Current treatments for the management of homozygous familial hypercholesterolemia: a systematic review and commentary.

AIMS: Homozygous familial hypercholesterolemia (HoFH) is a rare disorder characterized by markedly elevated circulating low-density lipoprotein cholesterol (LDL-C) from birth. This review aimed to critically evaluate treatments for HoFH with respect to their efficacy, safety, accessibility, overall context and position within the treatment pathway. METHODS: A mixed-methods review was undertaken to systematically identify and characterize primary interventional studies on HoFH, with a focus on LDL-C reduction as the primary outcome. Interventions assessed were ezetimibe, proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i), lomitapide, evinacumab, with or without LDL apheresis. RESULTS: Twenty-six seminal studies reporting unique patient data were identified. Four studies were randomized controlled trials (RCTs) with the remainder being single-arm trials or observational registries. Data extracted were heterogeneous and not suitable for meta-analyses. Two RCTs, assessed at being low risk of bias, demonstrated PCSK9i were safe and moderately effective. An RCT demonstrated evinacumab was safe and effective in all HoFH subgroups. Lomitapide was reported to be efficacious in a single-arm trial, but issues with adverse events, tolerability, and adherence were identified. An RCT on ezetimibe showed it was moderately effective when combined with a statin. LDL apheresis was reported as effective, but its evidence base was at very high risk of bias. All interventions lowered LDL-C, but the magnitude of this, and certainty in the supporting evidence, varied. CONCLUSION: In practice, multiple treatments are required to treat HoFH. The sequencing of these should be made on an individualized basis, with consideration made to the benefits of each intervention.

Homozygous familial hypercholesterolaemia (HoFH) is a rare genetic disorder that results in elevated cholesterol levels, which can cause premature cardiovascular events such as heart attacks and stroke. We performed a literature review to systematically identify and analyse studies reporting on newer treatments for HoFH which lower cholesterol levels, focussing on the overall advantages and disadvantages of each treatment. We identified 26 studies, including clinical trials and observational research, reporting on the interventions ezetimibe, proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i), lomitapide, evinacumab, and LDL apheresis. While all treatments showed promise in reducing cholesterol levels, none were sufficient to effectively treat HoFH on their own, and often the confidence in the results were limited by the methodological weaknesses of the studies. The evidence suggests that management of HoFH requires an individualized approach, with consideration given to the efficacy, safety, tolerability and accessibility of each treatment.

Deficiency of lncRNA MERRICAL abrogates macrophage chemotaxis and diabetes-associated atherosclerosis.

Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr-/- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage-enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL-deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr-/- mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage-enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

FAM222A, Part of the BET-Regulated Basal Endothelial Transcriptome, Is a Novel Determinant of Endothelial Biology and Angiogenesis-Brief Report.

BACKGROUND: BETs (bromodomain and extraterminal domain-containing epigenetic reader proteins), including BRD4 (bromodomain-containing protein 4), orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced proinflammatory, proatherosclerotic transcriptional responses; BET inhibitors, like JQ1, repress these effects and decrease atherosclerosis. While BET effects in pathogenic conditions have prompted therapeutic BET inhibitor development, BET action under basal conditions, including ECs, has remained understudied. To understand BET action in basal endothelial transcriptional programs, we first analyzed EC RNA-Seq data in the absence versus presence of JQ1 before using BET regulation to identify novel determinants of EC biology and function. METHODS: RNA-Seq datasets of human umbilical vein ECs without and with JQ1 treatment were analyzed. After identifying C12orf34, also known as FAM222A (family with sequence similarity 222 member A), as a previously unreported, basally expressed, potently JQ1-induced EC gene, FAM222A was studied in endothelial and angiogenic responses in vitro using small-interference RNA silencing and lentiviral overexpression, in vitro, ex vivo and in vivo, including aortic sprouting, matrigel plug assays, and murine neonatal oxygen-induced retinopathy. RESULTS: Resting EC RNA-Seq data indicate BETs direct transcriptional programs underlying core endothelial properties including migration, proliferation, and angiogenesis. BET inhibition in resting ECs also significantly induced a subset of mRNAs, including FAM222A-a unique BRD4-regulated gene with no reported EC role. Silencing endothelial FAM222A significantly decreased cellular proliferation, migration, network formation, aorta sprouting, and Matrigel plug vascularization through coordinated modulation of VEGF (vascular endothelial growth factor) and NOTCH mediator expression in vitro, ex vivo, in vivo; lentiviral FAM222A overexpression had opposite effects. In vivo, siFAM222A significantly repressed retinal revascularization in neonatal murine oxygen-induced retinopathy through similar angiogenic signaling modulation. CONCLUSIONS: BET control over the basal endothelial transcriptome includes FAM222A, a novel, BRD4-regulated, key determinant of endothelial biology and angiogenesis.

Impaired angiogenesis in diabetic critical limb ischemia is mediated by a miR-130b/INHBA signaling axis.

Patients with peripheral artery disease (PAD) and diabetes compose a high-risk population for development of critical limb ischemia (CLI) and amputation, although the underlying mechanisms remain poorly understood. Comparison of dysregulated microRNAs from diabetic patients with PAD and diabetic mice with limb ischemia revealed the conserved microRNA, miR-130b-3p. In vitro angiogenic assays demonstrated that miR-130b rapidly promoted proliferation, migration, and sprouting in endothelial cells (ECs), whereas miR-130b inhibition exerted antiangiogenic effects. Local delivery of miR-130b mimics into ischemic muscles of diabetic mice (db/db) following femoral artery ligation (FAL) promoted revascularization by increasing angiogenesis and markedly improved limb necrosis and amputation. RNA-Seq and gene set enrichment analysis from miR-130b-overexpressing ECs revealed the BMP/TGF-ß signaling pathway as one of the top dysregulated pathways. Accordingly, overlapping downregulated transcripts from RNA-Seq and miRNA prediction algorithms identified that miR-130b directly targeted and repressed the TGF-ß superfamily member inhibin-ß-A (INHBA). miR-130b overexpression or siRNA-mediated knockdown of INHBA induced IL-8 expression, a potent angiogenic chemokine. Lastly, ectopic delivery of silencer RNAs (siRNA) targeting Inhba in db/db ischemic muscles following FAL improved revascularization and limb necrosis, recapitulating the phenotype of miR-130b delivery. Taken together, a miR-130b/INHBA signaling axis may provide therapeutic targets for patients with PAD and diabetes at risk of developing CLI.

ATP-releasing SWELL1 channel in spinal microglia contributes to neuropathic pain.

Following peripheral nerve injury, extracellular adenosine 5'-triphosphate (ATP)-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

A miRNA/CXCR4 signaling axis impairs monopoiesis and angiogenesis in diabetic critical limb ischemia.

Patients with peripheral artery disease (PAD) and diabetes have the highest risk of critical limb ischemia (CLI) and amputation, yet the underlying mechanisms remain incompletely understood. MicroRNA (miRNA) sequencing of plasma from diabetic patients with or without CLI was compared to diabetic mice with acute or subacute limb ischemia to identify conserved miRNAs. miRNA-KO mice on high-fat diet were generated to explore the impact on CLI. Comparison of dysregulated miRNAs from diabetic individuals with PAD and diabetic mice with limb ischemia revealed conserved miR-181 family members. High-fat-fed, diabetic Mir181a2b2-KO mice had impaired revascularization in limbs due to abrogation of circulating Ly6Chi monocytes, with reduced accumulation in ischemic skeletal muscles. M2-like KO macrophages under diabetic conditions failed to produce proangiogenic cytokines. Single-cell transcriptomics of the bone marrow niche revealed that the reduced monocytosis in diabetic KO mice was a result of impaired hematopoiesis, with increased CXCR4 signaling in bone marrow Lineage-Sca1+Kit+ (LSK) cells. Exogenous Ly6Chi monocytes from nondiabetic KO mice rescued the impaired revascularization in ischemic limbs of diabetic KO mice. Increased Cxcr4 expression was mediated by the miR-181 target, Plac8. Taken together, our results show that MiR-181a/b is a putative mediator of diabetic CLI and contributes to changes in hematopoiesis, monocytosis, and macrophage polarization.

ATP-releasing SWELL1 channel in spinal microglia contributes to neuropathic pain.

Following peripheral nerve injury, extracellular ATP-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia.

Peripheral artery disease (PAD) is an occlusive disease of limb arteries. Critical limb ischemia (CLI) is an advanced form of PAD that is prognostically worse in subjects with diabetes and can result in limb loss, gangrene, and death, although the underlying signaling mechanisms that contribute to its development remain poorly understood. By comparing plasma samples from diabetic humans with PAD and mouse models of PAD, we identified miR-375 to be significantly downregulated in humans and mice during progression to CLI. Overexpression of miR-375 was pro-angiogenic in endothelial cells in vitro and induced endothelial migration, proliferation, sprouting, and vascular network formation, whereas miR-375 inhibition conferred anti-angiogenic effects. Intramuscular delivery of miR-375 improved blood flow recovery to diabetic mouse hindlimbs following femoral artery ligation (FAL) and improved neovessel growth and arteriogenesis in muscle tissues. Using RNA-sequencing and prediction algorithms, Kruppel-like factor 5 (KLF5) was identified as a direct target of miR-375 and siRNA knockdown of KLF5 phenocopied the effects of miR-375 overexpression in vitro and in vivo through regulatory changes in NF-kB signaling. Together, a miR-375-KLF5-NF-kB signaling axis figures prominently as a potential therapeutic pathway in the development CLI in diabetes.

Dysregulated Genes, MicroRNAs, Biological Pathways, and Gastrocnemius Muscle Fiber Types Associated With Progression of Peripheral Artery Disease: A Preliminary Analysis.

Background Peripheral artery disease (PAD) is associated with gastrocnemius muscle abnormalities. However, the biological pathways associated with gastrocnemius muscle dysfunction and their associations with progression of PAD are largely unknown. This study characterized differential gene and microRNA (miRNA) expression in gastrocnemius biopsies from people without PAD compared with those with PAD. Participants with PAD included those with and without PAD progression. Methods and Results mRNA and miRNA sequencing were performed to identify differentially expressed genes, differentially expressed miRNAs, mRNA-miRNA interactions, and associated biological pathways for 3 sets of comparisons: (1) PAD progression (n=7) versus non-PAD (n=7); (2) PAD no progression (n=6) versus non-PAD; and (3) PAD progression versus PAD no progression. Immunohistochemistry was performed to determine gastrocnemius muscle fiber types and muscle fiber size. Differentially expressed genes and differentially expressed miRNAs were more abundant in the comparison of PAD progression versus non-PAD compared with PAD with versus without progression. Among the top significant cellular pathways in subjects with PAD progression were muscle contraction or development, transforming growth factor-beta, growth/differentiation factor, and activin signaling, inflammation, cellular senescence, and notch signaling. Subjects with PAD progression had increased frequency of smaller Type 2a gastrocnemius muscle fibers in exploratory analyses. Conclusions Humans with PAD progression exhibited greater differences in the number of gene and miRNA expression, biological pathways, and Type 2a muscle fiber size compared with those without PAD. Fewer differences were observed between people with PAD without progression and control patients without PAD. Further study is needed to confirm whether the identified transcripts may serve as potential biomarkers for diagnosis and progression of PAD.

miR-181b regulates vascular endothelial aging by modulating an MAP3K3 signaling pathway.

Endothelial cell (EC) aging plays a vital role in the pathogenesis of cardiovascular disease (CVD). MicroRNAs have emerged as crucial regulators of target gene expression by inhibiting mRNA translation and/or promoting mRNA degradation. We identify an aging-related and oxidative stress-responsive microRNA, miR-181b, that inhibits endothelial cell apoptosis and senescence. In gain- or loss-of-function studies, miR-181b regulated the expression of key apoptosis markers (Bcl2, Bax, cleaved-Caspase3) and senescence markers (p16, p21, γH2AX) and the ratio of apoptotic cells (TUNEL-positive) and senescent cells (SA-ßgal-positive) in H2 O2 -induced ECs. Mechanistically, miR-181b targets MAP3K3 and modulates a MAP3K3/MKK/MAPK signaling pathway. MAP3K3 knockdown recapitulated the phenotype of miR-181b overexpression and miR-181b was dependent on MAP3K3 for regulating EC apoptosis and senescence. In vivo, miR-181b expression showed a negative correlation with increasing age in the mouse aorta. Endothelial-specific deficiency of miR-181a2b2 increased the target MAP3K3, markers of vascular senescence (p16, p21), and DNA double-strand breaks (γH2AX) in the aorta of aged mice. Collectively, this study unveils an important role of miR-181b in regulating vascular endothelial aging via an MAP3K3-MAPK signaling pathway, providing new potential therapeutic targets for antiaging therapy in CVD.

Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Axis in CD4+ T Cells.

BACKGROUND: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS: TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS: TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

Endothelial cell-specific deletion of a microRNA accelerates atherosclerosis.

BACKGROUND AND AIMS: Chronic vascular endothelial inflammation predisposes to atherosclerosis; however, the cell-autonomous roles for endothelial-expressing microRNAs (miRNAs) are poorly understood in this process. MiR-181b is expressed in several cellular constituents relevant to lesion formation. The aim of this study is to examine the role of genetic deficiency of the miR-181b locus in endothelial cells during atherogenesis. METHODS AND RESULTS: Using a proprotein convertase subtilisin/kexin type 9 (PCSK9)-induced atherosclerosis mouse model, we demonstrated that endothelial cell (EC)-specific deletion of miR-181a2b2 significantly promoted atherosclerotic lesion formation, cell adhesion molecule expression, and the influx of lesional macrophages in the vessel wall. Yet, endothelium deletion of miR-181a2b2 did not affect body weight, lipid metabolism, anti-inflammatory Ly6Clow or the pro-inflammatory Ly6Cinterm and Ly6Chigh fractions in circulating peripheral blood mononuclear cells (PBMCs), and pro-inflammatory or anti-inflammatory mediators in both bone marrow (BM) and PBMCs. Mechanistically, bulk RNA-seq and gene set enrichment analysis of ECs enriched from the aortic arch intima, as well as single cell RNA-seq from atherosclerotic lesions, revealed that endothelial miR-181a2b2 serves as a critical regulatory hub in controlling endothelial inflammation, cell adhesion, cell cycle, and immune response during atherosclerosis. CONCLUSIONS: Our study establishes that deficiency of a miRNA specifically in the vascular endothelium is sufficient to profoundly impact atherogenesis. Endothelial miR-181a2b2 deficiency regulates multiple key pathways related to endothelial inflammation, cell adhesion, cell cycle, and immune response involved in the development of atherosclerosis.

Isolation and culture of murine aortic cells and RNA isolation of aortic intima and media: Rapid and optimized approaches for atherosclerosis research.

BACKGROUND AND AIMS: Isolation of cellular constituents from the mouse aorta is commonly used for expression or functional analyses in atherosclerosis research. However, current procedures to isolate primary cells are difficult, inefficient, and require separate mice. RNA extraction from aortic intima and media for transcriptomic analysis is also considered difficult with mixed RNA yields. To address these gaps, we provide: 1) a rapid, efficient protocol to isolate and culture diverse cell types concomitantly from the mouse aorta using immunomagnetic cell isolation; and 2) an optimized aortic intimal peeling technique for efficient RNA isolation from the intima and media. METHODS AND RESULTS: Aortic cells were obtained using an enzymatic solution and different cell types were isolated by magnetic beads conjugated to antibodies targeting endothelial cells (CD31+), leukocytes (CD45+), and fibroblast cells (CD90.2+), and smooth muscle cells were isolated by negative selection. Our protocol allows the isolation of relatively large numbers of cells (10,000 cells per aorta) in a predictable manner with high purity (>90%) verified by cell-marker gene expression, immunofluorescence, and flow cytometry. These cells are all functionally active when grown in cell culture. We also provide a rapid method to collect aortic intima-enriched RNA from Ldlr-/- mice utilizing an intima peeling approach and assess transcriptomic profiling associated with accelerated lesion formation. CONCLUSIONS: This protocol provides an effective means for magnetic bead-based isolation of different cell types from the mouse aortic wall, and the isolated cells can be utilized for functional and mechanistic studies for a range of vascular diseases including atherosclerosis.

A miRNA cassette reprograms smooth muscle cells into endothelial cells.

Cellular reprogramming through targeting microRNAs (miRNAs) holds promise for regenerative therapy due to their profound regulatory effects in proliferation, differentiation, and function. We hypothesized that transdifferentiation of vascular smooth muscle cells (SMCs) into endothelial cells (ECs) using a miRNA cassette may provide a novel approach for use in vascular disease states associated with endothelial injury or dysfunction. miRNA profiling of SMCs and ECs and iterative combinatorial miRNA transfections of human coronary SMCs revealed a 4-miRNA cassette consisting of miR-143-3p and miR-145-5p inhibitors and miR-146a-5p and miR-181b-5p mimics that efficiently produced induced endothelial cells (iECs). Transcriptome profiling, protein expression, and functional studies demonstrated that iECs exhibit high similarity to ECs. Injected iECs restored blood flow recovery even faster than conventional ECs in a murine hindlimb ischemia model. This study demonstrates that a 4-miRNA cassette is sufficient to reprogram SMCs into ECs and shows promise as a novel regenerative strategy for endothelial repair.

MiR-30 promotes fatty acid beta-oxidation and endothelial cell dysfunction and is a circulating biomarker of coronary microvascular dysfunction in pre-clinical models of diabetes.

BACKGROUND: Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction. METHODS: The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs). RESULTS: We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid ß-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid ß-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium. CONCLUSIONS: MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart.

Deficiency of lncRNA SNHG12 impairs ischemic limb neovascularization by altering an endothelial cell cycle pathway.

SNHG12, a long noncoding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease has not been elucidated. Hind-limb ischemia studies using femoral artery ligation (FAL) in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain- and loss-of-function studies demonstrated that SNHG12 regulated angiogenesis - SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including IGF-2 mRNA-binding protein 3 (IGF2BP3, also known as IMP3). RNA-Seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IMP3. Knockdown of SNHG12 in mice undergoing FAL using injected gapmeRs) decreased angiogenesis, an effect that was more pronounced in a model of insulin-resistant db/db mice. RNA-Seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12 knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.

A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-ß1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600­1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.