PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMC^PCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMC^PCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMC^PCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMC^PCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.     

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.     

Reactive Oxygen Species: Modulators of Phenotypic Switch of Vascular Smooth Muscle Cells

Reactive oxygen species (ROS) are natural byproducts of oxygen metabolism in the cell. At physiological levels, they play a vital role in cell signaling. However, high ROS levels cause oxidative stress, which is implicated in cardiovascular diseases (CVD) such as atherosclerosis, hypertension, and restenosis after angioplasty. Despite the great amount of research conducted to identify the role of ROS in CVD, the image is still far from being complete. A common event in CVD pathophysiology is the switch of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. Interestingly, oxidative stress is a major contributor to this phenotypic switch. In this review, we focus on the effect of ROS on the hallmarks of VSMC phenotypic switch, particularly proliferation and migration. In addition, we speculate on the underlying molecular mechanisms of these cellular events. Along these lines, the impact of ROS on the expression of contractile markers of VSMCs is discussed in depth. We conclude by commenting on the efficiency of antioxidants as CVD therapies.     

The Effect of Activated FXIII,a Transglutaminase,on Vascular Smooth Muscle Cells

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca²⁺ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.     

Porphyromonas gingivalis Lipopolysaccharides Promote Proliferation and Migration of Human Vascular Smooth Muscle Cells through the MAPK/TLR4 Pathway

Atherosclerosis is a major cause of mortality worldwide. The initial change in atherosclerosis is intimal thickening due to muscle cell proliferation and migration. A correlation has been observed between periodontal disease and atherosclerosis. Here, we investigated the proliferation and migration of human aortic smooth muscle cells (HASMCs) using Porphyromonas gingivalis-derived LPS (Pg-LPS). To elucidate intracellular signaling, toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) of HASMCs were knocked down, and the role of these molecules in Pg-LPS-stimulated proliferation and migration was examined. The role of mitogen-activated protein kinase (MAPK) in HASMC proliferation and migration was further elucidated by MAPK inhibition. Pg-LPS stimulation increased the proliferation and migration of HASMCs and activated the TLR4/MyD88 pathway. TLR4 knockdown inhibited Pg-LPS stimulated HASMCs proliferation and migration. Pg-LPS stimulation led to the phosphorylation of P38 MAPK, JNK, and ERK, and MyD88 knockdown inhibited the phosphorylation of P38 MAPK and JNK but not ERK. P38 MAPK and SAPK/JNK inhibition did not suppress the proliferation of HASMCs upon Pg-LPS stimulation, but ERK inhibition significantly inhibited proliferation. SAPK/JNK and ERK inhibition suppressed Pg-LPS-stimulated migration of HASMCs. In conclusion, our findings suggest that Pg-LPS may promote atherosclerosis via the activation of MAPK through TLR4.     

Up-Regulation of Rhoa/Rho Kinase Pathway by Translationally Controlled Tumor Protein in Vascular Smooth Muscle Cells

Translationally controlled tumor protein (TCTP), a repressor for Na,K-ATPase has been implicated in the development of systemic hypertension, as proved by TCTP-over-expressing transgenic (TCTP-TG) mice. Aorta of TCTP-TG exhibited hypercontractile response compared to that of non-transgenic mice (NTG) suggesting dys-regulation of signaling pathways involved in the vascular contractility by TCTP. Because dys-regulation of RhoA/Rho kinase pathway is implicated in increased vascular contractility, we examined whether TCTP induces alterations in RhoA pathway in vascular smooth muscle cells (VSMCs). We found that TCTP over-expression by adenovirus infection up-regulated RhoA pathway including the expression of RhoA, and its downstream signalings, phosphorylation of myosin phosphatase target protein (MYPT-1), and myosin light chain (MLC). Conversely, lentiviral silencing of TCTP reduced the RhoA expression and Rho kinase signalings. Using immunohistochemical and Western blotting studies on aortas from TCTP-TG confirmed the elevated expression of RhoA and increase in p-MLC (phosphorylated MLC). In contrast, down-regulation of RhoA and p-MLC were found in aortas from heterozygous mice with deleted allele of TCTP (TCTP^+/−). We conclude that up-regulation of TCTP induces RhoA-mediated pathway, and that TCTP-induced RhoA plays a role in the regulation in vasculature. Modulation of TCTP may offer a therapeutic target for hypertension and in vascular contractility dysfunction.     

Injectable Gel Form of a Decellularized Bladder Induces Adipose-Derived Stem Cell Differentiation into Smooth Muscle Cells In Vitro

Biologic scaffolds composed of extracellular matrix components have been proposed to repair and reconstruct a variety of tissues in clinical and pre-clinical studies. Injectable gels can fill and conform any three-dimensional shape and can be delivered to sites of interest by minimally invasive techniques. In this study, a biological gel was produced from a decellularized porcine urinary bladder by enzymatic digestion with pepsin. The enzymatic digestion was confirmed by visual inspection after dissolution in phosphate-buffered saline solution and Fourier-transform infrared spectroscopy. The rheological and biological properties of the gel were characterized and compared to those of the Matrigel^TM chosen as a reference material. The storage modulus G’ reached 19.4 ± 3.7 Pa for the 30 mg/mL digested decellularized bladder gels after ca. 3 h at 37 °C. The results show that the gel formed of the porcine urinary bladder favored the spontaneous differentiation of human and rabbit adipose-derived stem cells in vitro into smooth muscle cells to the detriment of cell proliferation. The results support the potential of the developed injectable gel for tissue engineering applications to reconstruct for instance the detrusor muscle part of the human urinary bladder.