Evodiamine Attenuates PDGF-BB-Induced Migration of Rat Vascular Smooth Muscle Cells through Activating PPARγ

The uncontrolled migration of vascular smooth muscle cells (VSMCs) into the intima is a critical process in the development of atherosclerosis. Evodiamine, an indole alkaloid extracted from the Chinese medicine evodia, has been shown to inhibit tumor cell invasion and protect the cardiovascular system, but its effects on VSMCs remain unknown. In the present study, we investigated the inhibitory effects of evodiamine on the platelet-derived growth factor-BB (PDGF-BB)-induced VSMC migration using wound healing and transwell assays, and assessed its role in decreasing the protein levels of matrix metalloproteinases and cell adhesion molecules. More importantly, we found that evodiamine activated the expression and nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ). Inhibition of PPARγ activity by using its antagonist T0070907 and its specific siRNA oligonucleotides significantly attenuated the inhibitory effects of evodiamine on VSMC migration. Taken together, our results indicate a promising anti-atherogenic effect of evodiamine through attenuation of VSMC migration by activating PPARγ.     

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1β secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1β in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.     

Association between the Hypomethylation of Osteopontin and Integrin β3 Promoters and Vascular Smooth Muscle Cell Phenotype Switching in Great Saphenous Varicose Veins

Lower extremity varicose veins are a common condition in vascular surgery and proliferation of vascular smooth muscle cells (VSMCs) in the intima is a significant pathological feature of varicosity. However, the pathogenesis of varicose veins is not fully understood. Osteopontin (OPN) could promote the migration and adhesion of VSMCs through the cell surface receptor integrin β3 and the cooperation of OPN and integrin β3 is involved in many vascular diseases. However, the role of OPN and integrin β3 in varicosity remains unclear. In the current study, we found that the methylation levels in the promoter regions of OPN and integrin β3 genes in the VSMCs of varicose veins are reduced and the protein expression of OPN and integrin β3 are increased, compared with normal veins. Furthermore, it was observed that VSMCs in the neointima of varicose veins were transformed into the synthetic phenotype. Collectively, hypomethylation of the promoter regions for OPN and integrin β3 genes may increase the expression of these genes in varicosity, which is closely related to VSMC phenotype switching. Hypomethylation of the promoter regions for OPN and integrin β3 genes may be a key factor in the pathogenesis of varicosity.     

Dextromethorphan Reduces Oxidative Stress and Inhibits Uremic Artery Calcification

Medial vascular calcification has emerged as a key factor contributing to cardiovascular mortality in patients with chronic kidney disease (CKD). Vascular smooth muscle cells (VSMCs) with osteogenic transdifferentiation play a role in vascular calcification. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors reduce reactive oxygen species (ROS) production and calcified-medium–induced calcification of VSMCs. This study investigates the effects of dextromethorphan (DXM), an NADPH oxidase inhibitor, on vascular calcification. We used in vitro and in vivo studies to evaluate the effect of DXM on artery changes in the presence of hyperphosphatemia. The anti-vascular calcification effect of DXM was tested in adenine-fed Wistar rats. High-phosphate medium induced ROS production and calcification of VSMCs. DXM significantly attenuated the increase in ROS production, the decrease in ATP, and mitochondria membrane potential during the calcified-medium–induced VSMC calcification process (p < 0.05). The protective effect of DXM in calcified-medium–induced VSMC calcification was not further increased by NADPH oxidase inhibitors, indicating that NADPH oxidase mediates the effect of DXM. Furthermore, DXM decreased aortic calcification in Wistar rats with CKD. Our results suggest that treatment with DXM can attenuate vascular oxidative stress and ameliorate vascular calcification.     

Alternative C3 Complement System: Lipids and Atherosclerosis

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin α_Mβ₂ receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.     

Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification

Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD). To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs) stimulated calcium deposition in vascular smooth muscle cells (VSMCs) through excessive oxidative stress and phenotypic transition into osteoblastic cells. Since AGEs can induce apoptosis, in this study we investigated its role on VSMC apoptosis, focusing mainly on the underlying mechanisms. A rat VSMC line (A7r5) was cultured, and treated with glycolaldehyde-derived AGE-bovine serum albumin (AGE3-BSA). Apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. To quantify apoptosis, an enzyme-linked immunosorbent assay (ELISA) for histone-complexed DNA fragments was employed. Real-time PCR was performed to determine the mRNA levels. Treatment of A7r5 cells with AGE3-BSA from 100 µg/mL concentration markedly increased apoptosis, which was suppressed by Nox inhibitors. AGE3-BSA significantly increased the mRNA expression of NAD(P)H oxidase components including Nox4 and p22^phox, and these findings were confirmed by protein levels using immunofluorescence. Dihydroethidisum assay showed that compared with cBSA, AGE3-BSA increased reactive oxygen species level in A7r5 cells. Furthermore, AGE3-induced apoptosis was significantly inhibited by siRNA-mediated knockdown of Nox4 or p22^phox. Double knockdown of Nox4 and p22^phox showed a similar inhibitory effect on apoptosis as single gene silencing. Thus, our results demonstrated that NAD(P)H oxidase-derived oxidative stress are involved in AGEs-induced apoptosis of VSMCs. These findings might be important to understand the pathogenesis of vascular calcification in diabetes and CKD.     

Yohimbine,an α2-Adrenoceptor Antagonist,Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.     

An Inducible and Vascular Smooth Muscle Cell-Specific Pink1 Knockout Induces Mitochondrial Energetic Dysfunction during Atherogenesis

DNA damage and mitochondrial dysfunction are defining characteristics of aged vascular smooth muscle cells (VSMCs) found in atherosclerosis. Pink1 kinase regulates mitochondrial homeostasis and recycles dysfunctional organelles critical for maintaining energetic homeostasis. Here, we generated a new vascular-specific Pink1 knockout and assessed its effect on VSMC-dependent atherogenesis in vivo and VSMC energetic metabolism in vitro. A smooth muscle cell-specific and MHC-Cre-inducible flox’d Pink1^f/f kinase knockout was made on a ROSA26^+/0 and ApoE^−/− C57Blk6/J background. Mice were high fat fed for 10 weeks and vasculature assessed for physiological and pathogical changes. Mitochondrial respiratory activity was then assessed in wild-type and knockout animals vessels and isolated cells for their reliance on oxidative and glycolytic metabolism. During atherogenesis, we find that Pink1 knockout affects development of plaque quality rather than plaque quantity by decreasing VSMC and extracellular matrix components, collagen and elastin. Pink1 protein is important in the wild-type VSMC response to metabolic stress and induced a compensatory increase in hexokinase II, which catalyses the first irreversible step in glycolysis. Pink1 appears to play an important role in VSMC energetics during atherogenesis but may also provide insight into the understanding of mitochondrial energetics in other diseases where the regulation of energetic switching between oxidative and glycolytic metabolism is found to be important.     

Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.     

HuR and TIA1/TIAL1 Are Involved in Regulation of Alternative Splicing of SIRT1 Pre-mRNA

SIRT1 is a pleiotropic protein that plays critical and multifunctional roles in metabolism, senescence, longevity, stress-responses, and cancer, and has become an important therapeutic target across a range of diseases. Recent research demonstrated that SIRT1 pre-mRNA undergoes alternative splicing to produce different isoforms, such as SIRT1 full-length and SIRT1-ΔExon8 variants. Previous studies revealed these SIRT1 mRNA splice variants convey different characteristics and functions to the protein, which may in turn explain the multifunctional roles of SIRT1. However, the mechanisms underlying the regulation of SIRT1 alternative splicing remain to be elucidated. Our objective is to search for new pathways that regulate of SIRT1 alternative splicing. Here we describe experiments showing that HuR and TIA1/TIAL1, two kinds of RNA-binding proteins, were involved in the regulation of alternative splicing of SIRT1 pre-mRNA under normal and stress circumstances: HuR increased SIRT1-ΔExon8 by promoting SIRT1 exon 8 exclusion, whereas TIA1/TIAL1 inhibition of the exon 8 exclusion led to a decrease in SIRT1-ΔExon8 mRNA levels. This study provides novel insight into how the alternative splicing of SIRT1 pre-mRNA is regulated, which has fundamental implications for understanding the critical and multifunctional roles of SIRT1.     

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.     

BGP-15 Inhibits Hyperglycemia-Aggravated VSMC Calcification Induced by High Phosphate

Vascular calcification associated with high plasma phosphate (Pi) level is a frequent complication of hyperglycemia, diabetes mellitus, and chronic kidney disease. BGP-15 is an emerging anti-diabetic drug candidate. This study was aimed to explore whether BGP-15 inhibits high Pi-induced calcification of human vascular smooth muscle cells (VSMCs) under normal glucose (NG) and high glucose (HG) conditions. Exposure of VSMCs to Pi resulted in accumulation of extracellular calcium, elevated cellular Pi uptake and intracellular pyruvate dehydrogenase kinase-4 (PDK-4) level, loss of smooth muscle cell markers (ACTA, TAGLN), and enhanced osteochondrogenic gene expression (KLF-5, Msx-2, Sp7, BMP-2). Increased Annexin A2 and decreased matrix Gla protein (MGP) content were found in extracellular vesicles (EVs). The HG condition markedly aggravated Pi-induced VSMC calcification. BGP-15 inhibited Pi uptake and PDK-4 expression that was accompanied by the decreased nuclear translocation of KLF-5, Msx-2, Sp7, retained VSMC markers (ACTA, TAGLN), and decreased BMP-2 in both NG and HG conditions. EVs exhibited increased MGP content and decreased Annexin A2. Importantly, BGP-15 prevented the deposition of calcium in the extracellular matrix. In conclusion, BGP-15 inhibits Pi-induced osteochondrogenic phenotypic switch and mineralization of VSMCs in vitro that make BGP-15 an ideal candidate to attenuate both diabetic and non-diabetic vascular calcification.