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Suppression of type I collagen in human scleral fibroblasts treated with extremely low-frequency electromagnetic fields.

Purpose To investigate the expression differences of type I collagen (COL1A1) and its underlying mechanisms in human fetal scleral fibroblasts (HFSFs) that were treated with conditioned medium from retinal pigment epithelial (RPE) cells under extremely low-frequency electromagnetic fields (ELF-EMFs). Methods The ELF-EMFs used in this study were established by slidac and artificial coils. Growth of the treated HFSFs was evaluated by a cell-counting kit-8 assay. The expression of COL1A1 and matrix metalloproteinases-2 (MMP-2) in the treated HFSFs was detected by reverse transcription PCR (RT-PCR) and western blot, and the expression of transforming growth factor-β2 (TGF-β2) and basic fibroblast growth factor-2 (FGF-2) in RPE cells exposed to EMFs was detected by RT-PCR. The expression of COL1A1 and MMP-2 in HFSFs was further confirmed by immunofluorescence staining. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2 also called p44/p42 mitogen-activated protein kinases [MAPK]) and p38 in HFSFs was measured by western blot. Results We found that exposure to ELF-EMFs resulted in a decreased proliferation rate of HFSFs and that addition of RPE supernatant medium could enhance this effect. Compared with that of the control cells, a significant decrease in collagen synthesis was detected in HFSFs under ELF-EMFs. However, the expression of MMP-2 was upregulated, which could be further enhanced via an RPE supernatant additive. The activities of ERK1/2 and p38 were significantly increased in HFSFs exposed to ELF-EMFs, and this effect could be enhanced by RPE supernatant medium additive. Conclusions Our results suggested that ELF-EMFs can inhibit the expression of type I collagen in HFSFs and contribute to the remodeling of the sclera.

Side effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on public health, especially on ocular health, have recently attracted more attention than they have received in the past. Morphological alteration of the conjunctiva and reductions in the number of goblet cells could result from 50 Hz ELF-EMFs at 1.5 mT [1]. Several studies have also demonstrated that there are relationships between electromagnetic radiation (EMR) and cataract [2,3], corneal [3,4], retinal damage [4]. However, little is known about the biologic function of scleral changes exposed to ELF-EMFs.
T h e h u m a n s c l e r a i s c o m p o s e d m a i n ly of t y pe I collagen f ibr ils (50% to 70%), which mostly distribute among the equator and posterior pole region of the eyeball [5]. Scleral thinning involves a net loss of matrix, smaller diameter collagen fibrils in the sclera, a net loss of scleral tissue through reduced collagen synthesis, and increased degradation [6,7]. Scleral fibroblasts, which reside between the collagen fiber and bundle lamellae, could monitor changes of the surrounding extracellular matrix [8,9]. The proliferation of scleral fibroblasts changes with the decrease in proteoglycan synthesis, which could affect the biomechanics of sclera and induce elongation of the eye [10]. The remodeling of sclera and the extension of axial length could induce refractive errors, retinal degeneration, and/or detachment [11,12]. In our preliminary experiments, we found that ELF-EMFs could lengthen the axial length of guinea pig's eyes and induce a structure disorder of collagen fibrils in guinea pig scleral. Information on the biologic effects of ELF-EMFs on scleral fibroblasts, therefore, is of interest and has never been investigated.
It is well known that the retina is the source of ocular growth-regulating signals and that retinal pigment epithelial (RPE) cells are intimately involved in eye growth regulation [13]. Increasing evidence has shown that the retina could synthesize and secrete cytokines and enzymes (such as transforming growth factor-β [TGF-β] and basic fibroblast growth factor [bFGF]) to regulate the remodeling of sclera [14][15][16][17]. However, no report has addressed the biologic function of human fetal scleral fibroblasts (HFSFs) exposed to ELF-EMFs or HFSFs treated with RPE supernatant medium.
Matrix metalloproteinases (MMPs) are secreted by different kinds of cells, such as fibroblasts and inflammatory cells [18]. MMPs can degrade one or several components of extraceellular matrix (ECM) and promote ECM remodeling [19]. It is well recognized that high expression of MMP-2 could lead to collagen degradation [20], which plays a crucial role in the regulation of scleral pathological remodeling [21]. The MMP-2 gene was found to be involved in human refractive variation [22] and participated in altering the extracellular matrix in the posterior sclera [23]. An elevated level of active MMP-2 expression has been detected in induced tree shrews with myopia [24]. Moreover, MMP-2 might be involved in tissue remodeling through activating extracellular signal-regulated kinase 1/2 (ERK1/2, also called p44/p42 mitogen-activated protein kinase [MAPK]) and p38 MAPK [25,26]. EMF exposure could activate multiple downstream signaling pathways, such as ERK signaling [27]. We speculated that ELF-EMFs might upregulate MMP-2 expression and activity by activating ERK1/2 and p38 MAPK signal molecules.
In this study we used an EMF with 0.2 mT intensity, which is the upper safety limit for public exposure to nonionizing radiation in the guidelines of the International Commission on Non-Ionizing Radiation Protection [28]. Our objective was to detect the effect of ELF-EMFs on sclera by evaluating their influence on proliferation and content of type I collagen in cultures of HFSFs. We then investigated the effect of ELF-EMF exposure with and without RPE supernatant additive on the biologic function of HFSFs and explored the underlying mechanisms of RPE supernatant influence on HFSFs.

METHODS
Cell culture: HFSFs were obtained from Beijing Institute of Ophthalmology (Beijing, China), and RPE cells (ARPE19) were obtained from the American Type Culture Collection. HFSFs and RPE cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, CA) with 1% antibiotic/antimycotic (penicillin-streptomycin; Invitrogen) and 10% fetal bovine serum (FBS; Gibco), and incubated at 37 °C in a humidified incubator containing 5% CO 2 . The growth medium was changed every 3 days. When the cultures began to reach 80% confluence, cells were trypsinized for 1 min at 37 °C in 0.25% trypsin/EDTA, and then subcultured at a split ratio of 1:3 in 25-mm 2 plastic bottles.
Exposure system and Experiment design: ELF-EMFs were generated in a device constructed by winding 150 turns of insulated soft copper wire with a diameter of 1.5 mm. Coils were placed vertically, facing one another ( Figure 1). The magnetic flux density at the center of the coils was measured with an electromagnetic field radiation tester (EMF-827; Taiwan, China) and was adjusted by varying the coil current. Magnetic field measurements showed that under experimental conditionals, the magnetic field exposure system produced a stable flux density of 0.2 mT and a stable frequency of 50 Hz, with negligible vibration. Temperature was continuously monitored by Therm 2283-2 thermometers (Taylor Precision Instruments, Oak Brook, IL). None of the experimental conditions produced any measureable temperature increase in the cell culture medium. The exposure system was put in a humidified incubator at 37 °C, which contained 5% CO 2 . Exposed cells were placed inside the coils with a sinusoidal 50 Hz electromagnetic field at 0.2 mT for 24 h. Control cells (not exposed to ELF-EMFs) were cultured in another incubator without power coils. RPE cells (5×10 5 cells cultured in 25-mm 2 plastic bottles) cultured with DMEM without FBS were exposed to ELF-EMFs (0.2 mT) for 24 h. The supernatant medium of RPE was then collected and condensed by an ultra filter device (Millipore, Billerica, MA) by spinning the device at 4,000 × g for 25 min. The ultrafiltrate protein concentration was measured using the Bradford Protein Assay kit (Beyotime, Shanghai, China).
Cell proliferation assay: The proliferation rate of HFSFs treated with or without RPE supernatant medium while exposed to ELF-EMFs was evaluated with the cell counting kit-8 assay (Dojin Laboratories, Kumamoto, Japan). For the cell proliferation assay, 100 μl of passage HFSFs (2,000, 4,000, 6,000, 8,000, and 10,000 cells/well) were seeded into 96-well plates containing DMEM with 10% FBS. Cells were allowed to attach to the substratum for 12 h. The following day, the medium was replaced with fresh medium and the cells exposed to ELF-EMFs (0.2 mT, 50 Hz) were cultured in different humidified incubators (37 °C, containing 5% CO 2 ) for 24 h. Control cultures were kept in the same conditions as the exposed ones without field application. In a separate 96-well plate, HFSFs were seeded at an initial density of 6,000 cells/well at various concentrations of RPE supernatant (0.0075, 0.015, 0.03, 0.06, and 0.12 mg/ml) or the control condensed medium. The cultures were exposed to ELF-EMFs (0.2 mT, 50 Hz) at 37 °C with 5% CO 2 for 24 h. Control cultures were kept in the same conditions as the exposed ones without field application.The solution of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium and 1-methoxyphenazine methosulfate (Dojindo, Japan) was then added to each well, and cells were incubated for another 2 h. Finally absorbance was measured at 450 nm using a microplate reader.
Indirect immunofluorescence: Fibroblasts (1×10 5 cells) were grown on coverslips in six-well plates (Corning Ltd) to 50%-60% confluence. The cells were exposed to ELF-EMFs (50 Hz, 0.2 mT) produced by the coils and treated with RPE supernatant medium (0.015 mg/ml) for 24 h. Then, the cells were then washed three times with phosphate buffered saline (0.01 M PBS: KH 2 PO 4 , 0.20 g; Na 2 HPO 4 , 1.56 g; NaCl, 8.0 g; KCl 0.20 g), covered with 10% normal goat serum diluted in PBS, and incubated for 20 min at 37 °C. The slides were incubated at 4 °C overnight with primary antibodies (anticollagen type I diluted to 1:500 in PBS, anti-MMP-2 diluted to 1:100 in PBS; Abcam, Cambridge, UK). Cells incubated in PBS without primary antibodies were used as a negative control group. The antibody-treated and negative-controlled sample slides were washed with PBS and exposed to DyLight 488-conjugated anti-mouse immunoglobulin G (IgG) antibodies (Invitrogen Corp, Carlsbad, CA) at 37 °C for 60 min. The slides were washed three times with PBS, and cell nuclei were stained with 4', 6-diamidino-2-phenylindole (Invitrogen). Immunofluorescence images were taken using an inverted fluorescent microscope (Leica, Wetzlar, Germany).
Quantitative real-time PCR analysis: HFSFs and RPE cells were seeded in 25-mm 2 plastic bottles at 5×10 5 cells and cultured for 12 h. HFSFs cultures treated with RPE supernatant medium (0.015 mg/ml) were exposed to ELF-EMFs (50 Hz, 0.2 mT) produced by the coils. After exposure for 24 h, cells were harvested for total RNA extraction by using Trizol Reagent (Invitrogen). cDNAs were synthesized with 4 µg of total RNA, according to the manufacturer's protocol for the Reverse Transcription kit (Fermentas, Japan). Based on the sequences reported in the GenBank database (Table  1), COL1A1, MMP-2, TGF-β2, FGF-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) primers were designed, selected, and ordered using Primer-Premier 5 (Premier Biosoft Interpairs, Palo Alto, CA), BLAST, and Sangon (China), respectively. Quantitative real-time PCR was performed with an ABI7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). A typical reaction was performed in 25 µl, consisting of 2 µl cDNA, 12.5 µl 2X SYBR Green PCR buffer, and primer pairs (final 10 pmol each). The PCR temperature cycle was performed for 2 min at 95.0 °C, followed by 40 cycles with primer annealing for 30 s at the temperatures indicated in Table 1, and extension for 30 s at 72.0 °C. The change in threshold cycle (∆CT) was calculated by subtracting the average CT of GAPDH mRNA from the average CT of the target genes. All experiments were performed in triplicate. The comparative quantification values were obtained from the CT number at which the increase in signal was associated with an exponential growth of PCR products.
Western blot analysis: After exposure to ELF-EMFs (50 Hz, 0.2 mT) and treatment with or without RPE supernatant medium (0.015 mg/ml) or not, total cellular protein extract from HFSFs was obtained by lysing the cells in an ice-cold radioimmunoprecipitation assay buffer containing proteinase inhibitors (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% NP-40; 4 °C) and phosphatase inhibitors (R&D Systems). After centrifugation at 12,000 × g (30 min; 4 °C), the supernatants were collected and protein concentration measured using the bicinchoninic acid (BCA) Protein Assay It was then probed overnight at 4 °C with specific primary antibodies against collagen type I (1:5,000; Abcam), MMP-2 (1:1,000; Abcam), phosphor-ERK1/2, phosphor-p38, ERK1/2, p38, and β-actin (1:1,000; Cell Signaling Technology) and incubated in blocking buffer with ß-actin as an internal control. Immunoblots were then washed and incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit goat IgG HRP (1:1,000; Abmart, Shanghai, China). Membranes were developed with the ECL kit (Pierce, Thermo Scientific), according to the manufacturer's protocol, acquired with a ChemiDoc XR system (Bio-Rad Laboratories). The relative level of protein expression was expressed as the density ratio of the protein compared to β-actin in the same sample.
Data analysis: Statistical analysis was performed with SPSS 16.0 Statistical Software (SPSS, Inc., Chicago, IL). Data were expressed as the mean±standard deviation (SD) of at least three separate repeated experiments. The differences between exposed and untreated cells were tested using one-way analysis of variance or Dunnett's test. Statistical significance was accepted if p<0.05.

Changes in HFSFs proliferation:
There was a significant reduction in the proliferation rate of HFSF cells exposed to ELF-EMFs compared with control cultures (t=-3.26, p<0.05, Figure 2A). The inclusion of RPE supernatant medium resulted in a significantly reduced proliferation rate of HFSFs compared to that of the cells exposed to ELF-EMFs alone (t=-7.32, p<0.05, Figure 2B).

Electromagnetic fields and retinal cells can affect human scleral fibroblast cells COL1A1 and MMP-2 expression:
ELF-EMF exposure could significantly reduce the mRNA expression of COL1A1 in HFSFs (t=-4.083, p=0.05). The effect was more obvious at the mRNA level of HFSFs exposed to ELF-EMFs with RPE supernatant additive (t=-4.987, p<0.01, Figure 3A). ELF-EMF exposure also could significantly reduce the protein expression of COL1A1 in HFSFs, but no significant difference was found for protein levels between the treated HFSFs with RPE supernatant medium (0.015 mg/ml) and the control cells exposed to ELF-EMFs ( Figure 4). The mRNA level of MMP-2's was upregulated in cells exposed to ELF-EMFs (t=3.919, p<0.05), and RPE supernatant medium additive could enhance the ELF-EMF effect on the expression of MMP-2 (t=8.828, p<0.05, Figure  3B). The increased MMP-2 protein level correlated with the increased mRNA level (Figure 4). The expression of COL1A1 and MMP-2 in HFSFs was further confirmed by immunofluorescence ( Figure 5, Figure 6).
Electromagnetic fields can affect TGFβ2, FGF-2 mRNA expression in retinal pigment epitheliums: Relative TGF-β2 and FGF-2 mRNA levels were markedly changed in RPE cells after exposure to ELF-EMFs. Significantly increased TGF-β2 mRNA expression (t=4.45, p<0.05) and decreased FGF-2 mRNA expression (t=-5.81, p<0.05) were noted in RPE cells exposure to ELF-EMFs ( Figure 3C). COL1A1 and MMP-2 immunofluorescence staining changes after electromagnetic fields exposure: COL1A1 was confirmed to be expressed in the cytoplasm of HFSFs. After cells were exposed to ELF-EMFs (0.2 mT, 24 h), COL1A1 staining decreased. When cells were co-exposed to ELF-EMFs and RPE supernatant medium (0.015 mg/ml) additive, COL1A1 staining was much weaker ( Figure 5). However, following exposure to ELF-EMFs, the expression of MMP-2 showed more marked fluorescent signals localized in the cytoplasm of treated cells than in that of untreated cells. When cells were co-exposed to ELF-EMFs and RPE supernatant medium (0.015 mg/ml), MMP-2 staining appeared stronger than exposure to ELF-EMFs alone ( Figure 6).

Potential signal pathway of electromagnetic fields and retinal cells effects on human scleral fibroblast cells: Western blots
showed that the expression of p-p38 and p-ERK1/2 protein levels were significantly increased (Figure 7) after cells were exposed to ELF-EMFs (0.2 mT, 24 h). The expression level was higher when exposed to ELF-EMFs with RPE supernatant medium (0.015 mg/ml) additive (Figure 7).

DISCUSSION
Collagen synthesis in keloid fibroblasts has been shown to be inhibited when exposed to EMFs with 60 Hz for 10 days [29]. Collagen synthesis could be decreased in guinea pig's skin after exposure to 1 mT ELF-EMFs [30], and the expression of collagen I mRNA could be downregulated following EMF stimulation [31]. As an important component of sclera, type I collagen is involved in pathological scleral matrix remodeling. Our results demonstrated that exposure to ELF-EMFs downregulated mRNA and protein expression levels of COL1A1 in HFSFs. Moreover, under the same ELF-EMF exposure conditions, there was a significant difference in mRNA levels between cells treated with RPE supernatant medium and the control cells ( Figure 3A, Figure 4A, Figure 5). These results indicate that ELF-EMFs can suppress collagen synthesis in  the sclera and might induce sclera remodeling, which is a potential risk factor for the eye's elongation.
MMPs could affect scleral ECM during scleral remodeling by inhibiting collagen type I synthesis [32]. In this study, we found that, compared with the control cells, both the protein and mRNA expression levels of MMP-2 were upregulated with exposure to ELF-EMFs, and MMP-2 expression was further enhanced with the inclusion of RPE supernatant medium ( Figure 3B, Figure 4B, Figure 6). These data indicate that ELF-EMFs might act to enhance the initiation process of scleral remodeling by increasing the expression of MMP-2.
RPE cells can synthesize and secrete cytokines and enzymes to regulate the proliferation of scleral fibroblasts, regulate the production of collagen, and further affect the development of myopia [33]. We therefore detected the mRNA expression levels of TGF-β2 and FGF-2 in RPE cells and found the mRNA expression of TGF-β2 was enhanced but that of FGF-2 decreased after exposure to an EMF for 24 h. TGF-β could induce MMP production from fibroblasts by interfering with Smad and MAPK pathways in vitro [34].
TGF-β also plays an inhibitory role of human scleral fibroblasts attachment to collagen type I in vitro and modulates scleral cell-matrix interactions in vivo [35]. In this study, FGF-2 mRNA was decreased in RPE cells after 24 h of exposure to an EMF. These results suggest that ELF-EMFs may regulate sclera changes though regulating cytokines of RPE.
Scleral fibroblasts are involved in scleral remodeling and play an important role in maintaining eye size [8]. Our results showed that following exposure to electromagnetic field, HFSFs proliferation was significantly decreased, which implied that ELF-EMFs suppress collagen synthesis might be mediated by inhibit HFSFs proliferation, and retinal is the source of ocular growth-regulating signals [12]. Therefore, the decreased proliferation of HFSFs by EMFs might also via activate some signal pathways in RPE cells.
It has been reported that MAPKs can be activated in response to a variety of environmental stresses including radiation [36]. ERK1/2 and p38 MAPK play important roles in the collagen content exposed to ELF-EMFs [37]. Inactivation of the ERK1/2 MAPK signaling pathway can increase the expression of type I collagen in fibroblasts [38]. MAPKs can also regulate the expression of MMP-2; for example, MMP-2 can be stimulated by cyclic strain in endothelial cells in vitro, in part through both p38-and ERK-dependent pathways [39]. Inhibiting the activity of ERK could downregulate the expression of MMP-2 in fibroblasts [40]. Therefore, Figure 6. Electromagnetic fields and retinal cells can affect the expression of MMP-2. Expression of MMP-2 in HFSFs were cultured at ELF-EMFs with RPE supernatant medium or not. The control cells were cultured without ELF-EMFs and RPE supernatant additive. Then the cells were labeled with antibodies for MMP-2 (green). 4', 6-diamidino-2-phenylindole dyed the nucleus (blue). A-C: PBS was used instead of primary antibody as a negative control. D-F: Control cells were cultured without ELF-EMFs and RPE supernatant additive. G-I: Cells were cultured with ELF-EMFs exposure. MMP-2 showed more marked fluorescent signal than the control cells. J-K: Cells were coexposed to ELF-EMFs and RPE supernatant medium. MMP-2 staining became stronger than exposure to ELF-EMFs only. Scale bar=20 μm. we tried to identify whether ELF-EMFs can regulate the content change of collagen through the MAPK pathway. We found that the collagen content decreased in HFSFs in almost all experiments ( Figure 3A, Figure 4A, Figure 5). In contrast, increased expressions of ERK1/2 and p38 MAPK phosphorylation were detected when the cells were exposed to ELF-EMF and RPE supernatant medium. These results suggest that the suppression of collagen expression might be associated with the activation of the ERK1/2 and p38 MAPK pathways.
In conclusion, ELF-EMFs can regulate collagen synthesis in HFSFs by regulating the MAPK pathways and can contribute to scleral remodeling. Additional studies will be required to clarify which cytokines and/or enzymes participate in regulating collagen synthesis and MMP-2 activity in RPE supernatant medium exposed to ELF-EMFs.


Plaque regression and plaque stabilisation in cardiovascular diseases

Atherosclerosis is characterized by formation of plaques on the inner walls of arteries that threatens to become the leading cause of death worldwide via its sequelae of myocardial infarction and stroke. Endothelial dysfunction leads to cholesterol uptake and accumulation of in ﬂ ammatory markers within the plaque. The stability of a plaque eventually depends on the balance between vascular smooth muscle cells that stabilize it and the in ﬂ ammatory cells like macrophages and T lymphocytes that make it prone to rupture. The current approach to manage atherosclerosis focuses on the treatment of a ruptured plaque and efforts have been made to reduce the risk of plaque rupture by identifying vulnerable plaques and treating them before they precipitate into clinical events. New diagnostic approaches such as IVUS and CIMT ultrasound are now being preferred over traditional coronary angiography because of their better accuracy in measuring plaque volume rather than the level of stenosis caused. The present review highlights the literature available on two prevalent approaches to manage a vulnerable plaque, namely, plaque stabilization and plaque regression, and their validation through various treatment modalities in recent plaque management studies. Plaque stabilization focuses on stabilizing the content of plaque and strengthening the overlying endothelium, while plaque regression focuses on the overall reduction in plaque volume and to reverse the arterial endothelium to its normal functional state. Although earlier studies contemplated the practicality of plaque regression and focused greatly on stabilization of a vulnerable plaque, our review indicated that, aided by the use of superior diagnostics tools, more intensive lipid modifying therapies have resulted in actual plaque regression.

INTRODUCTION
Atherosclerosis is a systemic disease characterized by narrowing of blood vessels due to the formation of atheromatous plaque or atheroma (Plural: Atheromata). Atheroma is the deposition of fatty material and cholesterol within the inner wall of arteries that makes arterial lumen stenotic. [1] The major clinical manifestations of atherosclerosis are angina, myocardial infarction (MI), transient cerebral ischemic attacks, and strokes. [2] In the year 2008, out of 17.3 million cardiovascular (CV) deaths, MI alone was responsible for 7.3 million deaths and stroke alone caused 6.2 million deaths. [3]

Pathophysiology of atherosclerotic plaque
Arterial endothelial activation is the first step in the pathophysiology of atherosclerotic plaque where there is an increased expression of adhesion molecules which attracts and internalizes the circulating monocytes and leukocytes from the bloodstream [4] and gets converted into macrophages by macrophage-colony stimulating factor (M-CSF). [5] In addition, cholesterol uptake from apolipoprotein B containing lipoproteins such as low-density lipoprotein-cholesterol (LDL-C) and oxidation of native LDL-C by oxygen-free radicals stimulates the expression of several infl ammatory markers, which play a crucial role in development of plaque [ Figure 1]. [6] The stability of atherosclerotic plaque depends on the interplay of vascular smooth muscle cells (VSMCs) and infl ammatory cells (macrophages and T lymphocytes). The VSMCs synthesize structurally important collagens that provide

A B S T R A C T
Atherosclerosis is characterized by formation of plaques on the inner walls of arteries that threatens to become the leading cause of death worldwide via its sequelae of myocardial infarction and stroke. Endothelial dysfunction leads to cholesterol uptake and accumulation of infl ammatory markers within the plaque. The stability of a plaque eventually depends on the balance between vascular smooth muscle cells that stabilize it and the infl ammatory cells like macrophages and T lymphocytes that make it prone to rupture. The current approach to manage atherosclerosis focuses on the treatment of a ruptured plaque and efforts have been made to reduce the risk of plaque rupture by identifying vulnerable plaques and treating them before they precipitate into clinical events. New diagnostic approaches such as IVUS and CIMT ultrasound are now being preferred over traditional coronary angiography because of their better accuracy in measuring plaque volume rather than the level of stenosis caused. The present review highlights the literature available on two prevalent approaches to manage a vulnerable plaque, namely, plaque stabilization and plaque regression, and their validation through various treatment modalities in recent plaque management studies. Plaque stabilization focuses on stabilizing the content of plaque and strengthening the overlying endothelium, while plaque regression focuses on the overall reduction in plaque volume and to reverse the arterial endothelium to its normal functional state. Although earlier studies contemplated the practicality of plaque regression and focused greatly on stabilization of a vulnerable plaque, our review indicated that, aided by the use of superior diagnostics tools, more intensive lipid modifying therapies have resulted in actual plaque regression.
stability to the plaque while infl ammatory cells release matrix metalloproteinases (MMPs), which degrade collagen and extracellular matrix, potentially weakening the plaque. [7] In unstable plaque, the balance among these competing factors favors collagen breakdown rather than synthesis. With plaque rupture, lipid fragments and cellular debris are released into the vessel lumen resulting in the formation of a thrombus.

Diagnosis of plaque
While studying plaque regression, it is quintessential to identify accurate changes in plaque composition and volume  to treat ruptured plaque and to indicate plaque vulnerability in asymptomatic individuals. [8] A crucial factor impeding the diagnosis of plaque is the effect of arterial wall remodeling. In the early stages of plaque formation, plaque growth is eccentric, and due to expansive (positive) remodeling, there occurs compensatory outward enlargement of the vessel wall in order to accommodate considerable plaque volume without much compromise on the vessel lumen. [9] Coronary angiography is the traditional gold standard for atherosclerotic plaque assessment, but since its assessment is based on the imaging of arterial lumen stenosis, it fails to detect the effect of expansive remodeling [4] and hence, lacks the ability to measure accurate changes in atheroma volume.
Intravascular ultrasound (IVUS) depicts the lumen size as well as thickness of the entire vessel wall but is invasive in nature. Ultrasound measurement of the carotid intima media thickness (CIMT) is a non-invasive technique that accurately assesses changes in the wall thickness and, more importantly, correlates these changes with future clinical events. [10] High resolution magnetic resonance imaging (MRI) has emerged as a leading non-invasive imaging technique that has monitored changes in plaque volume. [11] However, MRI studies correlating changes in atheroma volume with reduction in events are lacking. [4] Other emerging diagnostic techniques of plaque imaging such as computed tomography (CT) luminal coronary stenosis assessment, ECG-gated single-photon emission computed tomography (SPECT), infrared spectroscopy, and intra-coronary thermography have effectively detected changes in anatomical and functional information in an unstable plaque, but these results are yet to be validated in larger studies. [5] Purpose of this review The current modalities of plaque management are based on the treatment of ruptured plaque that precipitates into acute clinical events. The present review focuses on the need of preventive approaches for plaque management by impeding the progression of plaque before rupture and stabilizing the composition of plaque to prevent rupture (plaque stabilization), and if possible, to reverse the growth of plaque altogether (plaque regression).

Search strategies used in this review
We identifi ed electronic databases, mainly MEDLINE, HighWire, Cochrane, and Google Scholar for articles from 2001 through December 2011 by using keywords "Atherosclerotic plaque," "plaque regression," "plaque stabilization," and "vulnerable plaque." In MEDLINE, we used the Medical Subject Heading (MeSH) terms: "Atherosclerotic plaque," AND the Subheadings "diagnosis," "pathophysiology," "prevention and control," and "therapy." Reference lists of screened articles were also searched to identify additional data.

PREDISPOSING FACTORS OF ATHEROSCLEROSIS
Dyslipidemia, hypertension, obesity, smoking, diabetes, genetic predisposition, homocysteinaemia, immune complexes, and infectious agents are few of the risk factors, which favor progression of atherosclerosis. [12,6] Risk factors for atherosclerosis as per the latest WHO Report on cardiovascular disease (CVD) [1] are shown in [ Table 1]. In the fi rst decade of life, atherosclerosis begins as "fatty streaks," [13] which progress to unstable or vulnerable plaques by the next two decades on continuous exposure to CV risk factors. [2] These vulnerable plaques may further cause obstruction of blood fl ow to vital organs, resulting in heart attack, stroke, and other CV complications.

T H E R A P E U T I C S T R A T E G I E S F O R ATHEROSCLEROTIC PLAQUE
Interventions that target lipid metabolism are the primary interventions for prevention of CV morbidity and mortality. [14] Other therapeutic strategies include anti-thrombotic agents, anti-oxidants, and diet management; these strategies have been used either alone or in combination with lipid-lowering agents. Based on the stage of plaque, there are different treatment options. For example, in a patient with acute coronary syndrome (ACS), the destabilized plaque would require a different treatment approach and strategy than the plaque that has not yet destabilized. The recent approaches to plaque management primarily focus on the need for early plaque detection and their treatment before any clinical event occurs. Hence, it is imperative to identify plaques at an early stage where they have not yet ruptured but are highly susceptible to rupture, commonly referred to as the vulnerable plaque. The characteristics of vulnerable plaque are enumerated in Table 2. [7,15] As evident in Table 2, a variety of intrinsic and extrinsic factors predisposes plaque to instability and acute disruption. In order to prevent complications of plaque rupture, it is important not only to identify vulnerable patients based on traditional risk factors but also to identify vulnerable plaques in asymptomatic individuals. [15] Plaque stabilization Plaque stabilization is an approach towards altering the structure, content, or function of the plaque and/or its overlying endothelium to either prevent or reduce the severity of plaque rupture. [16] Once the plaque has disrupted/eroded with overlying thrombus, stabilization is possible by percutaneous coronary intervention (PCI), with interventions such as lipid-lowering, anti-thrombotic, and anti-coagulant agents. [17] In the 1990s, several angiography trials on lipid lowering demonstrated significant reduction in clinical events despite a small amount of plaque regression. [18] A series of post-mortem and angiographic studies identifi ed that the probability of MI incidence was not refl ected by corresponding increase in arterial stenosis. [19] Hence, it was considered that the stability of the plaque rather than its absolute size determined the likelihood of rupture, making a change in plaque composition rather than plaque regression a worthwhile clinical goal. [7] The basic idea of plaque stabilization is to make the plaques more fi brous and reduce their lipid content. Several in vivo studies have reported the role of macrophages, MMP expression, and interstitial collagen content in plaque stability. [20] Decreased macrophage counts and MMP expression, and increased interstitial collagen content had led to increase the plaque stability.
Clinical studies of statin therapy have consistently demonstrated increase in hyperechogenicity index (suggesting an increase in fi brous tissue), and reductions in the plaque lipid pool, but only modest reductions in plaque volume. [21] These changes in plaque structure may have improved the endothelial function and reduced the platelet thrombogenicity, translating into improved outcomes in both Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering with Atorvastatin (MIRACL) [22] and the Pravastatin Acute Coronary Treatment (PACT) [23] early cholesterol reduction studies in ACS patients. Plaque stabilization may also lead to signifi cant reduction in the rate of progression of carotid intima-medial thickening, which may be due to either up-regulation of type III collagen synthesis [24] or anti-atherogenic effect. [25] Niacin and fi brates have shown increase in high-density lipoprotein cholesterol (HDL-C), either alone or in combination with simvastatin, leading to improved CV outcomes in high-risk groups with low HDL-C. [26] Other cholesterol-reducing agents, such as nicotinic acid and ezetimibe, may improve plaque stabilization by reducing the lipid pool and improving the endothelial function, but whether this will translate into improved outcomes in ACS is yet to be ascertained. [15] Plaque stabilization can also be achieved by various anti-thrombotic therapies, including anti-platelets and anti-coagulants. Anti-platelet therapies (aspirin, clopidogrel or prasugrel, intravenous anti-platelet drugs [glycoprotein IIb/IIIa inhibitors]) reduce platelet activation and aggregation, which are integral steps in the formation of a thrombus after plaque disruption. [27] The anti-coagulant therapies (unfractionated heparin, low molecular weight heparins, fondaparinux, and bivalirudin) target the clotting cascade to prevent deposition of fi brin strands in the clot. Both anti-platelet and anti-coagulant therapies heal the intimal tear and retard the endothelium activation, leading to plaque stabilization. Aspirin has anti-platelet and anti-inflammatory properties. It covalently binds cyclo-oxygenase and reduces interleukin-6, C-reactive protein (CRP), and macrophage colony stimulating factor. [15] Recent advances in the molecular biology of the platelets have demonstrated that the platelet integrin GP IIb/IIIa plays a pivotal role in the fi nal common pathway leading to platelet aggregation. [28] Clopidogrel and ticlopidine are two adenosine diphosphate receptor antagonists (Gp IIb/IIIa inhibitors) that reportedly inhibited platelet activation, degranulation, and release of pro-thrombotic and infl ammatory mediators and prevented activation of the glycoprotein (Gp) IIb/IIIa receptor. [15] These Gp IIb/IIIa inhibitors exhibited maximum benefi t in patients who underwent PCI for destabilized plaque. [29] Aspirin, alone or in combination with heparin and clopidogrel, served as a treatment option in ACS. In the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial, striking benefi t of the combination of clopidogrel and aspirin over aspirin alone was observed in patients with unstable angina and non-ST elevation myocardial infarction in terms of reduced clinical events. [30] The Heart Outcomes Prevention Evaluation (HOPE) study had shown ACE inhibitors such as ramipril to improve plaque stability by inhibiting endothelial dysfunction and oxygen-free radical production by angiotensin; by decreasing macrophage activity, or by inhibiting VSMC lipoxygenase activity. [25] The ACE inhibitors not only potentiate vasodilator bradykinin and decrease platelet adhesion but also inhibit pro-atherosclerotic agents like plasminogen activator inhibitor. There is enough accumulated evidence that chronic ACE inhibitor therapy stabilizes plaque and decreases vascular reactivity. [28] Calcium antagonists such as amlodipine have shown anti-atherogenic role in patients with ACS where they had stabilized plaques by interfering with the lipid oxidation process, reducing the foam cell formation, and signifi cantly increasing the trans-membrane calcium transport mechanism. Amlodipine also signifi cantly reduced the rate of CIMT and reduced ACS admissions in high CV risk groups. [31] Oral anti-oxidants also contribute to plaque stabilization by inhibiting LDL-C oxidation and stabilizing vascular reactivity. However, trials assessing the effects of treatment with the anti-oxidant probucol, either alone or in concert with lovastatin, on restenosis rates after PCI have yielded mixed results. [32,33] Oral treatment with corticosteroids improved clinical outcomes and reduced in-stent restenosis in patients with high CRP concentrations. [34] Diets rich in anti-oxidants have high concentrations of omega-3 class of essential fatty acids, which have been reported to reduce CV events. One such example of an anti-oxidant rich diet is the Mediterranean-type diet, [35] which has been reported to signifi cantly lower the saturated fat content and ACS events in CAD patients in the Lyon Diet Heart Study. Dietary intake of saltwater fi sh rich in omega-3 polyunsaturated fats confer cardio-protective effects mediated in part by reductions in platelet aggregation as well as other benefi cial effects on arterial-wall endothelial function.

Plaque regression
Plaque regression, as viewed today, delves over the prospect of treating vulnerable plaques beyond just stabilization. It emphasizes on aggressive and/or long-term therapy in removing lipids and necrotic material, restoring normal endothelial function and repair of denuded areas to bring about changes beyond just stabilization of vulnerable plaques. Although critics had earlier refuted the concept of plaque regression, recent studies by Nissen et al. [36][37][38] have showed atherosclerosis regression to be a realistic goal in some patients. In addition, earlier angiography studies, which were unable to show aggressive and long-term treatment (usually 1 and 2 years) with statins, has been the most widely studied approach to treat plaque regression. It has been shown to reduce LDL-C and elevate HDL-C levels; [4] the combination of which has been found to be an effective clinical strategy to regress atherosclerosis in the Arterial Biology for the Investigation for the Treatment Effects of Reducing Cholesterol series of studies. [39,40] Another retrospective analysis found that aggressive LDL-C lowering with statins significantly correlated with the reduction in coronary calcium-volume score by electron-beam CT, indicating that coronary artery calcifi cations can shrink. [4] Besides inhibition of HMG-CoA reductase, statins show pleiotropic effects and considerably reduce the plaque burden by: (1) diminution of the plaque lipid-rich core; (2) reduction in the infl ammation with decreased macrophage and foam cell formation; (3) promotion of fi brous cap thickening; and (4) decreasing platelet reactivity. [41] All these effects translate to an overall improvement in endothelial function, which can play a crucial role in preventing plaque formation altogether. In the REVERSAL trial, [37] high dose statin group had 0.4% decrease (regression) in the median atheroma volume in comparison to 2.7% increase (progression) in the moderate-dose group over an 18-month period.
A sub-analysis of the REVERSAL study by Nicholls and colleagues [42] reported that aggressive lipid-lowering therapy was effective in suppressing the development of coronary plaques associated with an increase in body mass index (BMI). In the ASTEROID study, 63.6% of patients experienced regression with a decrease in mean total atheroma volume by 7% after 24 months of 40 mg/day rosuvastatin treatment. [38] Notably, 84% of the patients in ASTERIOD study received a beta-blocker during the study, which may also have some effects on plaque regression. The most recent study focusing on intensive statin therapy for plaque regression was the SATURN trial, which compared the effect of rosuvastatin (40 mg daily) and atorvastatin (80 mg daily) in 1039 patients with coronary disease for 104 weeks. Results showed that maximal doses of both rosuvastatin and atorvastatin resulted in signifi cant plaque regression in majority of the patients, with a relatively higher percentage of patients showing regression with rosuvastatin. [43] Another potential intervention that can raise HDL-C was the inhibition of cholesteryl ester transfer protein (CETP).

Various trials, including Investigation of Lipid Level
Management to Understand Its Impact in Atherosclerotic Events (ILUMINATE), [44] Investigation of Lipid Level Management Using Coronary Ultrasound to Assess Reduction of Atherosclerosis by CETP Inhibition and HDL Elevation (ILLUSTRATE), [45] Radiance 1 (Rating Atherosclerotic Disease Change by Imaging with a New CETP Inhibitor) [46] have shown no improvements in coronary or carotid anatomy despite signifi cant increase in HDL-cholesterol levels in the statin-torcetrapib groups.
ApoA-1 oral peptides have been developed based on the observation that certain genetic polymorphisms were associated with high HDL-C levels. When administered to patients with ACS, 5 weekly infusions of a reconstituted version of ApoA-I resulted in marked plaque regression as assessed by coronary IVUS. [36] A major limitation of current ApoA-I mimetics was that they required parenteral administration because of gastrointestinal proteases that may prevent their effective oral use. [4] CONCLUSION The current literature on management of atherosclerotic plaque suggests that numerous associations have been made between systemic factors important in plaque evolution, thereby creating diverse avenues for disease prevention. Of equal importance is the parallel development of non-invasive imaging techniques that can accurately confi rm the effect of different treatment modalities. Improvements in our understanding of rupture-prone vulnerable plaques will help us choose the right strategy to manage plaque progression. While plaque stabilization mainly focuses on repairing the endothelial cap and strengthening collagen content, plaque regression mainly focuses on aggressive lipid management to remove necrotic components and restore normal endothelial function of the arterial wall. Plaque stabilization has already been proven to reduce clinical events and studies with statins, HDL-C increasing agents, anti-thrombotic agents, ACE inhibitors, calcium antagonists, oral anti-oxidants, and diet modifi cations have shown that reduction in clinical events need not be refl ected by reduced plaque volume. The major studies supporting plaque stabilization included MIRACL, PACT, CURE, HOPE, and Lyon Diet Heart Study. However, recent IVUS studies have shown the incompetence of angiography to precisely measure plaque volume. New insights into the possibility of plaque regression have been elicited by the ASTEROID, REVERSAL, and SATURN studies that utilized intensive statin therapy and demonstrated plaque regression in majority of study patients. However, these trials showed less than 10% regression in overall plaque volume only, which may not explain CV event reduction. This highlights the practical importance of plaque stabilization as the treatment approach to manage plaque disruption. Nonetheless, the growing interest in treating vulnerable plaque has substantially raised the bar on current treatment approaches and, if complemented by confi rmatory imaging techniques and markers, can possibly help regress plaque evolution and eradicate the possibility of atherosclerosis altogether.