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

Abstract

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.

Keywords: cardiovascular disease; phenotypic switch; reactive oxygen species; vascular smooth muscle cell.

Figure 1

Redox signaling pathways regulating vascular smooth muscle cell (VSMC) proliferation. Superoxide anion, O₂⁻, induces cell proliferation by activating the mitogen-activated protein kinase (MAPK), ERK1/2, or upregulating the transcription factor Id3. Hydrogen peroxide, H₂O₂, promotes VSMC proliferation by activating the p38 MAPK, the CypA chaperone protein and the proto-oncogenes c-myc, c-fos and c-jun. The inhibitory actions of H₂O₂ are elicited via the redox sensitive transcription factor gut-enriched Kruppel-like factor, GKLF. Figure key: arrow: activation, block arrow: inhibition, up-arrow: upregulation, question mark: potential crosstalk.

Figure 2

Platelet-derived growth factor-β (PDGF-β)-activated pathways mediating vascular smooth muscle cell (VSMC) migration. After platelet-derived growth factor receptor-β (PDGF-R ββ ) activation, NADPH oxidase-1 (Nox1)-released peroxide (H₂O₂) activates Slingshot1L (SSLH1) and LIM kinase (LIMK), cofilin phosphatase and kinase, respectively. The net result is cofilin_.dephosphorylation leading to actin reorganization and ultimately migration. Furthermore, PDGF-induced H₂O₂ activates the Src/ (phosphoinositide-dependent kinase-1) PDK1/ (p21-activated protein kinase) PAK1 signaling pathway mediating VSMC migration. Additionally, Src activation increases L-type voltage-dependent calcium channel (CaV1.2) activity leading to increased intracellular calcium (Ca²⁺) concentration, and consequently VSMC migration. In addition, ERK1/2, JNK and p38 mediate PDGF-induced migration. Whether the activation of these MAPKs is ROS-dependent is yet to be determined. Figure key: arrow: activation, question mark: potential crosstalk.

Figure 3

Reactive oxygen species (ROS) regulation of cellular signaling pathways involved in vascular smooth muscle cell (VSMC) fate and cell cycle progression. Superoxides anion, O₂⁻, -induces DNA synthesis, and consequently cell cycle progression, by activating ERK1/2. Alternatively, O₂⁻ mediates angiotensin II (Ang-II)-induced cell cycle progression via upregulating Id3 transcription factor, which in turn downregulates cell cycle proteins, including p27, p53 and p21, leading to cell cycle progression. Nitric oxide (NO)-induced ROS induce VSMC apoptosis associated with DNA synthesis inhibition. Hydrogen peroxide (H₂O₂) downregulates NADPH oxidase (NOX4), leading to cell senescence and DNA synthesis inhibition. H₂O₂ also attenuates cell cycle progression by upregulating the transcription factor gut-enriched Kruppel-like factor (GKLF) via p38. H₂O₂ regulates cell cycle proteins and proto-oncogenes to induce cell cycle progression or cell death. Melatonin upregulates sestrin2 leading to ROS inhibition, which decreases VSMC apoptosis. Basal ROS inhibition by catalase overexpression or antioxidants, (Pyrrolidinedithiocarbamate) PDTC or (N-acetylcysteine) NAC, initiates VSMC apoptosis.

Figure 4

Various factors determining the effect of ROS on VSMC fate. ROS-inducing stimulus, ROS concentrations, as well as ROS type, play a major role in VSMC response. ROS-initiated signaling pathways involving redox-sensitive genes underwrite VSMC cell fate regulation. The vascular beds from which VSMCs are isolated contribute to their differential responses to ROS.

Figure 5

Diverse vascular smooth muscle cell (VSMC) responses to different hydrogen peroxide (H₂O₂)-generating stimuli. Glucose oxidase/glucose (GO/G)- or diethylmaleate (DEM)-induced H₂O₂ leads to VSMC apoptosis, while angiotensin II (Ang II)-induced H₂O₂ causes VSMC hypertrophy. H₂O₂ released in response to xanthine/xanthine oxidase, platelet-derived growth factor (PDGF), or bradykinin promotes DNA synthesis and, consequently, VSMC proliferation. Treating human VSMCs with H₂O₂ induces cell senescence. Figure key: up-arrow: increment.

Figure 6

The impact of the vascular smooth muscle cell (VSMC) microenvironment on determining the effect of reactive oxygen species (ROS) on phenotypic switch. Oxidative stress seems to be crucial for maintaining the contractile phenotype of quiescent VSMCs and for the differentiation of embryonic contractile VSMCs. NAPDH oxidase (NOX4)-produced hydrogen peroxide (H₂O₂) activates the transcription complex serum response factor (SRF)/myocardin via p38. This complex translocates to the nucleus and upregulates the transcription of contractile markers, such as calponin and myosin heavy chain. NOX4 also seems to play a structural role in maintaining a contractile phenotype by binding to α-actin stress fibers, characteristic of this phenotype. In the context of atherosclerosis, ROS induces VSMC dedifferentiation by activating the NF-κB and/or Elk-1/SRF signaling pathways. NF-κB upregulates the transcription of the synthetic marker osteopontin, and associates with myocardin to repress the myocardin-dependent contractile gene expression of smooth muscle 22 (SM22). Alternatively, ROS promote Elk-1/SRF complex formation, which activates the transcription of synthetic markers, vimentin and osteopontin, via connective tissue growth factor (CTGF). The Elk-1/SRF complex, alternatively, downregulates the contractile markers smoothelin B and α-smooth muscle (αSM). In an osteogenic medium, ROS induce VSMCs, which induces transition to the osteoblast-like cell phenotype, characteristic of vessel calcification in advanced atherosclerosis. The ROS-induced osteoblast-like cell phenotype is mediated via AKT-activated Runx2, a key transcription factor for osteogenic differentiation. In diabetic VSMC, ROS induce a synthetic phenotype by decreasing calponin, probably via ERK1/2. Conversely, ROS provoke the contractile phenotypic switch of diabetic VSMC by upregulating microRNA-145, which in turn increases the activity of myocardin in an ERK1/2-dependent manner. Cyclic stretch evokes a VSMC synthetic phenotypic switch through NOX1-derived ROS release via myocyte enhancer factor 2B (MEF2B), resulting in the upregulation of osteopontin and the downregulation of contractile markers calponin1 and smoothelin B. Figure key: up-arrow: upregulation, down-arrow: downregulation, arrow: activation, block arrow: inhibition, question mark: potential crosstalk.