Integrative analysis of the lncRNA-miRNA-mRNA interactions in smooth muscle cell phenotypic transitions.

Objectives: We previously found that the pluripotency factor OCT4 is reactivated in smooth muscle cells (SMC) in human and mouse atherosclerotic plaques and plays an atheroprotective role. Loss of OCT4 in SMC in vitro was associated with decreases in SMC migration. However, molecular mechanisms responsible for atheroprotective SMC-OCT4-dependent effects remain unknown. Methods: Since studies in embryonic stem cells demonstrated that OCT4 regulates long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), making them candidates for OCT4 effect mediators, we applied an in vitro approach to investigate the interactions between OCT4-regulated lncRNAs, mRNAs, and miRNAs in SMC. We used OCT4 deficient mouse aortic SMC (MASMC) treated with the pro-atherogenic oxidized phospholipid POVPC, which, as we previously demonstrated, suppresses SMC contractile markers and induces SMC migration. Differential expression of lncRNAs, mRNAs, and miRNAs was obtained by lncRNA/mRNA expression array and small-RNA microarray. Long non-coding RNA to mRNA associations were predicted based on their genomic proximity and association with vascular diseases. Given a recently discovered crosstalk between miRNA and lncRNA, we also investigated the association of miRNAs with upregulated/downregulated lncRNA-mRNA pairs. Results: POVPC treatment in SMC resulted in upregulating genes related to the axon guidance and focal adhesion pathways. Knockdown of Oct4 resulted in differential regulation of pathways associated with phagocytosis. Importantly, these results were consistent with our data showing that OCT4 deficiency attenuated POVPC-induced SMC migration and led to increased phagocytosis. Next, we identified several up- or downregulated lncRNA associated with upregulation of the specific mRNA unique for the OCT4 deficient SMC, including upregulation of ENSMUST00000140952-Hoxb5/6 and ENSMUST00000155531-Zfp652 along with downregulation of ENSMUST00000173605-Parp9 and, ENSMUST00000137236-Zmym1. Finally, we found that many of the downregulated miRNAs were associated with cell migration, including miR-196a-1 and miR-10a, targets of upregulated ENSMUST00000140952, and miR-155 and miR-122, targets of upregulated ENSMUST00000155531. Oppositely, the upregulated miRNAs were anti-migratory and pro-phagocytic, such as miR-10a/b and miR-15a/b, targets of downregulated ENSMUST00000173605, and miR-146a/b and miR-15b targets of ENSMUST00000137236. Conclusion: Our integrative analyses of the lncRNA-miRNA-mRNA interactions in SMC indicated novel potential OCT4-dependent mechanisms that may play a role in SMC phenotypic transitions.

Unraveling the Role of Sex in Endothelial Cell Dysfunction: Evidence From Lineage Tracing Mice and Cultured Cells.

BACKGROUND: Biological sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (ECs) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC. METHODS: We used human and mouse aortic EC as well as EC-lineage tracing (Cdh5-CreERT2 Rosa-YFP [yellow fluorescence protein]) atherosclerotic Apoe-/- mice to investigate the biological sexual dimorphism of the EC functions in vitro and in vivo. Bioinformatics analyses were performed on male and female mouse aortic EC and human lung and aortic EC. RESULTS: In vitro, female human and mouse aortic ECs showed more apoptosis and higher cellular reactive oxygen species levels than male EC. In addition, female mouse aortic EC had lower mitochondrial membrane potential (ΔΨm), lower TFAM (mitochondrial transcription factor A) levels, and decreased angiogenic potential (tube formation, cell viability, and proliferation) compared with male mouse aortic EC. In vivo, female mice had significantly higher lipid accumulation within the aortas, impaired glucose tolerance, and lower endothelial-mediated vasorelaxation than males. Using the EC-lineage tracing approach, we found that female lesions had significantly lower rates of intraplaque neovascularization and endothelial-to-mesenchymal transition within advanced atherosclerotic lesions but higher incidents of missing EC lumen coverage and higher levels of oxidative products and apoptosis. RNA-seq analyses revealed that both mouse and human female EC had higher expression of genes associated with inflammation and apoptosis and lower expression of genes related to angiogenesis and oxidative phosphorylation than male EC. CONCLUSIONS: Our study delineates critical sex-specific differences in EC relevant to proinflammatory, pro-oxidant, and angiogenic characteristics, which are entirely consistent with a vulnerable phenotype in females. Our results provide a biological basis for sex-specific proatherosclerotic mechanisms.

Smooth muscle cells-specific loss of OCT4 accelerates neointima formation after acute vascular injury.

Introduction: There is growing evidence that smooth muscle cell (SMC) phenotypic transitions play critical roles during normal developmental and tissue recovery processes and in pathological conditions such as atherosclerosis. However, the molecular mechanisms responsible for these transitions are not well understood. Recently, we found that the embryonic stem cell/induced pluripotent stem cell (iPSC) factor OCT4, which was believed to be silenced in somatic cells, plays an atheroprotective role in SMC, and regulates angiogenesis after corneal alkali burn and hindlimb ischemia by mediating microvascular SMC and pericyte migration. However, the kinetics of OCT4 activation in arterial SMC and its role in acute pathological conditions are still unknown. Methods and Results: Here, using an Oct4-IRES-GFP reporter mouse model, we found that OCT4 is reactivated in the carotid artery 18 hours post-acute ligation-induced injury, a common in vivo model of the SMC phenotypic transitions. Next, using a tamoxifen-inducible Myh11-CreERT2 Oct4 knockout mouse model, we found that the loss of OCT4, specifically in SMC, led to accelerated neointima formation and increased tunica media following carotid artery ligation, at least in part by increasing SMC proliferation within the media. Bulk RNA sequencing analysis on the cultured SMC revealed significant down-regulation of the SMC contractile markers and dysregulation of the genes belonging to the regulation of cell proliferation and, positive and negative regulation for cell migration ontological groups following genetic inactivation of Oct4. We also found that loss of Oct4 resulted in suppression of contractile SMC markers after the injury and in cultured aortic SMC. Further mechanistic studies revealed that OCT4 regulates SMC contractile genes, ACTA2 and TAGLN, at least in part by direct binding to the promoters of these genes. Conclusion: These results demonstrate that the pluripotency factor OCT4 is quickly activated in SMC after the acute vascular injury and inhibits SMC hyperproliferation, which may be protective in preventing excessive neointima formation.

Endothelial-to-Mesenchymal Transition in Atherosclerosis: Friend or Foe?

Despite many decades of research, complications of atherosclerosis resulting from the rupture or erosion of unstable plaques remain the leading cause of death worldwide. Advances in cellular lineage tracing techniques have allowed researchers to begin investigating the role of individual cell types in the key processes regulating plaque stability, including maintenance of the fibrous cap, a protective collagen-rich structure that underlies the endothelium. This structure was previously thought to be entirely derived from smooth muscle cells (SMC), which migrated from the vessel wall. However, recent lineage tracing studies have identified endothelial cells (EC) as an essential component of this protective barrier through an endothelial-to-mesenchymal transition (EndoMT), a process that has previously been implicated in pulmonary, cardiac, and kidney fibrosis. Although the presence of EndoMT in atherosclerotic plaques has been shown by several laboratories using EC-lineage tracing mouse models, whether EndoMT is detrimental (i.e., worsening disease progression) or beneficial (i.e., an athero-protective response that prevents plaque instability) remains uncertain as there are data to support both possibilities, which will be further discussed in this review.

Most cancers carry a substantial deleterious load due to Hill-Robertson interference.

Cancer genomes exhibit surprisingly weak signatures of negative selection (Martincorena et al., 2017; Weghorn, 2017). This may be because selective pressures are relaxed or because genome-wide linkage prevents deleterious mutations from being removed (Hill-Robertson interference; Hill and Robertson, 1966). By stratifying tumors by their genome-wide mutational burden, we observe negative selection (dN/dS ~ 0.56) in low mutational burden tumors, while remaining cancers exhibit dN/dS ratios ~1. This suggests that most tumors do not remove deleterious passengers. To buffer against deleterious passengers, tumors upregulate heat shock pathways as their mutational burden increases. Finally, evolutionary modeling finds that Hill-Robertson interference alone can reproduce patterns of attenuated selection and estimates the total fitness cost of passengers to be 46% per cell on average. Collectively, our findings suggest that the lack of observed negative selection in most tumors is not due to relaxed selective pressures, but rather the inability of selection to remove deleterious mutations in the presence of genome-wide linkage.

Inhibition of DYRK1a Enhances Cardiomyocyte Cycling After Myocardial Infarction.

BACKGROUND: DYRK1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) contributes to the control of cycling cells, including cardiomyocytes. However, the effects of inhibition of DYRK1a on cardiac function and cycling cardiomyocytes after myocardial infarction (MI) remain unknown. METHODS: We investigated the impacts of pharmacological inhibition and conditional genetic ablation of DYRK1a on endogenous cardiomyocyte cycling and left ventricular systolic function in ischemia-reperfusion (I/R) MI using αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (RLTG) (denoted αDKRC::RLTG) and αMHC-Cre::Fucci2aR::DYRK1aflox/flox mice. RESULTS: We observed that harmine, an inhibitor of DYRK1a, improved left ventricular ejection fraction (39.5±1.6% and 29.1±1.6%, harmine versus placebo, respectively), 2 weeks after I/R MI. Harmine also increased cardiomyocyte cycling after I/R MI in αDKRC::RLTG mice, 10.8±1.5 versus 24.3±2.6 enhanced Green Fluorescent Protein (eGFP)+ cardiomyocytes, placebo versus harmine, respectively, P=1.0×10-3. The effects of harmine on left ventricular ejection fraction were attenuated in αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes. The conditional cardiomyocyte-specific genetic ablation of DYRK1a in αMHC-Cre::Fucci2aR::DYRK1aflox/flox (denoted DYRK1a k/o) mice caused cardiomyocyte hyperplasia at baseline (210±28 versus 126±5 cardiomyocytes per 40× field, DYRK1a k/o versus controls, respectively, P=1.7×10-2) without changes in cardiac function compared with controls, or compensatory changes in the expression of other DYRK isoforms. After I/R MI, DYRK1a k/o mice had improved left ventricular function (left ventricular ejection fraction 41.8±2.2% and 26.4±0.8%, DYRK1a k/o versus control, respectively, P=3.7×10-2). RNAseq of cardiomyocytes isolated from αMHC-Cre::Fucci2aR::DYRK1aflox/flox and αMHC-Cre::Fucci2aR mice after I/R MI or Sham surgeries identified enrichment in mitotic cell cycle genes in αMHC-Cre::Fucci2aR::DYRK1aflox/flox compared with αMHC-Cre::Fucci2aR. CONCLUSIONS: The pharmacological inhibition or cardiomyocyte-specific ablation of DYRK1a caused baseline hyperplasia and improved cardiac function after I/R MI, with an increase in cell cycle gene expression, suggesting the inhibition of DYRK1a may serve as a therapeutic target to treat MI.

Platinum Chemotherapy Induces Lymphangiogenesis in Cancerous and Healthy Tissues That Can be Prevented With Adjuvant Anti-VEGFR3 Therapy.

Chemotherapy has been used to inhibit cancer growth for decades, but emerging evidence shows it can affect the tumor stroma, unintentionally promoting cancer malignancy. After treatment of primary tumors, remaining drugs drain via lymphatics. Though all drugs interact with the lymphatics, we know little of their impact on them. Here, we show a previously unknown effect of platinums, a widely used class of chemotherapeutics, to directly induce systemic lymphangiogenesis and activation. These changes are dose-dependent, long-lasting, and occur in healthy and cancerous tissue in multiple mouse models of breast cancer. We found similar effects in human ovarian and breast cancer patients whose treatment regimens included platinums. Carboplatin treatment of healthy mice prior to mammary tumor inoculation increased cancer metastasis as compared to no pre-treatment. These platinum-induced phenomena could be blocked by VEGFR3 inhibition. These findings have implications for cancer patients receiving platinums and may support the inclusion of anti-VEGFR3 therapy into treatment regimens or differential design of treatment regimens to alter these potential effects.

Endothelial OCT4 is atheroprotective by preventing metabolic and phenotypic dysfunction.

AIMS: Until recently, the pluripotency factor Octamer (ATGCAAAT)-binding transcriptional factor 4 (OCT4) was believed to be dispensable in adult somatic cells. However, our recent studies provided clear evidence that OCT4 has a critical atheroprotective role in smooth muscle cells. Here, we asked if OCT4 might play a functional role in regulating endothelial cell (EC) phenotypic modulations in atherosclerosis. METHODS AND RESULTS: Specifically, we show that EC-specific Oct4 knockout resulted in increased lipid, LGALS3+ cell accumulation, and altered plaque characteristics consistent with decreased plaque stability. A combination of single-cell RNA sequencing and EC-lineage-tracing studies revealed increased EC activation, endothelial-to-mesenchymal transitions, plaque neovascularization, and mitochondrial dysfunction in the absence of OCT4. Furthermore, we show that the adenosine triphosphate (ATP) transporter, ATP-binding cassette (ABC) transporter G2 (ABCG2), is a direct target of OCT4 in EC and establish for the first time that the OCT4/ABCG2 axis maintains EC metabolic homeostasis by regulating intracellular heme accumulation and related reactive oxygen species production, which, in turn, contributes to atherogenesis. CONCLUSIONS: These results provide the first direct evidence that OCT4 has a protective metabolic function in EC and identifies vascular OCT4 and its signalling axis as a potential target for novel therapeutics.

Single-cell transcriptomic profile of human pulmonary artery endothelial cells in health and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is an insidious disease characterized by severe remodeling of the pulmonary vasculature caused in part by pathologic changes of endothelial cell functions. Although heterogeneity of endothelial cells across various vascular beds is well known, the diversity among endothelial cells in the healthy pulmonary vascular bed and the pathologic diversity among pulmonary arterial endothelial cells (PAEC) in PAH is unknown and previously unexplored. Here single-cell RNA sequencing technology was used to decipher the cellular heterogeneity among PAEC in the human pulmonary arteries isolated from explanted lungs from three patients with PAH undergoing lung transplantation and three healthy donor lungs not utilized for transplantation. Datasets of 36,368 PAH individual endothelial cells and 36,086 healthy cells were analyzed using the SeqGeq bioinformatics program. Total population differential gene expression analyses identified 629 differentially expressed genes between PAH and controls. Gene Ontology and Canonical Ingenuity analysis revealed pathways that are known to be involved in pathogenesis, as well as unique new pathways. At the individual cell level, dimensionality reduction followed by density based clustering revealed the presence of eight unique PAEC clusters that were typified by proliferative, angiogenic or quiescent phenotypes. While control and PAH harbored many similar subgroups of endothelial cells, PAH had greater proportions of angiogenic and proliferative subsets. These findings identify that only specific subgroups of PAH PAEC have gene expression different than healthy PAEC, and suggest these subpopulations lead to the pathologic functions leading to remodeling.

Endothelial TLR2 promotes proangiogenic immune cell recruitment and tumor angiogenesis.

Toll-like receptor 2 (TLR2) is implicated in various pathologies, mainly in terms of its function within innate immune cells. However, TLR2 is also present in endothelial cells. Here, we explored the physiological and pathophysiological roles of endothelial TLR2 signaling. We found that TLR2 was highly abundant in the endothelium within various tissues using TLR2-IRES-EGFP reporter mice and was required for proinflammatory endothelial cell function. Endothelial cells lacking TLR2 exhibited reduced proinflammatory potential at the protein, cell, and tissue levels. Loss of endothelial TLR2 blunted the inflammatory response to both exogenous and endogenous danger signals in endothelial cells in culture and in vivo. Endothelial TLR2 promoted tumor growth, angiogenesis, and protumorigenic immune cell recruitment in a mouse model of prostate cancer. Furthermore, the cell surface localization of P-selectin and the subsequent production of other critical cell adhesion molecules (such as E-selectin, ICAM-1 and VCAM-1) that recruit immune cells required endothelial TLR2. Our findings demonstrate that endothelial cells actively contribute to innate immune pathways and propose that endothelial TLR2 has a pathological role in proinflammatory conditions.

Perivascular cell-specific knockout of the stem cell pluripotency gene Oct4 inhibits angiogenesis.

The stem cell pluripotency factor Oct4 serves a critical protective role during atherosclerotic plaque development by promoting smooth muscle cell (SMC) investment. Here, we show using Myh11-CreERT2 lineage-tracing with inducible SMC and pericyte (SMC-P) knockout of Oct4 that Oct4 regulates perivascular cell migration and recruitment during angiogenesis. Knockout of Oct4 in perivascular cells significantly impairs perivascular cell migration, increases perivascular cell death, delays endothelial cell migration, and promotes vascular leakage following corneal angiogenic stimulus. Knockout of Oct4 in perivascular cells also impairs perfusion recovery and decreases angiogenesis following hindlimb ischemia. Transcriptomic analyses demonstrate that expression of the migratory gene Slit3 is reduced following loss of Oct4 in cultured SMCs, and in Oct4-deficient perivascular cells in ischemic hindlimb muscle. Together, these results provide evidence that Oct4 plays an essential role within perivascular cells in injury- and hypoxia-induced angiogenesis.

High-content phenotypic assay for proliferation of human iPSC-derived cardiomyocytes identifies L-type calcium channels as targets.

Over 5 million people in the United States suffer from heart failure, due to the limited ability to regenerate functional cardiac tissue. One potential therapeutic strategy is to enhance proliferation of resident cardiomyocytes. However, phenotypic screening for therapeutic agents is challenged by the limited ability of conventional markers to discriminate between cardiomyocyte proliferation and endoreplication (e.g. polyploidy and multinucleation). Here, we developed a novel assay that combines automated live-cell microscopy and image processing algorithms to discriminate between proliferation and endoreplication by quantifying changes in the number of nuclei, changes in the number of cells, binucleation, and nuclear DNA content. We applied this assay to further prioritize hits from a primary screen for DNA synthesis, identifying 30 compounds that enhance proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Among the most active compounds from the phenotypic screen are clinically approved L-type calcium channel blockers from multiple chemical classes whose activities were confirmed across different sources of human induced pluripotent stem cell-derived cardiomyocytes. Identification of compounds that stimulate human cardiomyocyte proliferation may provide new therapeutic strategies for heart failure.