3'UTR shortening of HAS2 promotes hyaluronan hyper-synthesis and bioenergetic dysfunction in pulmonary hypertension.

Pulmonary hypertension (PH) comprises a diverse group of disorders that share a common pathway of pulmonary vascular remodeling leading to right ventricular failure. Development of anti-remodeling strategies is an emerging frontier in PH therapeutics that requires a greater understanding of the interactions between vascular wall cells and their extracellular matrices. The ubiquitous matrix glycan, hyaluronan (HA), is markedly elevated in lungs from patients and experimental models with PH. Herein, we identified HA synthase-2 (HAS2) in the pulmonary artery smooth muscle cell (PASMC) layer as a predominant locus of HA dysregulation. HA upregulation involves depletion of NUDT21, a master regulator of alternative polyadenylation, resulting in 3'UTR shortening and hyper-expression of HAS2. The ensuing increase of HAS2 and hyper-synthesis of HA promoted bioenergetic dysfunction of PASMC characterized by impaired mitochondrial oxidative capacity and a glycolytic shift. The resulting HA accumulation stimulated pro-remodeling phenotypes such as cell proliferation, migration, apoptosis-resistance, and stimulated pulmonary artery contractility. Transgenic mice, mimicking HAS2 hyper-synthesis in smooth muscle cells, developed spontaneous PH, whereas targeted deletion of HAS2 prevented experimental PH. Pharmacological blockade of HAS2 restored normal bioenergetics in PASMC, ameliorated cell remodeling phenotypes, and reversed experimental PH in vivo. In summary, our results uncover a novel mechanism of HA hyper-synthesis and downstream effects on pulmonary vascular cell metabolism and remodeling.

Lipid phosphate phosphatase 3 in smooth muscle cells regulates angiotensin II-induced abdominal aortic aneurysm formation.

Genetic variants that regulate lipid phosphate phosphatase 3 (LPP3) expression are risk factors for the development of atherosclerotic cardiovascular disease. LPP3 is dynamically upregulated in the context of vascular inflammation with particularly heightened expression in smooth muscle cells (SMC), however, the impact of LPP3 on vascular pathology is not fully understood. We investigated the role of LPP3 and lysophospholipid signaling in a well-defined model of pathologic aortic injury and observed Angiotensin II (Ang II) increases expression of PLPP3 in SMCs through nuclear factor kappa B (NF-κB) signaling Plpp3 global reduction (Plpp3+/-) or SMC-specific deletion (SM22-Δ) protects hyperlipidemic mice from AngII-mediated aneurysm formation. LPP3 expression regulates SMC differentiation state and lowering LPP3 levels promotes a fibroblast-like phenotype. Decreased inactivation of bioactive lysophosphatidic acid (LPA) in settings of LPP3 deficiency may underlie these phenotypes because deletion of LPA receptor 4 in mice promotes early aortic dilation and rupture in response to AngII. LPP3 expression and LPA signaling influence SMC and vessel wall responses that are important for aortic dissection and aneurysm formation. These findings could have important implications for therapeutics targeting LPA metabolism and signaling in ongoing clinical trials.

MiR-34a-3p suppresses pulmonary vascular proliferation in acute pulmonary embolism rat by targeting DUSP1.

BACKGROUND: Acute pulmonary embolism (APE) is a prevalent reason of cardiovascular morbidity and mortality. Recent studies have underscored the positive effects of microRNAs (miRNAs) on many diseases. The present study aimed to identify the critical miRNA with differential expressions and explore its role in APE. METHODS: The critical miRNA with its target gene was screened by bioinformatics analysis. Their binding relationship was analyzed by TargetScan, Dual-luciferase reporter and RNA pull-down assays. A rat model of APE was established by self-blood coagulum. Human pulmonary artery smooth muscle cells (PASMCs) were exposed to platelet-derived growth factor (PDGF-BB) for excessive proliferation, and transfected with miR-34a-3p mimic. Mean pulmonary arterial pressure (mPAP) of rat was measured, and the pulmonary tissues were used for the pathological observation by Hematoxylin-Eosin (H&E) staining. Cell viability and proliferation were detected by Cell Counting Kit-8 (CCK-8) and EdU assays. The expressions of miR-34a-3p with its target genes (including dual-specificity phosphatase-1 (DUSP1)), neuron-derived orphan receptor-1 (NOR-1) and proliferating cell nuclear antigen (PCNA) were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) or/and Western blot. RESULTS: MiR-34a-3p expression was down-regulated in APE patients, which attenuated the increment of mPAP and thickening of the pulmonary arterial walls in APE rats, accompanied with regulation of NOR-1 and PCNA levels. MiR-34a-3p suppressed DUSP1 expression by directly binding to its 3'-untranslated region (UTR), and attenuated cell viability, proliferation, and the expressions of NOR-1 and PCNA in PDGF-BB-induced PASMCs by inhibiting DUSP1 expression. CONCLUSION: Up-regulated miR-34a-3p negatively regulates DUSP1 expression to inhibit PASMC proliferation, which, thus, may act on APE treatment by negatively regulating pulmonary vascular proliferation.

PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) Triggers Vascular Smooth Muscle Cell Senescence and Apoptosis: Implication of Its Direct Role in Degenerative Vascular Disease.

OBJECTIVE: PCSK9 (proprotein convertase subtilisin/kexin type 9) plays a critical role in cholesterol metabolism via the PCSK9-LDLR (low-density lipoprotein receptor) axis in the liver; however, evidence indicates that PCSK9 directly contributes to the pathogenesis of various diseases through mechanisms independent of its LDL-cholesterol regulation. The objective of this study was to determine how PCSK9 directly acts on vascular smooth muscle cells (SMCs), contributing to degenerative vascular disease. Approach and Results: We first examined the effects of PCSK9 on cultured human aortic SMCs. Overexpression of PCSK9 downregulated the expression of ApoER2 (apolipoprotein E receptor 2), a known target of PCSK9. Treatment with soluble recombinant human ApoER2 or the DNA synthesis inhibitor, hydroxyurea, inhibited PCSK9-induced polyploidization and other cellular responses of human SMCs. Treatment with antibodies against ApoER2 resulted in similar effects to those observed with PCSK9 overexpression. Inducible, SMC-specific knockout of Pcsk9 accelerated neointima formation in mouse carotid arteries and reduced age-related arterial stiffness. PCSK9 was expressed in SMCs of human atherosclerotic lesions and abundant in the "shoulder" regions of vulnerable atherosclerotic plaques. PCSK9 was also expressed in SMCs of abdominal aortic aneurysm, which was inversely related to the expression of smooth muscle α-actin. CONCLUSIONS: Our findings demonstrate that PCSK9 inhibits proliferation and induces polyploidization, senescence, and apoptosis, which may be relevant to various degenerative vascular diseases.

Inhibition of XIST attenuates abdominal aortic aneurysm in mice by regulating apoptosis of vascular smooth muscle cells through miR-762/MAP2K4 axis.

Abdominal aortic aneurysm (AAA) is a common chronic aortic degenerative disease. Long non-coding RNA X-inactive specific transcript (XIST) is associated with the progression of AAA, while the underlying mechanism is still unclear. We investigated the functional role of XIST in AAA. AAA mouse model was established by administration of Angiotensin II (Ang II). Primary mouse vascular smooth muscle cells (VSMCs) were separated from the abdominal aorta of Ang II-induced AAA mice, and then treated with Ang II. XIST was highly expressed in Ang II-treated VSMCs. Cell proliferation ability was decreased and apoptosis was increased in VSMCs following Ang II treatment. XIST knockdown reversed the impact of Ang II on cell proliferation and apoptosis in VSMCs. XIST promoted mitogen-activated protein kinase kinase 4 (MAP2K4) expression by sponging miR-762. XIST overexpression suppressed cell proliferation and apoptosis of Ang II-treated VSMCs by regulating miR-762/MAP2K4 axis. Finally, Ang II-induced AAA mouse model was established to verify the function of XIST in AAA. Inhibition of XIST significantly attenuated the pathological changes of abdominal aorta tissues in Ang II-induced mice. The expression of miR-762 was inhibited, and MAP2K4 expression was enhanced by XIST knockdown in the abdominal aorta tissues of AAA mice. In conclusion, these data demonstrate that inhibition of XIST attenuates AAA in mice, which attributes to inhibit apoptosis of VSMCs by regulating miR-762/MAP2K4 axis. Thus, this study highlights a novel ceRNA circuitry involving key regulators in the pathogenesis of AAA.

Cezanne is a critical regulator of pathological arterial remodelling by targeting ß-catenin signalling.

AIMS: Pathological arterial remodelling including neointimal hyperplasia and atherosclerosis is the main underlying cause for occluding arterial diseases. Cezanne is a novel deubiquitinating enzyme, functioning as a NF-кB negative regulator, and plays a key role in renal inflammatory response and kidney injury induced by ischaemia. Here we attempted to examine its pathological role in vascular smooth muscle cell (VSMC) pathology and arterial remodelling. METHODS AND RESULTS: Cezanne expression levels were consistently induced by various atherogenic stimuli in VSMCs, and in remodelled arteries upon injury. Functionally, VSMCs over-expressing wild-type Cezanne, but not the mutated catalytically-inactive Cezanne (C209S), had an increased proliferative ability and mobility, while the opposite was observed in VSMCs with Cezanne knockdown. Surprisingly, we observed no significant effects of Cezanne on VSMC apoptosis, NF-κB signalling, or inflammation. RNA-sequencing and biochemical studies showed that Cezanne drives VSMC proliferation by regulating CCN family member 1 (CCN1) by targeting ß-catenin for deubiquitination. Importantly, local correction of Cezanne expression in the injured arteries greatly decreased VSMC proliferation, and prevented arterial inward remodelling. Interestingly, global Cezanne gene deletion in mice led to smaller atherosclerotic plaques, but with a lower level of plaque stability. Translating, we observed a similar role for Cezanne in human VSMCs, and higher expression levels of Cezanne in human atherosclerotic lesions. CONCLUSION: Cezanne is a key regulator of VSMC proliferation and migration in pathological arterial remodelling. Our findings have important implications for therapeutic targeting Cezanne signalling and VSMC pathology in vascular diseases.

Characterization of Type 1 Angiotensin II Receptor Activation Induced Dual-Specificity MAPK Phosphatase Gene Expression Changes in Rat Vascular Smooth Muscle Cells.

Activation of the type I angiotensin receptor (AT1-R) in vascular smooth muscle cells (VSMCs) plays a crucial role in the regulation of blood pressure; however, it is also responsible for the development of pathological conditions such as vascular remodeling, hypertension and atherosclerosis. Stimulation of the VSMC by angiotensin II (AngII) promotes a broad variety of biological effects, including gene expression changes. In this paper, we have taken an integrated approach in which an analysis of AngII-induced gene expression changes has been combined with the use of small-molecule inhibitors and lentiviral-based gene silencing, to characterize the mechanism of signal transduction in response to AngII stimulation in primary rat VSMCs. We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression; several genes, including DUSP5, DUSP6, and DUSP10, were identified as upregulated genes in response to stimulation. Since various dual-specificity MAPK phosphatase (DUSP) enzymes are important in the regulation of mitogen-activated protein kinase (MAPK) signaling pathways, these genes have been selected for further analysis. We investigated the kinetics of gene-expression changes and the possible signal transduction processes that lead to altered expression changes after AngII stimulation. Our data shows that the upregulated genes can be stimulated through multiple and synergistic signal transduction pathways. We have also found in our gene-silencing experiments that epidermal growth factor receptor (EGFR) transactivation is not critical in the AngII-induced expression changes of the investigated genes. Our data can help us understand the details of AngII-induced long-term effects and the pathophysiology of AT1-R. Moreover, it can help to develop potential interventions for those symptoms that are induced by the over-functioning of this receptor, such as vascular remodeling, cardiac hypertrophy or atherosclerosis.

Dihydromyricetin ameliorates vascular calcification in chronic kidney disease by targeting AKT signaling.

Vascular calcification is highly prevalent in chronic kidney disease (CKD), and is characterized by transdifferentiation from contractile vascular smooth muscle cells (VSMCs) into an osteogenic phenotype. However, no effective and therapeutic option to prevent vascular calcification is yet available. Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis grossedentata, has been found to inhibit VSMCs proliferation and the injury-induced neointimal formation. However, whether DMY has an effect on osteogenic differentiation of VSMCs and vascular calcification is still unclear. In the present study, we sought to investigate the effect of DMY on vascular calcification in CKD and the underlying mechanism. DMY treatment significantly attenuated calcium/phosphate-induced calcification of rat and human VSMCs in a dose-dependent manner, as shown by Alizarin Red S staining and calcium content assay, associated with down-regulation of osteogenic markers including type I collagen (COL I), Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteocalcin (OCN). These results were further confirmed in aortic rings ex vivo. Moreover, DMY ameliorated vascular calcification in rats with CKD. Additionally, we found that AKT signaling was activated during vascular calcification, whereas significantly inhibited by DMY administration. DMY treatment significantly reversed AKT activator-induced vascular calcification. Furthermore, inhibition of AKT signaling efficiently attenuated calcification, which was similar to that after treatment with DMY alone, and DMY had a better inhibitory effect on calcification as compared with AKT inhibitor. The present study demonstrated that DMY has a potent inhibitory role in vascular calcification partially by inhibiting AKT activation, suggesting that DMY may act as a promising therapeutic candidate for patients suffering from vascular calcification.

Osteoprotegerin regulates vascular function through syndecan-1 and NADPH oxidase-derived reactive oxygen species.

Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar-Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes.

Baicalin attenuates angiotensin II-induced blood pressure elevation and modulates MLCK/p-MLC signaling pathway.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin, is an important flavonoid in Scutellaria baicalensis Georgi extracts, which exhibits therapeutic effects on anti-hypertension, but its underlying mechanisms remain to be further explored. Therefore, we investigated the effects and molecular mechanisms of Baicalin on anti-hypertension. In vivo studies revealed that Baicalin treatment significantly attenuated the elevation in blood pressure, the pulse propagation and thickening of the abdominal aortic wall in C57BL/6 mice infused with Angiotensin II (Ang II). Moreover, RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 537 differentially expressed transcripts and multiple enriched signaling pathways (including vascular smooth muscle contraction and calcium signaling pathway). Consistently, we found that Baicalin pretreatment significantly alleviated the Ang II induced constriction of abdominal aortic ring, while promoted NE pre-contracted vasodilation of abdominal aortic ring at least partly dependent on L-type calcium channel. In addition, Ang II stimulation significantly increased cell viability and PCNA expression, while were attenuated after Baicalin treatment. Moreover, Baicalin pretreatment attenuated Ang II-induced intracellular Ca2+ release, Angiotensin II type 1 receptor (AT1R) expression and activation of MLCK/p-MLC pathway in vascular smooth muscle cells (VSMCs). The present work further addressed the pharmacological and mechanistic insights on anti-hypertension of Baicalin, which may help better understand the therapeutic effect of Scutellaria baicalensis Georgi on anti-hypertension.

Human Vascular Smooth Muscle Function and Oxidative Stress Induced by NADPH Oxidase with the Clinical Implications.

Among reactive oxygen species, superoxide mediates the critical vascular redox signaling, resulting in the regulation of the human cardiovascular system. The reduced form of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) is the source of superoxide and relates to the crucial intracellular pathology and physiology of vascular smooth muscle cells, including contraction, proliferation, apoptosis, and inflammatory response. Human vascular smooth muscle cells express NOX1, 2, 4, and 5 in physiological and pathological conditions, and those enzymes play roles in most cardiovascular disorders caused by hypertension, diabetes, inflammation, and arteriosclerosis. Various physiologically active substances, including angiotensin II, stimulate NOX via the cytosolic subunits' translocation toward the vascular smooth muscle cell membrane. As we have shown, some pathological stimuli such as high glucose augment the enzymatic activity mediated by the phosphatidylinositol 3-kinase-Akt pathway, resulting in the membrane translocation of cytosolic subunits of NOXs. This review highlights and details the roles of human vascular smooth muscle NOXs in the pathophysiology and clinical aspects. The regulation of the enzyme expressed in the vascular smooth muscle cells may lead to the prevention and treatment of human cardiovascular diseases.

HDAC6 Activates ERK in Airway and Pulmonary Vascular Remodeling of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a multisystemic respiratory disease that is associated with progressive airway and pulmonary vascular remodeling due to the increased proliferation of bronchial smooth muscles cells (BSMCs) and pulmonary arterial smooth muscle cells (PASMCs) and the overproduction of extracellular matrix (e.g., collagen). Cigarette smoke (CS) and several mediators, such as PDGF (platelet-derived growth factor) and IL-6, play critical roles in COPD pathogenesis. HDAC6 has been shown to be implicated in vascular remodeling. However, the role of airway HDAC6 signaling in pulmonary vascular remodeling in COPD and the underlying mechanisms remain undetermined. Here, we show that HDAC6 expression is upregulated in the lungs of patients with COPD and a COPD animal model. We also found that CS extract (CSE), PDGF, and IL-6 increase the protein levels and activation of HDAC6 in BSMCs and PASMCs. Furthermore, CSE and these stimulants induced deacetylation and phosphorylation of ERK1/2 and increased collagen synthesis and BSMC and PASMC proliferation, which were outcomes that were prevented by HDAC6 inhibition. Inhibition of ERK1/2 also diminished the CSE-, PDGF-, and IL-6-caused elevation in collagen levels and cell proliferation. Pharmacologic HDAC6 inhibition with tubastatin A prevented the CS-stimulated increases in the thickness of the bronchial and pulmonary arterial wall, airway resistance, emphysema, and right ventricular systolic pressure and right ventricular hypertrophy in a rat model of COPD. These data demonstrate that the upregulated HDAC6 governs the collagen synthesis and BSMC and PASMC proliferation that lead to airway and vascular remodeling in COPD.

Diabetes Impaired Ischemia-Induced PDGF (Platelet-Derived Growth Factor) Signaling Actions and Vessel Formation Through the Activation of Scr Homology 2-Containing Phosphatase-1.

Objective: Critical limb ischemia is a major complication of diabetes characterized by insufficient collateral vessel development and proper growth factor signaling unresponsiveness. Although mainly deactivated by hypoxia, phosphatases are important players in the deregulation of proangiogenetic pathways. Previously, SHP-1 (Scr homology 2-containing phosphatase-1) was found to be associated with the downregulation of growth factor actions in the diabetic muscle. Thus, we aimed to gain further understanding of the impact of SHP-1 on smooth muscle cell (SMC) function under hypoxic and diabetic conditions. Approach and Results: Despite being inactivated under hypoxic conditions, high glucose level exposure sustained SHP-1 phosphatase activity in SMC and increased its interaction with PDGFR (platelet-derived growth factor receptor)-ß, thus reducing PDGF proangiogenic actions. Overexpression of an inactive form of SHP-1 fully restored PDGF-induced proliferation, migration, and signaling pathways in SMC exposed to high glucose and hypoxia. Nondiabetic and diabetic mice with deletion of SHP-1 specifically in SMC were generated. Ligation of the femoral artery was performed, and blood flow was measured for 4 weeks. Blood flow reperfusion, vascular density and maturation, and limb survival were all improved while vascular apoptosis was attenuated in diabetic SMC-specific SHP-1 null mice as compared to diabetic mice. Conclusions: Diabetes and high glucose level exposure maintained SHP-1 activity preventing hypoxia-induced PDGF actions in SMC. Specific deletion of SHP-1 in SMC partially restored blood flow reperfusion in the diabetic ischemic limb. Therefore, local modulation of SHP-1 activity in SMC could represent a potential therapeutic avenue to improve the proangiogenic properties of SMC under ischemia and diabetes.

Salt-Inducible Kinase 3 Promotes Vascular Smooth Muscle Cell Proliferation and Arterial Restenosis by Regulating AKT and PKA-CREB Signaling.

Objective: Arterial restenosis is the pathological narrowing of arteries after endovascular procedures, and it is an adverse event that causes patients to experience recurrent occlusive symptoms. Following angioplasty, vascular smooth muscle cells (SMCs) change their phenotype, migrate, and proliferate, resulting in neointima formation, a hallmark of arterial restenosis. SIKs (salt-inducible kinases) are a subfamily of the AMP-activated protein kinase family that play a critical role in metabolic diseases including hepatic lipogenesis and glucose metabolism. Their role in vascular pathological remodeling, however, has not been explored. In this study, we aimed to understand the role and regulation of SIK3 in vascular SMC migration, proliferation, and neointima formation. Approach and Results: We observed that SIK3 expression was low in contractile aortic SMCs but high in proliferating SMCs. It was also highly induced by growth medium in vitro and in neointimal lesions in vivo. Inactivation of SIKs significantly attenuated vascular SMC proliferation and up-regulated p21CIP1 and p27KIP1. SIK inhibition also suppressed SMC migration and modulated actin polymerization. Importantly, we found that inhibition of SIKs reduced neointima formation and vascular inflammation in a femoral artery wire injury model. In mechanistic studies, we demonstrated that inactivation of SIKs mainly suppressed SMC proliferation by down-regulating AKT (protein kinase B) and PKA (protein kinase A)-CREB (cAMP response element-binding protein) signaling. CRTC3 (CREB-regulated transcriptional coactivator 3) signaling likely contributed to SIK inactivation-mediated antiproliferative effects. Conclusions: These findings suggest that SIK3 may play a critical role in regulating SMC proliferation, migration, and arterial restenosis. This study provides insights into SIK inhibition as a potential therapeutic strategy for treating restenosis in patients with peripheral arterial disease.

The ameliorative effect of terpinen-4-ol on ER stress-induced vascular calcification depends on SIRT1-mediated regulation of PERK acetylation.

Endoplasmic reticulum (ER) stress-mediated phenotypic switching of vascular smooth muscle cells (VSMCs) is key to vascular calcification (VC) in patients with chronic kidney disease (CKD). Studies have shown that activation/upregulation of SIRT1 has a protective effect on CKD-VC. Meanwhile, although terpinen-4-ol has been shown to exert a protective effect against cardiovascular disease, its role and underlying mechanism in VC remain unclear. Herein, we explored whether terpinen-4-ol alleviates ER stress-mediated VC through sirtuin 1 (SIRT1) and elucidated its mechanism to provide evidence for its application in the clinical prevention and treatment of VC. To this end, a CKD-related VC animal model and ß-glycerophosphate (ß-GP)-induced VSMC calcification model were established to investigate the role of terpinen-4-ol in ER stress-induced VC, in vitro and in vivo. Additionally, to evaluate the involvement of SIRT1, mouse and VSMC Sirt1-knockdown models were established. Results show that terpinen-4-ol inhibits calcium deposition, phenotypic switching, and ER stress in VSMCs in vitro and in vivo. Furthermore, pre-incubation of VSMCs with terpinen-4-ol or a SIRT1 agonist, decreased ß-GP-induced calcium salt deposition, increased SIRT1 protein level, and inhibited PERK-eIF2α-ATF4 pathway activation, thus, alleviating VC. Similar results were observed in VSMCs induced to overexpress SIRT1 via lentivirus transcription. Meanwhile, the opposite results were obtained in SIRT1-knockdown models. Further, results suggest that SIRT1 physically interacts with, and deacetylates PERK. Specifically, mass spectrometry analysis identified lysine K889 as the acetylation site of SIRT1, which regulates PERK. Finally, inhibition of SIRT1 reduced the effect of terpinen-4-ol on the deacetylation of PERK in vitro and in vivo and weakened the inhibitory effect of terpinen-4-ol against ER stress-mediated VC. Cumulatively, terpinen-4-ol was found to inhibit post-translational modification of PERK at the K889 acetylation site by upregulating SIRT1 expression, thereby ameliorating VC by regulating ER stress. This study provides insights into the underlying molecular mechanism of terpinen-4-ol, supporting its development as a promising therapeutic agent for CKD-VC.

Does Src Kinase Mediated Vasoconstriction Impair Penumbral Reperfusion?

Despite successful recanalization, a significant number of patients with ischemic stroke experience impaired local brain tissue reperfusion with adverse clinical outcome. The cause and mechanism of this multifactorial complication are yet to be understood. At the current moment, major attention is given to dysfunction in blood-brain barrier and capillary blood flow but contribution of exaggerated constriction of cerebral arterioles has also been suggested. In the brain, arterioles significantly contribute to vascular resistance and thus control of perfusion. Accordingly, pathological changes in arteriolar wall function can, therefore, limit sufficient reperfusion in ischemic stroke, but this has not yet received sufficient attention. Although an increased vascular tone after reperfusion has been demonstrated in several studies, the mechanism behind it remains to be characterized. Importantly, the majority of conventional mechanisms controlling vascular contraction failed to explain elevated cerebrovascular tone after reperfusion. We propose here that the Na,K-ATPase-dependent Src kinase activation are the key mechanisms responsible for elevation of cerebrovascular tone after reperfusion. The Na,K-ATPase, which is essential to control intracellular ion homeostasis, also executes numerous signaling functions. Under hypoxic conditions, the Na,K-ATPase is endocytosed from the membrane of vascular smooth muscle cells. This initiates the Src kinase signaling pathway that sensitizes the contractile machinery to intracellular Ca2+ resulting in hypercontractility of vascular smooth muscle cells and, thus, elevated cerebrovascular tone that can contribute to impaired reperfusion after stroke. This mechanism integrates with cerebral edema that was suggested to underlie impaired reperfusion and is further supported by several studies, which are discussed in this article. However, final demonstration of the molecular mechanism behind Src kinase-associated arteriolar hypercontractility in stroke remains to be done.

miR-33a-5p Suppresses ox-LDL-Stimulated Calcification of Vascular Smooth Muscle Cells by Targeting METTL3.

Oxidized low-density lipoprotein (ox-LDL) accumulation in the vascular wall plays a pivotal role in the development of atherosclerosis and vascular calcification. However, few studies focus on the regulatory roles of microRNAs in ox-LDL stimulated vascular calcification. The aim of the present study was to investigate how miR-33a-5p regulated vascular calcification stimulated by ox-LDL. In the present study, miR-33a-5p was downregulated during vascular smooth muscle cells (VSMCs) calcification and upon ox-LDL treatment. ox-LDL significantly stimulated VSMCs calcification, while miR-33a-5p overexpression by its mimics transfection inhibited alkaline phosphatase (ALP) activity, mineralization and marker genes associated with VSMCs calcification even in the presence of ox-LDL. Methyltransferase like 3 (METTL3) was the target gene of miR-33a-5p. METTL3 was upregulated during VSMCs calcification and upon ox-LDL treatment. When VSMCs were transfected with miR-33a-5p mimics, METTL3 was downregulated. METTL3 downregulation by siRNA method decreased VSMCs calcification even in the presence of ox-LDL. Taken together, these results suggest miR-33a-5p suppresses VSMCs calcification stimulated by ox-LDL via targeting METTL3, highlighting the critical role of miR-33a-5p/METTL3 in vascular calcification.

Adapter Protein RapGEF1 Is Required for ERK1/2 Signaling in Response to Elevated Phosphate in Vascular Smooth Muscle Cells.

The sodium-dependent phosphate transporter, SLC20A1, is required for elevated inorganic phosphate (Pi) induced vascular smooth muscle cell (VSMC) matrix mineralization and phenotype transdifferentiation. Recently, elevated Pi was shown to induce ERK1/2 phosphorylation through SLC20A1 by Pi uptake-independent functions in VSMCs, suggesting a cell signaling response to elevated Pi. Previous studies identified Rap1 guanine nucleotide exchange factor (RapGEF1) as an SLC20A1-interacting protein and RapGEF1 promotes ERK1/2 phosphorylation through Rap1 activation. In this study, we tested the hypothesis that RapGEF1 is a critical component of the SLC20A1-mediated Pi-induced ERK1/2 phosphorylation pathway. Co-localization of SLC20A1 and RapGEF1, knockdown of RapGEF1 with siRNA, and small molecule inhibitors of Rap1, B-Raf, and Mek1/2 were investigated. SLC20A1 and RapGEF1 were co-localized in peri-membranous structures in VSMCs. Knockdown of RapGEF1 and small molecule inhibitors against Rap1, B-Raf, and Mek1/2 eliminated elevated Pi-induced ERK1/2 phosphorylation. Knockdown of RapGEF1 inhibited SM22α mRNA expression and blocked elevated Pi-induced downregulation of SM22α mRNA. Together, these data suggest that RapGEF1 is required for SLC20A1-mediated elevated Pi signaling through a Rap1/B-Raf/Mek1/2 cell signaling pathway, thereby promoting ERK1/2 phosphorylation and inhibiting SM22α gene expression in VSMCs.

Tryptanthrin Regulates Vascular Smooth Muscle Cell Phenotypic Switching in Atherosclerosis by AMP-Activated Protein Kinase/Acetyl-CoA Carboxylase Signaling Pathway.

ABSTRACT: Atherosclerosis (AS) is one of the most severe cardiovascular diseases involved in the phenotypic switching of vascular smooth muscle cells (VSMCs). Tryptanthrin is a natural product with broad biological activities. However, the effect of tryptanthrin on atherosclerotic progression is unclear. The aim of this study was to determine the role of tryptanthrin in AS and explore the potential mechanism. In vitro, primary VSMCs were stimulated with platelet-derived growth factor-BB (PDGF) to induce cell dedifferentiation. Treatment with tryptanthrin (5 µM or 10 µM) suppressed the proliferation and recovered the contractility of VSMCs in the presence of PDGF. The contractile proteins (α-smooth muscle actin, calponin, and SM22α) were increased, and the synthetic protein vimentin was decreased by tryptanthrin in PDGF-induced VSMCs. ApoE-/- mice fed with high-fat diet were used as an in vivo model of AS. Similarly, gavage administration of tryptanthrin (50 mg/kg or 100 mg/kg) attenuated VSMC phenotypic changes from a contractile to a synthetic state in aortic tissues of AS mice. The serum lipid level, atherosclerotic plaque formation, and arterial intimal hyperplasia were attenuated by tryptanthrin. Furthermore, tryptanthrin increased the expression levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) both in vitro and in vivo. Administration of compound C, an AMPK inhibitor, reversed the inhibitory effect of tryptanthrin on VSMC dedifferentiation in vitro. Thus, we demonstrate that tryptanthrin protects against AS progression through the inhibition of VSMC switching from a contractile to a pathological synthetic phenotype by the activation of AMPK/ACC pathway. It provides novel insights into AS prevention and treatment.

MiR-4787-5p Regulates Vascular Smooth Muscle Cell Apoptosis by Targeting PKD1 and Inhibiting the PI3K/Akt/FKHR Pathway.

ABSTRACT: Vascular smooth muscle cell (VSMC) dysfunction is the main cause of aortic dissection (AD). In this study, we focused on the role and mechanism of miR-4787-5p in regulating VSMC apoptosis. Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of miR-4787-5p in aorta tissues of AD (n = 10) and normal aortic tissues of donors (n = 10). Cell apoptosis was tested by TUNEL assay and Annexin V FITC/PI staining flow cytometry. The expression of PC1 and the PI3K/Akt/FKHR signaling pathway associated proteins in VSMCs was measured by Western blot. We found that the miR-4787-5p was highly expressed in aorta tissues of AD compared with 10 healthy volunteers. Meanwhile, PI3K/Akt/FKHR signaling pathway was inactive in the aortic tissue of AD. The overexpression of miR-4787-5p significantly induced VSMC apoptosis, and miR-4787-5p knockdown showed the opposite results. In addition, polycystic kidney disease 1 gene, which encodes polycystin-1 (PC1), was found to be a direct target of miR-4787-5p in the VSMCs and this was validated using a luciferase reporter assay. Overexpression of PC1 by a lentivirus packaging PC1-overexpression plasmid (LV-PC1) plasmids markedly eliminated the promotion of miR-4787-5p overexpression on VSMC apoptosis. Finally, it was found that miR-4787-5p deactivated the PI3K/Akt/FKHR pathway, as demonstrated by the down-regulation of phosphorylated (p-)PI3K, p-Akt, and p-FKHR. In conclusion, these findings confirm an important role for the miR-4787-5p/polycystic kidney disease 1 axis in AD pathobiology.