Recent Highlights of ATVB

As primary causes of myocardial infarction, stroke, atherosclerosis, in-stent restenosis, and aneurysm, aortic diseases pose a serious threat to human health. Arterial remodeling is considered to be an important mechanism underlying the development of arterial diseases. Arterial remodeling refers to structural and functional alterations in arterial wall in response to vascular injury or aging. Vascular smooth muscle cells (SMCs), a major component of the arterial wall, exhibit remarkable phenotypic plasticity and can dedifferentiate from a contractile state to a synthetic state, along with increased proliferation and migration abilities.¹ Phenotypically modified SMCs play a major role in arterial remodeling.¹ Recent publications in ATVB have delineated new pathways or factors that are involved in SMC phenotypic modulation and arterial remodeling. These studies have provided valuable insights for better understanding of regulatory mechanisms responsible for vascular remodeling. This article highlights studies on this topic that have published in ATVB recently.

Transcriptional regulation

Transcription factors, together with their coactivators, are essential for regulating SMC phenotypic modulation and vascular remodeling, as manifested by the spatial and temporal expression of specific sets of genes. Several novel regulators have been identified to regulate the transcription activation or inactivation of SMC-specific genes in vascular remodeling. Olfm2 (Olfactomedin 2), a regulatory protein originally identified to be important for eye development during embryogenesis, modifies SMC phenotype and promotes vascular remodeling through interacting with SRF (serum derived factor), which in turn inhibits myocardin binding to SRF,² leading to suppression of SMC marker gene transcription. Conversely, proangiogenic factor Aggf1 interacts with myocardin to stabilize the SRF-myocardin complex in SMCs, thus inhibiting arterial remodeling.³ In addition, ZFP148, a Kruppel-type transcription factor regulating macrophage polarization, inhibits SMC marker expression in aneurysm in an NF-1-dependent manner.⁴

Phenotypic modulation leads to highly proliferative and migratory SMCs which are the major components of remodeled arteries.¹ Coactivator β-catenin promotes SMC proliferation and neointimal hyperplasia following vascular injury.⁵ In contrast, TCF7L2 negatively regulates vascular remodeling because it suppresses SMC proliferation.⁶ Although endothelial and SMC MEF2c are essential for angiogenesis and SMC differentiation, respectively,^{7, 8} a recent study using an SMC lineage-tracking system shows that endothelial MEF2c inhibits SMC migration into subendothelial intima, an essential process of vascular remodeling.⁹ Similarly, endothelial GATA (GATA zinc finger transcription factor family)-6 regulates SMC proliferation in neointimal hyperplasia via paracrine PDGF-B signaling.¹⁰ MEF2c and GATA6 appear to be mainly involved in endothelial-SMC interaction in vascular remodeling.

In addition to SMC phenotypic modulation and subsequent SMC proliferation/migration, SMC apoptosis is also involved in vascular remodeling by countering the SMC accumulation in arterial wall.¹¹ Yu et al find that FOXO3a induces SMC apoptosis, which attenuates vascular remodeling through mediating MMP13 expression.¹² BMAL1, a transcription factor regulating circadian rhythm and promoting aneurysm formation, regulates arterial wall remodeling and degeneration by binding to Timp4 promoter and thus repressing Timp4 transcription and activating MMPs in SMCs.¹³

Taken together, these recent studies identify multiple novel transcription factors or coactivators critically important for regulating several cellular processes involved in vascular remodeling and disease development. However, further work is required to address how vascular remodeling is orchestrated by these factors. For example, is a master regulator responsible for the interplay of these proteins?

Epigenetic regulation

Emerging evidence has shown that vascular SMCs undergo epigenetic alterations including DNA and histone modifications during phenotypic modulation and vascular remodeling.^14–16 Epigenetic modifications play important roles in transcriptional regulation of SMC-specific genes. Recent studies show that inhibition of DNA methyltransferase 1 blocks the methylation of demethylation enzyme Tet2 promoter and thus enhances Tet2 expression, which in turn stimulates myocardin expression through inducing myocardin promoter demethylation in SMCs, leading to a suppression of arterial remodeling in atherosclerosis.¹⁷ This is the first demonstration of a novel role of DNA methyltransferase1/Tet2/myocardin axis in SMC phenotypic modulation and vascular remodeling. Another study reveals that overexpression of epigenetic reader BRD4 (bromodomain protein 4) promotes coronary arterial remodeling in pulmonary arterial hypertension by triggering an imbalance between SMC proliferation and apoptosis.¹⁸ This study provides a novel mechanism explaining the prevalence of coronary artery disease in patients with pulmonary arterial hypertension. Furthermore, telomerase reverse transcriptase (TERT) regulates SMC proliferation and neointima formation in restenosis through augmenting chromatin accessibility to E2F1 and facilitating histone acetylation on E2F1 target genes in the SMCs,¹⁹ suggesting that TERT is a potent epigenetic regulator for SMC proliferation and vascular remodeling.

O-GlcNAcylation, a nutrient-sensitive sugar modification, involves the epigenetic regulation of gene expression.²⁰ O-GlcNAcylation modification regulates inflammation-induced vascular injury by adding N-acetyl-glucosamine residue (GlcNAc) on A20 with extracellular GlcN or Thiamet G. The zinc-finger protein A20, also known as TNFAIP3 (tumor necrosis factor α-induced protein 3), regulates inflammation, immune systems, and TNF-α–induced apoptosis, specifically NF-κB signaling.²¹ A20 knockdown eliminates the inhibitory effect of glucosamine on neointimal formation in vivo. This finding demonstrates that O-GlcNAcylation may be a beneficial epigenetic modification hindering the development of inflammation-related vascular diseases.²² More efforts are needed to elucidate the relationship between epigenome and genome, which may help understand the pathophysiology of vascular remodeling in vascular diseases.

Post-transcriptional regulation

MicroRNAs (miRNAs) are a class of small (21–25 nucleotides) non-coding RNA molecules that mediate translational repression or mRNA degradation of protein-coding genes in mammals.²³ A few new miRNAs have been identified as key post-transcriptional regulators for SMC phenotypic transition and arterial remodeling. MiR-34a induces SMC growth arrest and promotes SMC mineralization in vascular calcification through targeting AXL receptor tyrosine kinase and SIRT1.²⁴ In addition to miR-34a, miR-125b in SMCs appears to be also a potential regulator of vascular calcification. MiR-125b can be secreted by vascular SMCs, and serum miR-125b levels are inversely correlated with the severity of uremic vascular calcification in patients with end-stage renal diseases.²⁵ However, the roles of miR-125b in SMCs and vascular remodeling need to be further established by gain- or loss-of-function studies.

Besides a role in vascular calcification, several miRNAs are implicated in vascular remodeling in other disease states. MiR-21 inhibits the development of thoracic aortic aneurysm in mice through altering TGFβ/Smad3 signaling,²⁶ whereas miR-33 enhances abdominal aortic aneurysm (AAA) via inflammatory signaling pathways in SMCs.²⁷ HnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), on the other hand, reduces SMC proliferation and inhibits neointimal formation through modulating biosynthesis of miR-124.²⁸ Moreover, miR-143/145, the fate regulators of SMCs, are recently found to play essential roles in myogenic responsiveness. MiR-143/145 knockout mice exhibit aggravated neointimal hyperplasia in mesenteric artery lesions resulting from AngII-induced hypertension.²⁹ This study defines the importance of miR-143/145 mechanistically in myogenic contraction and in the development of vascular injury in myogenic active vessels during hypertension.

Unlike miRNAs, long non-coding RNAs (lncRNAs) are a large (> 200 nucleotides) and diverse class of transcribed RNAs. LncRNAs do not encode proteins and may regulate gene expression at post-transcriptional level via various mechanisms including serving as miRNA sponges.³⁰ A recent study identifies lncRNA MALAT1 as a novel regulator of SMC proliferation and migration in arterial stiffness.³¹ The study also provides a transcriptomic landscape of human aortic and coronary SMCs in response to extracellular matrix stiffness. Benefitted from their large quantities present in the transcriptome, identification of the functional lncRNAs may provide potential therapeutic targets for treating vascular diseases.

Receptor signaling pathways

Molecular interactions between ligands and receptors trigger signaling cascades essential for numerous cellular processes. Multiple receptors and/or ligands have been delineated to regulate different aspects of SMC phenotypic modulation and vascular remodeling. Transmembrane orphan receptor endosialin regulates SMC transition into synthetic and proinflammatory phenotypes in atherosclerosis.³² P2Y₁₂, a receptor originally identified in platelets, is induced in SMCs and enhances SMC motility in HFD-induced atherosclerosis through promoting cofilin dephosphorylation and actin disassembly.³³ Likewise, CD36, a thrombospondin receptor mediating inflammatory responses in macrophages and platelets, promotes SMC proliferation in neointimal hyperplasia by inactivating STAT3 signaling.³⁴ In addition, serotonin, a monoamine neurotransmitter, promotes SMC proliferation and vascular remodeling via 5-HT_1B receptor/Nox1/Nrf2 cascade, contributing to the development of pulmonary arterial hypertension.³⁵ Conversely, IGF1R in SMCs negatively regulates SMC proliferation and migration and thus impedes the progression of advanced atherosclerosis.³⁶

In addition to SMC phenotypic transition and proliferation/migration, ligand-receptor interactions are also found to regulate vascular calcification. For examples, estrogen steroid hormone 17β-estradiol increases SMC mineralization and atherosclerotic calcification through suppressing estrogen receptor β expression in both male and female mice.³⁷ Collagen receptor discoidin domain receptor 1 (DDR1) accelerates SMC mineralization and atherosclerotic vascular calcification in diabetes mellitus in a PI3K/Akt/Runx2 signaling axis-dependent manner.³⁸ In contrast, LDL receptor-related protein 1 (LRP1) promotes contractile SMC phenotype and protects against aneurysm development by modulating calcium signaling in SMCs.³⁹ How these different ligand-receptor interactions work together to regulate SMC phenotypes in vascular remodeling as seen in atherosclerosis and aneurysm development requires further investigation.

Mitochondrial signaling pathways

Mitochondria are highly dynamic organelles that play critical roles in regulating energy metabolism and oxidative stress in cells.⁴⁰ Mitochondrial dysfunction is closely associated with SMC phenotypic modulation in vascular remodeling.⁴¹ However, the key regulators and the underlying mechanisms for regulating mitochondria-related SMC function in vascular remodeling are not adequately identified. Yan et al have shown that paraoxonase suppresses oxidative stress in SMCs and protects against AAA formation.⁴² However, HMGB2 is found to promote the production of reactive oxygen species and serve as a positive regulator of SMC proliferation and migration in in-stent restenosis.⁴³ The paraoxonase/HMGB2 axis may play a key role in maintaining the balance between oxidized and reduced states of SMCs during vascular remodeling. Unlike HMGB2, CaMKII promotes SMC migration and neointimal hyperplasia through increasing mitochondrial mobility in SMCs, as reported in an elegant study by Nguyen et al.⁴⁴ MtDNA damage can promote atherosclerosis,⁴⁵ but the endogenous regulatory factors are less well understood. Yu et al show that mitochondrial helicase Twinkle increases mtDNA integrity and copy number in SMCs, which inhibits SMC apoptosis and thus stabilizes atherosclerotic plaques.⁴⁶

Other novel regulators and signaling pathways

Exosomes are extracellular vesicles released from the cells and participate in regulation of cellular processes.⁴⁷ SMC-derived exosomes loaded with prothrombin reduce SMC apoptosis and vascular calcification,⁴⁸ highlighting a new mediator between SMCs and vascular remodeling. In addition, a recent study has reported that antioxidant resveratrol attenuates angiotensin II-induced AAA in mice that is associated with changes of angiotensin-converting enzyme 2 (ACE2). In vitro studies support a novel signaling pathway of resveratrol increasing ACE2 in human aortic SMCs through a sirtuin-1-mediated pathway.⁴⁹ These results are consistent with a previous study showing that ACE2 deficiency exacerbates Ang II-mediated vascular remodeling due to increased reactive oxygen species and SMC apoptosis.⁵⁰ Moreover, a later study supports the protective role of resveratrol against arterial aging by increasing ACE2 and SIRT1 among other factors, ⁵¹ in line with the findings in angiotensin II-induced AAA. ⁴⁹ The later study suggests that resveratrol protects against vascular aging by reducing activity of the prorenin receptor (PRR)-ACE-Ang II axis and increasing activity of the ACE2-Ang-(1–7)-Ang II type 2 receptor (ATR2)-Mas receptor (MasR) axis.

Protein kinases are key regulators of cellular activation processes. Studies from our and other laboratories reveal that Janus kinase 3 (JAK3) and phosphoinositide 3-kinase-gamma (PI3Kγ) promote SMC proliferation and contribute to neointimal formation, respectively. ^{52, 53}. JAK3 promotes SMC proliferation through activating signal transducer and activator of transcription 3 (STAT3) and c-Jun N-terminal kinase (JNK), two signaling pathways critical for SMC proliferation and vascular remodeling. Knockdown of JAK3 attenuates injury-induced neointima formation by reducing neointimal SMC proliferation. JAK3 appears to be also involved in SMC survival in rat carotid artery balloon injury model. ⁵² Since Inhibition of JAK3 affects the activities of a number of PI3K downstream effectors including Akt in immune cells,⁵⁴ studying the potential crosstalk between JAK3 and PI3Kγ in SMCs may further unravel the mechanisms underlying vascular remodeling. In addition to JAK3 and PI3Kγ, checkpoint kinase 1 (CHK1) regulates vascular remodeling in pulmonary arterial hypertension through inducing SMC cell cycle progression,⁵⁵ suggesting a close correlation between SMC cell cycle control and SMC phenotypes in vascular remodeling.

Interleukin (IL)-33 is well established as a proinflammatory cytokine acting on vascular endothelial cells and pulmonary epithelial cells.^{56, 57} However, IL-33 is required for SMC migration and vascular remodeling in response to arterial injury, and IL-33 expression is mediated by nuclear factor of activated T cells 1 (NFATc1)/ E2F transcription factor 1 (E2F1)/ LIM and cysteine-rich domains protein 1 (LMCD1) axis.⁵⁸ Therefore, the roles and mechanisms of IL-33 in pathogenesis of SMC-related vascular diseases need particular emphasis.

Summary

SMC phenotypic transition-triggered arterial remodeling is a hallmark process that follows vascular injury. Recent identification of the important regulators and signaling pathways involved in this process is likely to provide novel insights into the disease development, which may lead to new therapeutic strategies in the prevention and treatment of proliferative vascular diseases.

Due to divergent biological factors causing sex-specific protective and harmful effects between males and females, many cardiovascular diseases show milder phenotypes in females than in males in their young ages. Thus, most studies on vascular remodeling have been performed in only one sex, mainly on males. This is partially due to the lack of SMC-specific Cre mouse lines for both males and females in recent years. However, National Institutes of Health guidelines requires researchers to consider sex difference as a biological variable in preclinical animal studies. ATVB has also published several articles to emphasize the importance of studying and reporting sex and sex difference.^59–62 Therefore, it is suggested to include both males and females in designing, executing, and reporting experiments studying SMC phenotypes in vascular remodeling and other related arterial pathology. Development of SMC-specific Cre lines that can cause Cre-Lox recombination in both male and female mice is likely to enhance the study of SMC phenotypes in vascular remodeling and related pathology in females.⁶³