Substrate stiffness promotes vascular smooth muscle cell calcification by reducing the levels of nuclear actin monomers.

Background: Vascular calcification (VC) is a prevalent independent risk factor for adverse cardiovascular events and is associated with diabetes, hypertension, chronic kidney disease, and atherosclerosis. However, the mechanisms regulating the osteogenic differentiation of vascular smooth muscle cells (VSMC) are not fully understood.

Methods: Using hydrogels of tuneable stiffness and lysyl oxidase-mediated stiffening of human saphenous vein ex vivo, we investigated the role of substrate stiffness in the regulation of VSMC calcification.

Combined role for YAP-TEAD and YAP-RUNX2 signalling in substrate-stiffness regulation of cardiac fibroblast proliferation.

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Here we characterise a novel mechanism involving a combined activation of Yes-associated protein (YAP) targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD). We demonstrate that cardiac fibroblast proliferation is enhanced by interaction with a stiff ECM compared to a soft ECM.

METTL3 Regulates Angiogenesis by Modulating let-7e-5p and miRNA-18a-5p Expression in Endothelial Cells.

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MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles.

MicroRNAs (miRs) regulate complex processes, including angiogenesis, by targeting multiple mRNAs. miR-24-3p-3p directly represses eNOS, GATA2, and PAK4 in endothelial cells (ECs), thus inhibiting angiogenesis during development and in the infarcted heart. miR-24-3p is widely expressed in cardiovascular cells, suggesting that it could additionally regulate angiogenesis by acting on vascular mural cells.

Nuclear actin regulates cell proliferation and migration via inhibition of SRF and TEAD.

Actin dynamics regulate cell behaviour in response to physiological signals. Here we demonstrate a novel role for nuclear actin in inhibiting cell proliferation and migration. We demonstrate that physiological signals that elevate cAMP, which is anti-mitogenic in vascular smooth muscle cells, increases nuclear actin monomer levels.

miR-15a/-16 Inhibit Angiogenesis by Targeting the Tie2 Coding Sequence: Therapeutic Potential of a miR-15a/16 Decoy System in Limb Ischemia.

MicroRNA-15a (miR-15a) and miR-16, which are transcribed from the miR-15a/miR-16-1 cluster, inhibit post-ischemic angiogenesis. MicroRNA (miRNA) binding to mRNA coding sequences (CDSs) is a newly emerging mechanism of gene expression regulation. We aimed to (1) identify new mediators of the anti-angiogenic action of miR-15a and -16, (2) develop an adenovirus (Ad)-based miR-15a/16 decoy system carrying a luciferase reporter (Luc) to both sense and inhibit miR-15a/16 activity, and (3) investigate Ad.

Role of TPBG (Trophoblast Glycoprotein) Antigen in Human Pericyte Migratory and Angiogenic Activity.

Objective- To determine the role of the oncofetal protein TPBG (trophoblast glycoprotein) in normal vascular function and reparative vascularization. Approach and Results- Immunohistochemistry of human veins was used to show TPBG expression in vascular smooth muscle cells and adventitial pericyte-like cells (APCs). ELISA, Western blot, immunocytochemistry, and proximity ligation assays evidenced a hypoxia-dependent upregulation of TPBG in APCs not found in vascular smooth muscle cells or endothelial cells.

Nerve growth factor gene therapy improves bone marrow sensory innervation and nociceptor-mediated stem cell release in a mouse model of type 1 diabetes with limb ischaemia.

Aims/hypothesis: Sensory neuropathy is common in people with diabetes; neuropathy can also affect the bone marrow of individuals with type 2 diabetes. However, no information exists on the state of bone marrow sensory innervation in type 1 diabetes. Sensory neurons are trophically dependent on nerve growth factor (NGF) for their survival.

Dual Role of CREB in The Regulation of VSMC Proliferation: Mode of Activation Determines Pro- or Anti-Mitogenic Function.

Vascular smooth muscle cell (VSMC) proliferation has been implicated in the development of restenosis after angioplasty, vein graft intimal thickening and atherogenesis. We investigated the mechanisms underlying positive and negative regulation of VSMC proliferation by the transcription factor cyclic AMP response element binding protein (CREB). Incubation with the cAMP elevating stimuli, adenosine, prostacyclin mimetics or low levels of forksolin activated CREB without changing CREB phosphorylation on serine-133 but induced nuclear translocation of the CREB co-factors CRTC-2 and CRTC-3.

Protein kinase CK2 inhibition suppresses neointima formation via a proline-rich homeodomain-dependent mechanism.

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs.

Divergent Regulation of Actin Dynamics and Megakaryoblastic Leukemia-1 and -2 (Mkl1/2) by cAMP in Endothelial and Smooth Muscle Cells.

Proliferation and migration of vascular smooth muscle cells (VSMCs) or endothelial cell (ECs) promote or inhibit, respectively, restenosis after angioplasty, vein graft intimal thickening and atherogenesis. Here we investigated the effects of cAMP-induced cytoskeletal remodelling on the serum response factor (SRF) co-factors Megakaryoblastic Leukemia-1 and -2 (MKL1 and MKL2) and their role in controlling VSMC and EC proliferation and migration. Elevation of cAMP using forskolin, dibutyryl-cAMP (db-cAMP), BAY60-6583 or Cicaprost induced rapid cytoskeleton remodelling and inhibited proliferation and migration in VSMCs but not EC.

Targeting Wnt/β-Catenin Activated Cells with Dominant-Negative N-cadherin to Reduce Neointima Formation.

Approximately 50% of coronary artery bypass grafts using the autologous saphenous vein fail within 10 years due to intimal thickening. This study examined whether a gene therapy approach that selectively kills Wnt/β-catenin/T cell factor (TCF) activated vascular smooth muscle cells (VSMCs) using dominant-negative N-cadherin (dn-N-cadherin) reduced intimal thickening. Cultured human VSMCs infected with an adenovirus (Ad) encoding dn-N-cadherin via the TCF promoter (Ad-TOP-dn-N-cadherin) specifically expressed dn-N-cadherin in response to activation of the Wnt/β-catenin/TCF pathway.

Regulation of Epigenetic Modifiers, Including KDM6B, by Interferon-γ and Interleukin-4 in Human Macrophages.

Background: Interferon-γ (IFN-γ) or interleukin-4 (IL-4) drives widely different transcriptional programs in macrophages. However, how IFN-γ and IL-4 alter expression of histone-modifying enzymes involved in epigenetic regulation and how this affects the resulting phenotypic polarization is incompletely understood.

Methods And Results: We investigated steady-state messenger RNA levels of 84 histone-modifying enzymes and related regulators in colony-stimulating factor-1 differentiated primary human macrophages using quantitative polymerase chain reaction.

Cigarette smoke extract profoundly suppresses TNFα-mediated proinflammatory gene expression through upregulation of ATF3 in human coronary artery endothelial cells.

Endothelial dysfunction caused by the combined action of disturbed flow, inflammatory mediators and oxidants derived from cigarette smoke is known to promote coronary atherosclerosis and increase the likelihood of myocardial infarctions and strokes. Conversely, laminar flow protects against endothelial dysfunction, at least in the initial phases of atherogenesis. We studied the effects of TNFα and cigarette smoke extract on human coronary artery endothelial cells under oscillatory, normal laminar and elevated laminar shear stress for a period of 72 hours.

PI16 is a shear stress and inflammation-regulated inhibitor of MMP2.

Raised endothelial shear stress is protective against atherosclerosis but such protection may be lost at sites of inflammation. We found that four splice variants of the peptidase inhibitor 16 (PI16) mRNA are among the most highly shear stress regulated transcripts in human coronary artery endothelial cells (HCAECs), in vitro but that expression is reduced by inflammatory mediators TNFα and IL-1β. Immunohistochemistry demonstrated that PI16 is expressed in human coronary endothelium and in a subset of neointimal cells and medial smooth muscle cells.

Zoledronate upregulates MMP-9 and -13 in rat vascular smooth muscle cells by inducing oxidative stress.

Background: Bisphosphonates, including zoledronate, target osteoclasts and are widely used in the treatment of osteoporosis and other bone resorption diseases, despite side effects that include damaging the stomach epithelium. Beneficial and adverse effects on other organ systems, including the cardiovascular system, have also been described and could impact on the use of bisphosphonates as therapeutic agents. Vascular smooth muscle cells (VSMCs) are major constituents of the normal vascular wall and have a key role in intimal thickening and atherosclerosis, in part by secreting MMPs that remodel the extracellular matrix and cleave cell surface proteins or secreted mediators.

The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP.

Aims: Inhibition of vascular smooth muscle cell (VSMC) proliferation by intracellular cAMP prevents excessive neointima formation and hence angioplasty restenosis and vein-graft failure. These protective effects are mediated via actin-cytoskeleton remodelling and subsequent regulation of gene expression by mechanisms that are incompletely understood. Here we investigated the role of components of the growth-regulatory Hippo pathway, specifically the transcription factor TEAD and its co-factors YAP and TAZ in VSMC.

p75(NTR)-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia.

The communication between vascular endothelial cells (ECs) and pericytes in the microvasculature is fundamental for vascular growth and homeostasis; however, these processes are disrupted by diabetes. Here we show that modulation of p75(NTR) expression in ECs exposed to high glucose activates transcription of miR-503, which negatively affects pericyte function. p75(NTR) activates NF-κB to bind the miR-503 promoter and upregulate miR-503 expression in ECs.

Gestational diabetes mellitus impairs fetal endothelial cell functions through a mechanism involving microRNA-101 and histone methyltransferase enhancer of zester homolog-2.

Objective: Gestational diabetes mellitus (GDM) produces fetal hyperglycemia with increased lifelong risks for the exposed offspring of cardiovascular and other diseases. Epigenetic mechanisms induce long-term gene expression changes in response to in utero environmental perturbations. Moreover, microRNAs (miRs) control the function of endothelial cells (ECs) under physiological and pathological conditions and can target the epigenetic machinery.

cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1.

Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1.

Relationship of MMP-14 and TIMP-3 expression with macrophage activation and human atherosclerotic plaque vulnerability.

Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissue inhibitor of metalloproteinases-3 (TIMP-3) overexpression is protective. MMP-14(hi) TIMP-3(lo) rabbit foam cells are more invasive and more prone to apoptosis than MMP-14(lo) TIMP-3(hi) cells. We investigated the implications of these findings for human atherosclerosis.

Effects of adenosine on lymphangiogenesis.

Background: The lymphatic system controls tissue homeostasis by draining protein-rich lymph to the vascular system. Lymphangiogenesis, the formation of lymphatic vessels, is a normal event in childhood but promotes tumor spread and metastasis during adulthood. Blocking lymphangiogenesis may therefore be of therapeutic interest.

EC4, a truncation of soluble N-cadherin, reduces vascular smooth muscle cell apoptosis and markers of atherosclerotic plaque instability.

Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect.

Local inhibition of microRNA-24 improves reparative angiogenesis and left ventricle remodeling and function in mice with myocardial infarction.

Myocardial infarction (MI) is the leading cause of death worldwide. MicroRNAs regulate the expression of their target genes, thus mediating a plethora of pathophysiological functions. Recently, miRNA-24 emerged as an important but controversial miRNA involved in post-MI responses.