4 weeks. At baseline, there was no difference in aortic diameter in smLRP1-/- vs smLRP1+/+ mice. Following 4 weeks of infusion, smLRP1-/- exacerbated AngII-induced ascending AA development (growth of ascending aortic diameter: smLRP1+/+ saline: 0.10 ± 0.03 mm; smLRP1-/- saline 0.09 ± 0.03 mm; smLRP1+/+ AngII: 0.20 ± 0.03 mm; smLRP1-/- AngII: 0.50 ± 0.10 mm, P<0.01). In addition, AngII-infused smLRP1-/- mice exhibited superior mesenteric artery dilation that was quantified by a 7T Bruker ClinScan magnetic resonance imager (smLRP1+/+ saline: 0.41 ± 0.01 mm; smLRP1-/- saline 0.49 ± 0.05 mm; smLRP1+/+ AngII: 0.36 ± 0.02 mm; smLRP1-/- AngII: 1.14 ± 0.34 mm, P<0.02). Immunostaining of ascending aortas in smLRP1-/- mice revealed increased elastin breaks (P<0.05) without macrophage infiltration. The superior mesenteric artery displayed elevated intima and media macrophage infiltration (P<0.05). Interestingly, smLRP1-/- did not augment AngII-induced abdominal AA development.

Conclusion:
LRP1 deficiency in vascular smooth muscle cells exacerbates development of AngII-induced ascending aortic and superior mesenteric artery aneurysms, without contributing to development of abdominal aortic aneurysms.
Abstract 103: Cross-Talk Between Macrophages, Smooth Muscle Cells and Endothelial Cells in Response to Cigarette Smoke: The Effects on MMP2 and -9
Authors: Ghosh Abhijit , Pechota Angela , Neevel Andrew , Golota Natalie , Upchurch Gilbert R , Eliason Jonathan L
Published: March 2022

Introduction:
Abdominal Aortic Aneurysm (AAA) development is linked to smoking and is characterized by extracellular matrix remodeling by MMP2 and -9. We hypothesized that MMP2 and -9 secretions and regulation in response to cigarette smoke is modulated by interactions between resident cells within the aorta, namely, aortic smooth muscles, endothelial cells, and infiltrating macrophages and could influence AAA formation and/or progression.

Methods:
Aqueous extract of cigarette smoke (AEC) was applied to male rat aortic smooth muscle (RASMC), endothelial (RAEC) or RAW cells in serum free medium (SFM). After 24h the cells were washed, fresh SFM was added and collected after 24h. This conditioned media (CM) was applied to the cells for 24h, again washed, fresh SFM added, and then incubated for 72h, at which point the media was saved for MMP2 and -9 analysis by zymography while the cellular protein was used to determine the ratio (pS/pJ) of phospho-Stat3 (pStat3) and phospho-Jak2 (pJak2) by western blot.

Results:
Unstimulated CM from RAW cells increased MMP2 by 24% (p=0.0357), MMP9 by 282.1% (p=0.0004) and pS/pJ by 136.5% in RASMC. AEC-CM from RAW and RAEC cells increased MMP9 by 200.5% (p=0.0004) and 17.1% (p=0.0301) respectively in RASMC and corresponding increases in pS/pJ (305.7% and 227.6%, respectively).

AEC-CM derived from RAW cells induced RAEC to produce moderate amounts of MMP2 (16.5%, p=0.0140) and MMP9 (29.5%, p=0.0207) and a 137.0% increase in pS/pJ.

RAW cells receiving unstimulated CM from RASMC and RAEC cells produced significant amounts of MMP9 (127.7%, p=0.0026 and 236.8%, p=0.0020, respectively) and increased levels of pS/pJ ratios (45.2% and 1282.8%, respectively). Moreover, AEC-CM from RASMC and RAEC induced significant production of MMP9 from RAW cells (154.8%, p=0.0001 and 162.4%, p=0.0022, respectively) and increase in pS/pJ ratios (1348.2%, and 1493.9%, respectively) over baseline.

Conclusion:
This is the first in vitro study demonstrating that cigarette smoke results in differential interactions between aortic smooth muscle cells, endothelial cells, and macrophage lineage cells resulting in enhanced levels of MMP2, MMP9, pStat3 and pJak2. These interactions may play an important role in AAA formation under in vivo conditions.