Cloning of a Novel Prolyl 4-Hydroxylase Subunit Expressed in the Fibrous Cap of Human Atherosclerotic Plaque

Smooth muscle cells (SMC) are known to migrate and proliferate to form a stabilizing fibrous cap that encapsulates atherosclerotic plaques. It has been shown that CD98hc, a transmembrane protein with a known role in amino acid transport and integrin signaling, is involved in proliferation and survival of various cell types including SMC. Based on these data, we hypothesized that CD98hc deficiency selectively in SMC would have pathogenic effects on atherosclerosis development and plaque composition. To test this, we utilized mice with SMC-specific deletion of the CD98hc ( CD98hc fl/fl SM22Cre + ) to determine the effects of CD98hc deficiency on SMC function in the context of atherosclerosis. We performed in vitro proliferation and survival/apoptosis assays to investigate the role of CD98hc in the proliferation and survival of primary mouse aortic vascular smooth muscle cells. We found that VSMC isolated from whole aortas of CD98hc -/- animals displayed approximately 60% reduced cell counts compared to control (41 ± 8.2% of control) after 5 days in culture. EdU assays in vivo showed a defect in the ability of CD98hc -/- SMC to proliferate, with 25% reduction in EdU-positive VSMC compared to controls (2.3 ± 0.2% vs 3 ± 0.2%). In addition, caspase-3 staining of SMC in vitro displayed a 41% increase in propensity of CD98hc -/- SMC to undergo apoptosis compared to controls (7.9 ± 0.6% vs 5.6 ± 0.5%). Furthermore, the absence of CD98hc in SMC caused a sharp increase in phosphorylated p-38, which was partially abrogated towards control levels when the cells were treated with PDGF-BB to induce proliferation. Long-term atherosclerosis study using SMC-CD98hc -/- /LDLR -/- mice showed that atherosclerotic plaque morphology was altered with increased necrotic core area (25.8 ± 1.9% vs 10.9 ± 1.6% necrotic core area per plaque area) due to a reduction in infiltration of SMC within the plaque (2.1 ± 0.4% vs 4.3 ± 0.4% SM22α positive area per plaque area) compared to control LDLR -/- mice. These data support an important role for CD98hc and its regulation of p-38 MAP kinase signaling in aortic vascular smooth muscle cell proliferation and survival. We conclude that CD98hc is critical for the formation of fibrous cap that is important in maintaining the stability of atherosclerotic plaque.

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Occurrence and characterization of lipofuscin and ceroid in human atherosclerotic plaque

Ultrastructural Pathology ◽

10.1080/01913123.2018.1544953 ◽

2018 ◽

Vol 42 (6) ◽

pp. 477-488 ◽

Cited By ~ 1

Author(s):

Ida Perrotta

Keyword(s):

Atherosclerotic Plaque ◽

Human Atherosclerotic Plaque

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In silico analyses of metagenomes from human atherosclerotic plaque samples

Microbiome ◽

10.1186/s40168-015-0100-y ◽

2015 ◽

Vol 3 (1) ◽

Cited By ~ 36

Author(s):

Suparna Mitra ◽

Daniela I. Drautz-Moses ◽

Morten Alhede ◽

Myat T. Maw ◽

Yang Liu ◽

...

Keyword(s):

Atherosclerotic Plaque ◽

In Silico ◽

Human Atherosclerotic Plaque ◽

In Silico Analyses

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Differentiation and Role of Macrophages in the Early Human Atherosclerotic Plaque

New Aspects of Metabolism and Behaviour of Mesenchymal Cells during the Pathogenesis of Arteriosclerosis ◽

10.1007/978-3-322-99112-6_8 ◽

1991 ◽

pp. 61-72

Author(s):

A. Roessner ◽

E. Vollmer ◽

E. Jaeger ◽

J. Rauterberg ◽

W. Böcker

Keyword(s):

Atherosclerotic Plaque ◽

Human Atherosclerotic Plaque ◽

Early Human

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Differentiation and Role of Macrophages in the Early Human Atherosclerotic Plaque

Current Topics in Pathology - Recent Progress in Atherosclerosis Research ◽

10.1007/978-3-642-76849-1_3 ◽

1993 ◽

pp. 59-71 ◽

Cited By ~ 5

Author(s):

A. Roessner ◽

E. Vollmer ◽

E. Jaeger ◽

J. Rauterberg ◽

W. Böcker

Keyword(s):

Atherosclerotic Plaque ◽

Human Atherosclerotic Plaque ◽

Early Human

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Abstract 13: Inhibition of Nudt6 Stabilizes Advanced Atherosclerotic Plaques

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.13 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Hong Jin ◽

Yuhuang Li ◽

Ekaterina Chernogubova ◽

Alexandra Bäcklund ◽

Stina Sellberg ◽

...

Keyword(s):

Carotid Artery ◽

Atherosclerotic Plaque ◽

Plaque Rupture ◽

Survival Rates ◽

Atherosclerotic Plaques ◽

Protein Coding ◽

Fibrous Cap ◽

Asymptomatic Patients ◽

Rupture Model

Natural antisense transcripts (NATs), a non-coding RNA subclass, being transcribed in antisense direction to protein coding genes, are an intriguing novel class of targetable modulators, exerting crucial effects on gene expression. Aim of the current study was to investigate the contribution of NATs to atherosclerotic plaque vulnerability. Using laser capture micro-dissection, we isolated fibrous caps tissue of carotid artery plaques from 20 symptomatic patients with ruptured lesions vs. 20 samples from asymptomatic patients with stable lesions. A human transcriptome array (HTA; GeneChip 2.0 ) was used to profile the expression of all currently annotated RNA transcripts. Nucleoside diphosphate-linked moiety X motif 6 (NUDT6) was identified as one of the most significantly up-regulated transcripts in fibrous caps of ruptured lesions. Interestingly, NUDT6 is an established antisense RNA targeting the fibroblast growth factor 2 (FGF2). Of importance, FGF2 was among the most significantly down-regulated transcripts in ruptured lesions, corresponding to elevated NUDT6 expression. In situ hybridization in both, human and mouse carotid atherosclerotic plaques, confirmed substantially higher expression levels of NUDT6 in ruptured lesions compared to stable. In addition, in situ hybridization revealed a distinct co-localization with smooth muscle cells (SMCs) in advanced plaques. Overexpression of NUDT6 in cultured human carotid artery SMCs effectively limited FGF2 on the mRNA as well as protein level. Furthermore, reduction of NUDT6 via siRNA stimulated proliferation and blocked apoptosis in SMCs. In an inducible atherosclerotic plaque rupture model using incomplete ligation and cuff placement on common carotid arteries of male apoE -/- mice, NUDT6 inhibition with gapmeRs was able to significantly improve SMC survival rates, leading to thicker fibrous caps, and to reduce the plaque rupture rate compared to scramble-gapmeR control-treated mice (22% vs . 63%, p = 0.03). The present study presents NUDT6 as a novel crucial antisense regulator of fibrous cap stability through steering SMC survival via targeting its sense RNA transcript FGF2.

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