extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases

Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat’s leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.

Introduction
Prostasin, matriptase, airway trypsin-like protease, and mast cell β-tryptase are trypsin-like proteases associated with airway mucosa. The present study profiles inhibitor susceptibility and mechanisms of inactivation of purified forms of these proteases. Prostasin (product of PRSS8), also called channel-activating protease, is a type I transmembrane protease that is anchored to airway cell surfaces via glycosylphosphatidyl inositol [1,2]. By activating the epithelial sodium channel (ENaC), prostasin increases Na + reabsorption by luminal epithelium, thereby regulating hydration. Cell surface-prostasin resists inactivation by endogenous antiproteases [3]. Knockdown of expression [4] and pharmacological inhibition decrease ENaCmediated Na + flux and are therapeutic strategies in cystic fibrosis [5,6], which may be associated with overactive ENaC. However, prostasin expression by alveolar epithelium is required for normal fluid clearance [7]. For proposed functions like regulating tight junctions and preserving epithelial barrier integrity [8], prostasin may depend on matriptase (product of ST14) [9,10]. In contrast to prostasin, matriptase is a type II transmembrane protease with non-catalytic extracellular domains and is regulated by hepatocyte growth factor activator inhibitor-1 [11]. Matriptase and prostasin activation may be co-dependent [10,12]. Therapeutic inhibition of matriptase has been proposed in conditions, like malignancy, in which it is overexpressed and may promote tumor cell tissue invasion [13]. On the other hand, matriptase is critical for skin and gut barrier function and is protective in models of colitis [14].
Less is known about airway trypsin-like protease (HAT in humans, product of TMPRSS11D) [15][16][17], which is a type II transmembrane protease. HAT is shed as an active, soluble protease into airways, where it promotes mucus production and is a target for inhibition in bronchitis [18]. β-tryptases (products of TPSAB1 and TPSB2) are produced by epithelium-infiltrating mast cells in asthma [19], in which tryptase inactivation is potentially therapeutic [20]. β-tryptase assembles into a toroidal tetramer, which affords protection from proteinaceous inactivators of trypsin-like proteases. It is stored in secretory granules and released from stimulated mast cells. Secreted β-tryptases can stimulate epithelial cell growth, cytokine production, and recruitment of inflammatory cells [21,22].
The inhibitors analyzed in this work were selected for therapeutic potential as topical inhibitors of tryptic proteases in the airway lumen. Most of these compounds have been tested in preclinical models of human disease or in humans, although neither the therapeutically relevant target nor the potential bystander targets of these inhibitors is known with certainty. Two of the inhibitors, nafamostat and camostat, are guanidinobenzoates that may be cleaved by proteases and can be suicide inhibitors [23][24][25]. Nafamostat has been used clinically as an anticoagulant in humans, in whom the targets are thought to be proteases of the clotting cascade. It is an inhibitory substrate for pancreatic trypsin, with which it forms a stable acyl-enzyme intermediate [23], and it is a particularly potent inhibitor of mast cell tryptases [26]. Nafamostat reduces inflammation in rats with colitis and mice with experimental asthma, possibly by inactivating tryptases [27,28], and reduces tryptase-induced itching in mice [29]. Camostat, on the other hand, attenuates kidney fibrosis in a rat model of chronic renal failure [30]. Given to hypertensive rats, camostat reduces blood pressure and improves kidney function [31], possibly by targeting renal prostasin. When applied to airway mucosa, camostat durably inhibits ENaC in guinea pig trachea, enhances mucociliary clearance in sheep bronchi [6], and increases transepithelial nasal potential difference in humans with cystic fibrosis, by mechanisms speculated to involve inhibition of prostasin [32].
Benzamidine and BABIM, by contrast, are competitive, reversible inhibitors. Benzamidine is a general tryptic protease inhibitor that reduces tryptase-induced enhancement of muscle contraction in isolated bronchi [33]. BABIM is a bifunctional aromatic amidine that inhibits tryptase and trypsin [34,35]. In sheep, topical BABIM blocks asthma-like airway responses, including increased resistance in allergen-challenged airway, potentially by inhibiting tryptase [20]. Aprotinin is a proteinaceous general inhibitor of tryptic serine proteases that blocks the substrate-binding site. Because of its size, it is ineffective versus proteases with restricted active sites [36]. It has been used as a drug in humans to treat pancreatitis and to limit blood loss in surgery [37]. Clinical targets may include kallikreins and activated proteases associated with complement activation and hemostasis. Aprotinin and related inhibitors are proposed as inhaled therapeutic inhibitors in cystic fibrosis [5]. When applied intratracheally, aprotinin attenuates epithelial ion transport in guinea pigs [38], and when applied to human airway epithelial monolayers, aprotinin reduces ENaC function [6] and surface tryptic activity [3].

Determination of IC 50
To compare potency of irreversible (or slowly reversible) inhibitors (for which K i values may be inapplicable) with potency of competitive, reversible inhibitors, we determined the concentration of inhibitor that achieves 50% inactivation (IC 50 ) of soluble forms of the following human airway tryptic proteases: prostasin, matriptase, HAT, and β-tryptase. IC 50 was determined by assaying initial rates of hydrolysis of 1-mM QAR in 50-mM Tris-HCl (pH 7.6) containing 120-mM NaCl 0.05% Tween 20, and 100 μg/ml bovine lung heparin (Sigma-Aldrich) at 37°C over a range of inhibitor concentrations (shown in Table 1). Serial absorbance measurements detecting products of substrate hydrolysis were performed at 410 nm in wells of 96-well, flat-bottom, polystyrene Costar plates (Corning, Tewksbury, MA) in a kinetic, temperaturecontrolled spectrophotometer (Synergy 2, BioTek Instruments, Winooski, VT). Microplates were covered with TempPlate Optical Film (USA Scientific, Ocala, FL) during spectrophotometric measurements to minimize evaporation.

Measurement of duration of inhibition by nafamostat
The durability of nafamostat inhibition was tested for the two proteases (matriptase and βtryptase) for which inhibition was nearly stoichiometric. To permit assessment of reversibility, the proteases and inhibitors were combined in equimolar concentrations in the presence of substrate QAR under conditions in which enzyme activity was nearly but not completely inhibited. Recovery of activity was monitored over 4-5 hours and compared with activity of uninhibited enzyme.

Assessment of product inhibition
Catalytic hydrolysis of nafamostat yields free 6-amidino-2-naphthol, which is an aromatic amidine related to several of the compounds tested in this study (see Fig 1) with potential to inhibit tryptic serine proteases. Thus, it could cause "product inhibition" and potentially account for prolonged inhibition of protease activity by nafamostat. To test this possibility, we used substrate QAR to compare the IC 50 of nafamostat and 6-amidino-2-naphthol versus matriptase and β-tryptase.
Active site titration of protease active sites using nafamostat As a calibration control, bovine trypsin was active site-titrated by an established colorimetric method as described [39]. Briefly, 1-to 3-microliter aliquots of trypsin (30 mg/ml) were added to 0.5 ml of 1-mM p-nitrophenyl-p'-guanidinobenzoate in 0.1 M Tris-HCl (pH 8.2) containing 0.02 M CaCl 2 in a quartz cuvette at 37°C while monitoring the "burst" increase in Absorbance at 405 nm on a Genesys 10S UV-Vis spectrophotometer equipped with a Peltier cuvette jacket (Thermo Fisher Scientific, Waltham, MA). The concentration of trypsin active sites was deduced using a molar extinction coefficient for 4-nitrophenol of 1.8 x 10 4 Absorbance Units M -1 cm -1 .
The same preparation of trypsin then was active site-titrated by the more sensitive fluorometric approach using nafamostat. To establish the relationship between fluorescence emission (in mV) and concentration for 6-amidino-2-naphthol, which is the fluorescent leaving group generated by formation of the acyl intermediate by trypsin's attack on nafamostat [23], serial dilutions of 6-amidino-2-naphthol in PBS were prepared. Fluorescence was measured using excitation and emission wavelengths of 320 nm and 490 nm, respectively, by injecting 0.1-ml aliquots into the 16-microliter flow cell of a Jasco FP-2020 Plus Fluorescence Detector (gain = 1000, attenuation = 8; Jasco Incorporated, Easton MD) at 0.1 ml/min to minimize bleaching. Flow was controlled by an AKTA Purifier 10 chromatography system (GE Healthcare Biosciences, Pittsburgh, PA). Fluorescence readings were blanked by injecting PBS. Molarity of active sites in solutions of trypsin was estimated and compared with values obtained by the colorimetric p-nitrophenyl-p'guanidinobenzoate approach by adding aliquots of enzyme stock solutions to nafamostat in PBS and measuring increase in fluorescence. Active site concentrations in preparations of matriptase and β-tryptase were determined fluorometrically in similar fashion.

Culturing human airway cells
Human CFBE41o-bronchial epithelial cells [40,41] were grown to confluence and differentiated in air-liquid interface culture as previously described [3]. Briefly, cells were seeded to confluence at 3x10 5 cells/well onto Costar Transwell membranes (0.33 cm 2 , 0.4-μm pores; Corning; Lowell, MA) coated with bovine serum albumin (1 mg/ml; Invitrogen, Carlsbad, CA), human fibronectin (30 μg/ml; BD Biosciences; San Jose, CA), and bovine collagen type II (10 μg/ml; BD Biosciences) in LHC basal medium (Invitrogen) and grown in minimal essential medium with Earle's salt supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin G, and 300 μg/ml hygromycin at 37°C in a 5% CO 2 incubator. Apical medium was removed 48 h after seeding to initiate air-interface culture. Basolateral medium was changed every 48 h for 12 days. Except as specified, culture media and supplements were from Invitrogen.

Assay of protease activity on the surface of airway epithelial cell monolayers
Tryptic protease activity on the apical surface of confluent CFBE41o-bronchial epithelial cell monolayers was measured by methods previously described [3]. We employed these cells because they form differentiated, electrically resistant monolayers that contain apical prostasin and matriptase and surface aprotinin-inhibitable tryptic activity [3]. Briefly, cells were washed in PBS after 12 days of air-interface culture on Transwell inserts. The apical side of the monolayer was immersed in 0.2 ml of PBS alone, PBS containing tryptic substrate QAR (1 mM) alone as control, or PBS containing QAR plus inhibitor (0.1 mM aprotinin, 0.01 mM nafamostat, or 0.01 mM camostat). The basolateral surface was bathed in PBS alone. Tryptic activity in aliquots of apical conditioned medium was monitored for 5 h at 37°C by spectrophotometry at 410 nm using a Synergy 2 SL Microplate Reader (BioTek Instruments).

Statistical methods
IC 50 values and associated confidence intervals were determined using the Hill slope curve-fitting utility implemented in Prism (GraphPad Software, La Jolla, CA). Differences in surface tryptic activity of cells incubated in the presence of substrate alone versus substrate plus inhibitor were compared using 1-tailed Student's t-tests.

Inhibitor susceptibility of tryptic airway proteases
The in vitro, steady state susceptibility of four human airway proteases (HAT, matriptase, prostasin and β-tryptase) to inactivation varied depending on the inhibitor studied. ). In such plots, activity is 0% when log 10 ([inhibitor]/[enzyme]) = 0 for an inhibitor that inactivates a protease with full potency and 1:1 stoichiometry. The normalized IC 50 for such an inhibitor would be log 10 (0.5) = -0.3. The observed IC 50 data are shown in Table 2.
As revealed in Fig 2, nafamostat achieved complete and nearly 1:1 stoichiometric inactivation of matriptase and tryptase, but less potently inhibited prostasin and was weak versus HAT, for which~10,000-fold excess of inhibitor over enzyme was required to achieve complete inhibition. For both matriptase and tryptase, nafamostat was the most potent of all anti-proteases studied. Camostat inactivated all enzymes but overall was substantially less potent than nafamostat, and exhibited the least ability of all of the inhibitors to discriminate between the tryptic airway proteases examined in this study. Although camostat, like nafamostat, is a guanidinobenzoate (see Fig 1), it was weakest towards matriptase whereas nafamostat was the strongest, suggesting that the leaving groups (prior to release) for these cleavable inhibitors are major determinants of specificity and potency. BABIM, which is an aromatic amidine but is not hydrolysable like nafamostat and camostat, was a complete and potent inhibitor of matriptase, but was less so for the other enzymes. Aprotinin exhibited nearly 1:1 stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and completely ineffective versus tryptase. Benzamidine was universally weak and did not fully inactivate any of the proteases at stoichiometries as high as 1,000,000:1.
Inhibition by 6-amidino-2-naphthol does not explain high potency of nafamostat versus matriptase and β-tryptase As shown in Fig 4, the IC 50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >10 4fold higher for matriptase and β-tryptase than the IC 50 of nafamostat itself. Although one potential mechanism of high potency of a cleavable inhibitor is generation of a leaving group that is a more potent competitive inhibitor than is the uncleaved parent compound, the very high IC 50 of 6-amidino-2-naphthol relative to that of nafamostat itself suggests that this is not the case. Therefore, inhibition by the non-acyl component of hydrolyzed nafamostat does not explain nafamostat's high potency upon cleavage by these enzymes and, given the very low rates of nafamostat turnover, 6-amidino-2-naphthol would be unlikely to achieve the concentrations needed to reduce matriptase and β-tryptase activity. Inhibition of matriptase and β-tryptase by nafamostat spontaneously reverses At nearly stoichiometric ratios of nafamostat to enzyme, inhibition of tryptase and β-tryptase slowly reverses, as seen in Fig 5. This behavior is consistent with nafamostat acting as a suicide inhibitor, with formation of a covalent (acyl) intermediate that is eventually broken by hydrolysis, with full release of the inhibitor and restoration of the free serine hydroxyl and enzymatic activity. The observed reversibility is not consistent with tight, non-covalent competitive inhibition, which should be stable over time, assuming that the inhibitor and protease are chemically and enzymatically stable. The rate of recovery of proteolytic activity under conditions in which the enzyme starts is in slight molar excess of nafamostat is 1-2% per hour, indicative of recovery kinetics and deacylation rates that are very slow from a pharmaceutical standpoint. Furthermore, in conditions for which there is a substantial molar excess of nafamostat, no reversibility is observed (not shown), presumably because any regenerated enzyme is promptly inactivated by formation of an acyl intermediate with a fresh molecule of nafamostat.
Sensitive active site-titration assay of matriptase and β-tryptase using nafamostat Given the observed nearly 1:1 stoichiometry and slow reversal of inhibition of matriptase and β-tryptase by nafamostat, we sought to detect release of 6-amidino-2-naphthol and to explore the possibility of using nafamostat as a "burst titrant" of active sites in preparations of these enzymes. We did not assess this possibility for prostasin or HAT because inhibition for these proteases did not approach 1:1 stoichiometry. Estimates of active site concentration in our stock preparation of trypsin were similar whether obtained using the colorimetric approach with p-nitrophenyl-p'-guanidinobenzoate or the fluorometric approach with nafamostat (0.83 versus 0.62 mM, respectively). However, the nafamostat method, as implemented using the inline flow cell of the Jasco FP-2020 Plus Fluorescence Detector, was far more sensitive, requiring dilution of trypsin stock solution~1,000-fold. The fluorometric nafamostat method, being capable of detecting active sites in the 1-nM range (see Fig 6) using sample volumes of 0.1 ml or less, offered the prospect of minimizing consumption of enzymes that are difficult or costly to produce. Application of the fluorometric nafamostat approach to matriptase and β-tryptase (Fig 6) suggests its utility as a sensitive method to quantify active sites. Titration of our preparation of matriptase identified an active-site concentration of 0.12 ± 0.02 μM (mean ± SD, N = 4), predicting that the preparation is 22% active using a denominator based on the manufacturer's estimate of protein concentration. Based on the active site estimate, the specific activity of matriptase for substrate QAR using our standard assay conditions is 3 x 10 7 Absorbance Units 410 nm min -1 M -1 . Assay of β-tryptase active sites in similar fashion yielded an active site concentration of 2.1 ± 0.1 μM (mean ± SD, N = 3), which is 110% of the value based on the manufacturer's estimate of protein concentration. Based on the active site estimate, the specific activity of β-tryptase for substrate GPK using our standard assay conditions is 2.5 x 10 7 Absorbance Units 410 nm min -1 M -1 . Nafamostat itself has minimal fluorescence at excitation and emission wavelengths optimized for 6-amidino-2-naphthol [23]. The observed low-level baseline fluorescence in stock solutions of nafamostat (as seen in Fig 6) may be due to non- Inhibition of apical tryptic enzyme activity in epithelial cells cultured at an air-liquid interface Fig 7 reveals that apical, cell surface, QAR-hydrolyzing protease activity is almost completely inhibited by aprotinin (100 μM), consistent with our prior study [3], while establishing that Inhibition of Airway Tryptic Proteases nafamostat and camostat (10 μM) are nearly as effective as aprotinin. In conjunction with our prior studies showing little if any active matriptase on the apical surface of CFBE41o-cells cultured in the same manner [3], the present data are consistent with prostasin (and not HAT or β-tryptase) being the principal source of apical tryptic activity.

Discussion
This study focuses on four proteases that share three major attributes: 1) they are trypsin-like, 2) they are found in human airway epithelium and 3) they are proposed as targets for inhibition to treat allergic or infectious airway disorders associated with inflammation and hypersecretion. This first direct comparison of these proteases reveals that each has a distinct profile of susceptibility to the inhibitors shown in Fig 1, despite sharing a capacity to cleave peptides after arginine residues. Several inhibitors examined here have been used to target specific airway tryptic proteases in vivo. Although these inhibitors exhibit a broad range of potency, none is selective for any one of the proteases examined (as shown in Figs 2 and 3. Among the implications of these findings is that pathology-modifying phenotypes resulting from application of these inhibitors potentially may arise from inactivation of proteases other than those that were targeted. The findings also raise the possibility of undesired bystander effects resulting from inactivation of these and other tryptic proteases. On the other hand, some of the inhibitors, such as nafamostat for tryptase and matriptase-and aprotinin for prostasin-were Inhibition of Airway Tryptic Proteases exceptionally potent, raising the possibility of developing more selective inhibitors with retained potency.
In the case of β-tryptase and matriptase, the findings show that nafamostat's high potency relates in part to actions as a suicide substrate. This results in formation of a covalently bound, inactivating intermediate that is stable for hours in aqueous solution. In this regard, nafamostat's bifunctionality could influence potency. As shown in Fig 1, nafamostat has the potential to occupy the tryptic primary specificity pocket using either its guanidino or its amidino end, but not both simultaneously. These docking modes have different consequences. Binding via the guanidino end positions nafamostat's carbonyl carbon to be attacked by the protease's active site serine Oγ to yield the 4-guanidino-benzoylated "acyl" enzyme. This is a substratelike interaction that leaves a bound fragment that cannot be competitively displaced by substrate. By contrast, docking with the amidino end in the specificity pocket is a competitive, reversible interaction that neither positions nafamostat for hydrolytic attack nor results in formation of an acyl intermediate. In the examples of matriptase and β-tryptase, the nearly 1:1 stoichiometry of inactivation by nafamostat, combined with the evidence of 6-amidino- Inhibition of surface tryptic activity on bronchial epithelial cells. Monolayers of CFBE41o-cells cultured on Transwell inserts at an air-liquid interface to promote differentiation, including apical-basolateral polarization, were assayed for surface tryptic activity by adding substrate QAR to apical bathing medium, with or without addition of camostat, nafamostat or aprotinin, followed by spectrophotometric monitoring of cleaved substrate at 410 nm. *P <0.05 and **P <0.01 versus change in absorbance in QAR medium without inhibitor.
doi:10.1371/journal.pone.0141169.g007 2-naphthol release and the finding that inhibition by 6-amidino-2-naphthol itself is comparatively weak, suggest that the binding mode with the guanidino end in the primary specificity pocket is highly favored. This is less likely to be the case for HAT and prostasin, towards which nafamostat is less potent.
It can be noted from the structures in Fig 1 that camostat lacks nafamostat's duality. Binding via its guanidino end is likely its only productive mode of action as an inhibitor, and predicts that its interactions necessarily involve formation of an acyl intermediate. However, the finding that camostat is much less potent than nafamostat as an inhibitor of matriptase and β-tryptase reveals that the mere presence of a 4-guanidino-benzoate moiety susceptible to nucleophilic attack to form a covalent intermediate does not guarantee high potency. Nonetheless, inhibition by camostat is complete at higher ratios of inhibitor to enzyme and is likely to be as durable as inhibition by nafamostat, given that the 4-guanidino benzoate moiety ends up covalently linked to prostasin to form the acyl enzyme complex captured and identified in prostasin crystallized either with camostat [25] or with nafamostat [24].
Nafamostat's ability to form an inhibitory complex at low concentration via a cleaved intermediate is undoubtedly responsible for the report of a highly favorable (sub-nanomolar) dissociation constant (K i ) when tested versus human β-tryptase [26]. However, as the present results suggest that nafamostat is a suicide substrate and burst titrant for tryptase, K i calculations, which assume a competitive and reversible mode of inhibition, are inapplicable to nafamostat inhibition of tryptase, for which the resulting data would depend on enzyme concentration. Similar considerations apply to camostat, which yielded apparent K i values that were >100-fold higher for prostasin than for matriptase [6], in contrast to our data in Table 2 showing that camostat is actually less potent versus matriptase than versus prostasin based on determination of concentration-normalized IC 50 , which is a more appropriate comparator for a suicide substrate. The discrepancy probably relates to the need to use concentrations of prostasin in tryptic substrate cleavage assays that are high relative to the concentrations needed of matriptase, which is an intrinsically more efficient enzyme [3]. Regardless, our data suggest that release of fluorogenic 6-amidino-4-naphthol from nafamostat by the catalytic serine of matriptase and β-tryptase is an active-site titration method that is much more sensitive than colorimetric methods. However, the findings also suggest that nafamostat will be less useful in this regard for HAT and prostasin, for which stoichiometries are less favorable.
The kinetic comparisons of prostasin, matriptase and HAT with the native, soluble β-tryptase tetramer were conducted with recombinant forms of these enzymes expressed as soluble catalytic domains. This is a matter of necessity since the assays developed to detect the activity of membrane-bound tryptic proteases are ill-suited to IC 50 determinations because of low concentrations of surface enzyme, the need for prolonged incubations, and the presence of other peptidases in living cells. A potential caveat to the work reported here is that inhibitor susceptibility of membrane-anchored type I (prostasin) and type II proteases (matriptase and HAT) may differ from that of soluble forms of the enzymes due to active site blockade or allosteric effects. However, matriptase and prostasin can exist natively in shed, soluble forms, and HAT's shed form may be the native enzyme's dominant form [18]. Furthermore, the small inhibitors profiled here are unlikely to be subject to steric constraints that might limit ability of a large, proteinaceous inhibitor to inactivate a surface-bound enzyme. Indeed, even aprotinin, which is the largest inhibitor in the present study, sharply reduces apical surface tryptic activity in cultured human airway epithelial cells, which contain prostasin and matriptase in membraneattached forms [3].
Although one of the principal findings of this study is the relative lack of inhibitor specificity towards four of the tryptic enzymes that have been considered as candidates for therapeutic inactivation in airway diseases, our data provide grounds for considering that prostasin is the principal source of apical surface tryptic activity in the human airway epithelial cell monolayers generating the data in Fig 7. The reasoning involves combining the present data generated using small inhibitors with prior studies using anti-matriptase single chain antibody [3], which is potent and selective for matriptase but inhibited only a small portion of aprotinin-sensitive tryptic activity on the apical surface of airway epithelial cells. Clearly, β-tryptases cannot be a source aprotinin-sensitive activity, because human β-tryptases completely resist aprotinin, even though aprotinin inhibits most trypsin-like serine proteases. The basis of tryptase's unusual resistance to aprotinin is a restricted active site [36]. Compared to prostasin and matriptase, HAT is much less sensitive to aprotinin and nafamostat, but all enzymes are similarly sensitive to camostat. Thus, the data are consistent with prostasin being a major source of airway surface tryptic activity and the major source of aprotinin-sensitive activity on the apical surface of cultured airway cells.

Author Contributions
Conceived and designed the experiments: SN WWR GHC. Performed the experiments: SN WWR. Analyzed the data: SN WWR GHC. Contributed reagents/materials/analysis tools: DCG. Wrote the paper: GHC.


extfieldsofstudy: Biology Medicine

text:
Natural Products as Tools for Defining How Cellular Metabolism Influences Cellular Immune and Inflammatory Function during Chronic Infection

Chronic viral infections like those caused by hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause disease that establishes an ongoing state of chronic inflammation. While there have been tremendous improvements towards curing HCV with directly acting antiviral agents (DAA) and keeping HIV viral loads below detection with antiretroviral therapy (ART), there is still a need to control inflammation in these diseases. Recent studies indicate that many natural products like curcumin, resveratrol and silymarin alter cellular metabolism and signal transduction pathways via enzymes such as adenosine monophosphate kinase (AMPK) and mechanistic target of rapamycin (mTOR), and these pathways directly influence cellular inflammatory status (such as NF-κB) and immune function. Natural products represent a vast toolkit to dissect and define how cellular metabolism controls cellular immune and inflammatory function.

Introduction
Natural products are pure compounds or mixtures, and they are often derived from primary and secondary metabolites of cells, tissues, and secretions from microorganisms (bacteria, archae, fungi), plants, and animals. They are found in marine and terrestrial environments. Pharmacognosy is the study of natural products, which is studied throughout the world through organizations such as the American Society of Pharmacognosy [1] and the Gesellschaft für Arzniepflanzen (GA society) [2].
Two-thirds of the population of the world uses alternative therapies, including botanical medicines, as their primary source of health care [3]. Natural products continue to play a critical role in drug discovery; a significant portion (28%) of the new drugs approved between 1981 and 2010 were of natural product origin [4]. Moreover, the 2015 Nobel Prize in Medicine was awarded to three scientists who discovered Invermectin and Artemisinin, two natural products with potent anti-infective properties. Thus, natural products represent a vast chemical toolbox of potent medicines with demonstrated health-promoting actions. However, a consistent issue with research on natural products, with the exception of a few drugs, is the lack of clarity surrounding their mechanisms of action. Following ingestion and absorption, natural products produce biological responses in mammalian cells by several mechanisms including direct antioxidant functions, interaction with membranes, and alterations in mitochondrial function, signal transduction, and gene expression. This review will focus on the emerging evidence that polyphenolic natural products alter cellular metabolism, which has profound impacts on cellular inflammatory status and function,

Chronic Inflammation and the Road to HCV-Induced Liver Disease
Once HCV infects a hepatocyte, it is sensed by TLR3 [11] and RIG-I [12]. This leads to induction of the cellular antiviral (i.e., production of IFN stimulated genes (ISGs) and pro-inflammatory (i.e., production of inflammatory cytokines and chemokines) responses. Chemokines like CXCL8 [13] and CXCL10 [14] are produced by HCV-infected hepatocytes, resulting in recruitment of immune cells to the liver, which result in the killing of hepatocytes.
For example, activated CD8+ T cells, NK cells, and NK T cells kill virus-infected cells via several means such as Fas/TRAIL-mediated apoptosis, the release of granzymes and perforin, and secretion of type II IFN [15,16]. Kupffer cells, which are the liver-resident macrophages, take up apoptotic bodies released from dying hepatocytes, thereby accelerating hepatocyte death via Fas-mediated apoptosis [17]. Kupffer cells also secrete TGF-β, inducing differentiation of hepatic stellate cells into proliferative myofibroblasts, which then secrete type I collagen as part of the general wound healing response to liver injury [17]. This inflammatory response is also augmented by liver sinusoidal endothelial cells, which play important roles in antiviral and inflammatory responses to virus infection, by direct sensing of viral pathogen associated molecular patterns (PAMPS) [18]. In summary, the primary sensing of HCV RNA by hepatocytes engages anti-viral and pro-inflammatory responses that recruit multiple immune cell types to the site of infection to amplify the inflammatory response. Since the majority of acute HCV infections are not cleared, the inflammatory response is perpetuated.

Chronic Inflammation: The Case for HIV
Despite effective virus control by ART, many HIV+ patients live an average of 10-30 years less than the HIV-uninfected public [19]. A favored explanation is that this is due, in part, to dysregulated immune function that arises from chronic HIV infection. This chronic immune activation (CIA) is associated with various inflammatory diseases and both AIDS-defining and non-AIDS defining cancers [20]. One theory of how CIA manifests posits that the initial reduction in CD4 T cells in the gut releases PAMPs to trigger toll-like receptor (TLR)-mediated activation of immune cells [21].
By the end of this year, it is estimated that more than 50% of HIV-infected patients in the United States will be over the age of 50 [22]. In the aging HIV+ population, there are increases in co-morbidities such as osteoporosis, diabetes, and cardiovascular disease, which normally occur later in life due to natural aging [23,24]. Despite years of viral suppression by potent ART, a substantial proportion of HIV+ patients present with CIA, which is associated with disease progression and mortality [25]. Thus, while ART suppresses HIV replication, many patients' CD4+ T cells fail to fully rebound to the level of non-infected patients [26], and this failure to is associated with a high rate of clinical events [27] and disease progression [28], and is thought to contribute to the shortened life expectancy in HIV infection [29] and accelerated aging. Immune-based factors, such as immunosenescence, characterized by continual immune cell death and turnover caused by uncontrolled, systemic, and low-grade inflammation, accelerates many of the aging phenotypes naturally observed in the HIV-negative elderly [30]: in HIV+ subjects with CD4 T cells that do not rebound during ART, their T cells are more likely to display immunosenescent phenotypes [31]; and these premature phenotypes are predictive of more rapid disease progression [32].

Metabolic Modulators That Impact Immune and Inflammatory Responses
An emerging body of literature has demonstrated the importance of cell metabolism in regulating the functionality and inflammatory status of the immune system [33][34][35]. In the following sections, we describe ways in which natural or synthetic drug modulation of key metabolic signaling enzymes influences immune cell function. We will focus on mTOR and AMPK, since they are key players in immune cell activation, inflammatory function, and fate decisions [34,36].

Immune Cell Activation
Immune cells exhibit marked changes in metabolism and metabolic signaling pathways when they transition from quiescence to activation. These metabolic changes also directly influence immune cell fate and functions. For example, resting/quiescent T cells produce energy in the form of ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OX PHOS) through the mitochondrial electron transport chain. This process requires the breakdown (i.e., catabolism) of glucose, fatty acids, and amino acids, which are essential components for cell growth. In stark contrast, activated T cells engage the Warburg effect [37]; cells break down glucose by aerobic glycolysis to produce ATP. Even though glycolysis is an inefficient way to make ATP as compared to OX PHOS, the prevailing thought is that glycolysis is preferred for T cell activation because it is an anabolic (i.e., cell building) form of metabolism which generates ATP while preserving amino acids, nucleotides, and fatty acids that are essential precursors for cell growth [38]. Exactly how these metabolic switches occur and how they affect immune cell activation and effector cell generation has become an area of active research [33,35,39,40].

Natural Products to Quell Chronic Inflammation in HCV and HIV Infection
There is a huge body of literature on many natural products and how they impact cellular inflammation and inflammatory signaling pathways. The reader is referred to recent reviews on the subject [41][42][43]. The following discussion focuses on three structural distinct polyphenolic compounds: curcumin, resveratrol, and silymarin ( Figure 1). To assist with making certain points, we will also describe the actions of a few other natural products.
Viruses 2015, 7, page-page subject [41][42][43]. The following discussion focuses on three structural distinct polyphenolic compounds: curcumin, resveratrol, and silymarin ( Figure 1). To assist with making certain points, we will also describe the actions of a few other natural products. Figure 1. Structures of silybin A and silybin B, resveratrol, and curcumin. Note that silybin A and silybin B comprise the two major flavonolignans in the extract known as silymarin. Silibinin is an equimolar mix of silybin A and silybin B. Not shown are the other 5 flavonolignans that comprise the extract silymarin [44]. The structures share the features of containing multiple phenol groups. Hence, these compounds are all polyphenol-containing natural products.
Curcumin is a diarlyheptanoid, distantly related to flavonoids because it possesses two phenol moieties and is therefore a polyphenolic natural product. It is derived from the rhizomes of Curcuma longa, a plant in the ginger family. It has diverse anti-inflammatory actions including direct suppression of pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, and CXCL8 [42]. Curcumin also appears to activate antioxidant responses by activation of the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which induces the expression of antioxidant genes including heme oxygenase 1, glutathione S-transferases (GST), and NAD(P)H quinone oxidoreductase 1. The pathways that account for these effects are diverse and include modulation of transcription factors including NF-κB, β-catenin, and Signal Transducer and Activator of Transcription 3 (Stat3) [42].
Resveratrol is a polyphenolic natural product (a stilbene) found in several plant species including red grapes. Resveratrol is thought to interact with several different putative cellular protein targets including Sirt1, cAMP phosphodiesterases, F1-ATPase [43]. Like curcumin, resveratrol shows strong anti-inflammatory activity, including suppression of COX2, NF-κB, and FOXO action [43].
Silymarin shows broad-spectrum anti-inflammatory activity, which, in addition to its antiviral activity, may have direct relevance for both HCV-and HIV-induced inflammation. In fact, there is evidence that the anti-inflammatory activity of silymarin occurs at various levels and targets multiple pathways. First, silymarin inhibits pro-inflammatory signaling pathways including NF-κB [47,48,57,58]. Second, silymarin inhibits the expression of multiple pro-inflammatory cytokines and chemokines (e.g., CXCL1, CXCL2, CXCL8, CXCL10, IL-1, TNF-α [57]), all of which are dysregulated Curcumin is a diarlyheptanoid, distantly related to flavonoids because it possesses two phenol moieties and is therefore a polyphenolic natural product. It is derived from the rhizomes of Curcuma longa, a plant in the ginger family. It has diverse anti-inflammatory actions including direct suppression of pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, and CXCL8 [42]. Curcumin also appears to activate antioxidant responses by activation of the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which induces the expression of antioxidant genes including heme oxygenase 1, glutathione S-transferases (GST), and NAD(P)H quinone oxidoreductase 1. The pathways that account for these effects are diverse and include modulation of transcription factors including NF-κB, β-catenin, and Signal Transducer and Activator of Transcription 3 (Stat3) [42].
Resveratrol is a polyphenolic natural product (a stilbene) found in several plant species including red grapes. Resveratrol is thought to interact with several different putative cellular protein targets including Sirt1, cAMP phosphodiesterases, F1-ATPase [43]. Like curcumin, resveratrol shows strong anti-inflammatory activity, including suppression of COX2, NF-κB, and FOXO action [43].
Silymarin shows broad-spectrum anti-inflammatory activity, which, in addition to its antiviral activity, may have direct relevance for both HCV-and HIV-induced inflammation. In fact, there is evidence that the anti-inflammatory activity of silymarin occurs at various levels and targets multiple pathways. First, silymarin inhibits pro-inflammatory signaling pathways including NF-κB [47,48,57,58]. Second, silymarin inhibits the expression of multiple pro-inflammatory cytokines and chemokines (e.g., CXCL1, CXCL2, CXCL8, CXCL10, IL-1, TNF-α [57]), all of which are dysregulated in CIA and during HIV-1 disease progression. Third, silymarin suppresses T-cell activation and proliferation [47,48,58], which is elevated in HIV-associated CIA.
Exemplified by the three compounds described above, a common theme among hundreds of polyphenolic natural products is that they almost invariably suppress inflammation (albeit it to differing degrees depending on the structure of the compound, the experimental system, dose, and duration of exposure). For example, at least a dozen natural products have been shown to effectively inhibit inflammatory bowel disease in mouse models through suppression of NF-κB and NF-κB regulated cytokines and chemokines (reviewed in [41]). This raises the question of how can so many structurally diverse compounds cause the same effects in cells? As far as defining the mechanistic underpinnings for the anti-inflammatory actions of natural products, there are plenty of examples of many natural products that inhibit a particular pathway, kinase, transcription factor, or modulate cellular gene and protein expression. There is also the frequent misconception in the literature that inhibition of an inflammatory pathway indicates that pathway is the target (i.e., the natural product binds to the protein). While many natural products do have bona fide protein targets to which they bind, the interaction may occur far upstream of the measured biological effect. Thus, there is great imprecision and a lack of a unified view of explaining how natural products suppress inflammation. As we shall see in the next section, the unifying effects that explain the anti-inflammatory effects of many natural products may lie at the level of altering cellular metabolism and cellular signaling pathways that receive metabolic inputs and cues.

AMPK, and mTOR: Cellular Signaling Pathways That Link Cellular Metabolism to Function
Mammalian cells express sentinel kinases and transcription factors that sense nutritional status in the microenvironment. For example, AMPK is a master regulator of cellular metabolism. AMPK is a tri-molecular complex of a catalytic α subunit (with serine/threonine kinase function) and regulatory β and γ subunits [59] (Figure 2). AMPK is activated by phosphorylation on Thr-172 by two major routes. One is via an upstream kinase known as Serine/threonine kinase 11 (STK11; a.k.a. liver kinase B1 (LKB1)), while the other upstream kinase is the calmodulin-dependent kinase kinases, CaMKKβ. LKB1 activates AMPK by phosphorylation in response to energy stress (i.e., decreased cellular ATP/ADP ratios), while CaMKKβ activates AMPK in response to treatments that increase intracellular Ca 2+ (Figure 2). Thus, AMPK is regulated by adenine nucleotides and calcium ions. The downstream effects of AMPK activation are numerous and serve to realign cellular energetics; more specifically, to restore the reductions in cellular ATP. Thus, catabolic responses (i.e., energy sparing) are activated while anabolic (i.e., energy consuming/cell building) processes are curtailed. Downstream effects occur through multiple transcription factors and changes in cellular metabolism including increasing glucose uptake, glycolysis, fatty acid uptake, fat oxidation, and autophagy, and to decrease anabolic processes such as protein, cholesterol, and fatty acid synthesis [59].
The mTOR pathway [60,61] is one downstream pathway that is inhibited by activated AMPK [62]. mTOR is a major hub for several metabolic inputs and cues, and consists of two complexes, mTOR containing complex 1 and 2 (mTORC1 and mTORC2). By sensing energy status, mTOR affords switching between anabolic (in a nutrient-rich environment) to catabolic (during nutrient depletion/stress) processes. Thus, when nutrients are abundant, mTOR kinase activity leads to the phosphorylation of several downstream targets that promote cell growth and survival, including mRNA and protein synthesis. mTOR complex 1 (mTORC1) consists of mTOR, GβL, DEPTOR and Raptor [63,64] and is regulated by a small GTPase, Rheb, which when bound by GTP, potently activates mTORC1 kinase activity. mTOR complex 2 (mTORC2) is composed of mTOR, Rictor, GβL, Sin1, PRR5/Protor-1, and DEPTOR [65,66]. mTORC2 promotes cellular survival by activating Akt and other pathways [67]. Thus, for the purpose of this review, natural products can be viewed as metabolic modulators that activate AMPK and suppress mTOR, which ultimately converge of suppression of pro-inflammatory signaling, exemplified by blockade of NF-κB ( Figure 2).
Viruses 2015, 7, page-page Figure 2. Metabolic Rules of Engagement of Natural Products with Cells. Some of the earliest cellular responses to natural product exposure include the alteration of mitochondrial function which increases the AMP:ATP ratio, leading to activation of AMPK by phosphorylation (1). Natural products like salicylate can also directly bind to and activate AMPK (2). AMPK activation suppresses Rheb, an upstream activator of mTOR signaling. Thus, AMPK activation by natural products can lead to mTOR inhibition. Natural products like rapamycin can also directly bind to and inhibit mTOR signaling (3). The alteration of mitochondrial energetics may be involved in natural product induction of DDIT4, a novel upstream inhibitor of mTOR (4). Finally, activation of AMPK and inhibition of mTOR by natural products may be directly involved in the suppression of pro-inflammatory signaling such as that mediated by NF-κB (5). For mTOR, there is evidence that IKK interacts with mTORC1 to mediate the connection of mTORC1 to NF-κB, while for AMPK, the route to NF-κB is not presently clear. Citations of studies supporting these interactions are provided in text. Note that the tri-molecular complexes of IKK (IKKα, IKKβ, and IKKγ), and the bimolecular complex of NF-κB are simplified as single proteins. Moreover, only the mTORC1 and canonical NF-κB pathways are shown.
Nuclear factor kappa B (NF-κB) is the classical pro-inflammatory transcription factor that controls the expression of hundreds of pro-inflammatory cytokines and chemokines as well as other inflammatory and immune molecules such major histocompatibility complex proteins, transcription factors, and inhibitors of apoptosis [68]. NF-κB is not a single protein but rather a complex of homo and heterodimeric complexes of proteins [69]. Inactive NF-κB maintains a cytoplasmic localization by associating with inhibitory proteins of the IκB family [70]. Stimulation of cells via cytokines, PAMPs, or immune cell receptor activation leads to NF-κB activation by phosphorylation of IκB proteins, which leads to their degradation (Figure 2). This allows NF-κB to enter the nucleus and exert its transcriptional effects. IκB phosphorylation is controlled by the multiprotein IκB-kinase (IKK) complex that contains 2 catalytic subunits, IKKα (IKK1) and IKKβ (IKK2), as well as the regulatory subunit IKKγ (a.k.a NEMO for NF-κB essential modifier). There are two pathways that lead to NF-κB activation: canonical and alternate. Activation of NF-κB via  (1); Natural products like salicylate can also directly bind to and activate AMPK (2); AMPK activation suppresses Rheb, an upstream activator of mTOR signaling. Thus, AMPK activation by natural products can lead to mTOR inhibition. Natural products like rapamycin can also directly bind to and inhibit mTOR signaling (3); The alteration of mitochondrial energetics may be involved in natural product induction of DDIT4, a novel upstream inhibitor of mTOR (4); Finally, activation of AMPK and inhibition of mTOR by natural products may be directly involved in the suppression of pro-inflammatory signaling such as that mediated by NF-κB (5). For mTOR, there is evidence that IKK interacts with mTORC1 to mediate the connection of mTORC1 to NF-κB, while for AMPK, the route to NF-κB is not presently clear. Citations of studies supporting these interactions are provided in text. Note that the tri-molecular complexes of IKK (IKKα, IKKβ, and IKKγ), and the bimolecular complex of NF-κB are simplified as single proteins. Moreover, only the mTORC1 and canonical NF-κB pathways are shown.
Nuclear factor kappa B (NF-κB) is the classical pro-inflammatory transcription factor that controls the expression of hundreds of pro-inflammatory cytokines and chemokines as well as other inflammatory and immune molecules such major histocompatibility complex proteins, transcription factors, and inhibitors of apoptosis [68]. NF-κB is not a single protein but rather a complex of homo and heterodimeric proteins [69]. Inactive NF-κB maintains a cytoplasmic localization by associating with inhibitory proteins of the IκB family [70]. Stimulation of cells via cytokines, PAMPs, or immune cell receptor activation leads to NF-κB activation by phosphorylation of IκB proteins, which leads to their degradation ( Figure 2). This allows NF-κB to enter the nucleus and exert its transcriptional effects. IκB phosphorylation is controlled by the multiprotein IκB-kinase (IKK) complex that contains 2 catalytic subunits, IKKα (IKK1) and IKKβ (IKK2), as well as the regulatory subunit IKKγ (a.k.a NEMO for NF-κB essential modifier). There are two pathways that lead to NF-κB activation: canonical and alternate. Activation of NF-κB via the canonical pathway involves phosphorylation of IκB-α by the IKKα/IKKβ/IKKγ complex, followed by degradation of IκB-α, permitting NF-κB (as p65/cRel and p50 dimers) to translocate to the nucleus (Figure 2). The alternate pathway involves another upstream molecule, NF-κB inducing kinase (NIK), which activates IKKα homodimers, which phosphorylate NF-κB (as a RelB and p100 dimer), leading to nuclear translocation [71,72].

Control of Immune and Inflammatory Responses by Natural Product Modulation of Metabolic Signaling by AMPK and mTOR
Galegine (related to guanidine), a natural product from the plant Galega officinalis is a potent AMPK activator used in the treatment of diabetes in the early part of the 20th century. Metformin (dimethylbiguanidine), also a potent AMPK activator, was subsequently developed as an anti-diabetic drug and is still used widely today. AMPK activation and the subsequent increased uptake of glucose, and reduction of fatty acid and triglyceride synthesis might be involved in how metformin improves insulin sensitivity. Interestingly, metformin activates AMPK by increasing the cellular ADP:ATP ratio, and this appears to arise through suppression of mitochondrial function [73].
In terms of AMPK's role in modulating immunity, a recent study found that metformin maintains anti-tumor CD8 T cells [74]. Specifically, metformin increased the number of CD8+ tumor infiltrating lymphocytes (TIL), which were found to be resistant to apoptosis and exhaustion, i.e., the cells were mainly PD-1 negative and Tim 3 positive, which are markers of tumor-rejecting effector memory cells. Thus, metformin supported the number and functionality of T cells in the tumor microenvironment, where T cells often become exhausted and therefore cannot act against tumor cells.
Rapamycin was discovered on the Pacific Island of Rapa Nui in 1975 [75]. Although originally discovered as a novel antifungal agent, experiments on mammalian cells revealed rapamycin interacts with protein FK-binding protein 12 (FKBP12) [61] to inhibit mTOR [76]. While primarily used as an immunosuppressant, recent data highlight the compound's ability to alter inflammation and immune responses in novel and exciting ways. For example, administration of rapamycin during immunization of mice with influenza virus completely changed the immune response in favorable ways [35]. Specifically, rapamycin altered the B cell response, resulting in a novel repertoire of antibodies that provided cross-strain protection against lethal influenza infection. A different mTOR inhibitor, RAD001 (Everolimus, PubChem Compound Identification number CID 6442177), was shown to increase the response to the influenza vaccine in elderly individuals. In this study, RAD001 treatment was associated with a reduction in the number of CD4 and CD8 T lymphocytes expressing the programmed death-1 (PD-1) receptor, which inhibits T cell signaling and is more highly expressed with age [77]. Furthermore, mTOR inhibition by rapamycin stimulates the production of memory CD8+ T cells in both murine and non-human primate infection and vaccination experiments [33]. These data suggest that modulation of cellular metabolism via mTOR is able to enhance immune responses to vaccine-or infection-induced challenges.
In addition to suppressing inflammation in many in vitro cell culture and in vivo animal models, natural products like curcumin, resveratrol and silymarin also share the function of regulating AMPK and mTOR. For example, resveratrol treatment of multiple cell types activates AMPK [43], although this effect, associated with decreased ATP levels, tends to be observed only with high concentrations of resveratrol (>50 µM) [78,79]. These data suggest that at high concentrations, resveratrol inhibits mitochondrial function, reducing cellular ATP levels, leading to AMPK activation ( Figure 2). Curcumin also appears to regulate mitochondrial function when the compound induces apoptosis of tumor cells [80]. Of note, resveratrol also regulates cyclic AMP levels, which can regulate AMPK activation by distinct mechanisms. The reader is referred to a recent review for further details [43].
Silibinin, which is a mixture of silybin A and silybin B (Figure 1), and is the major component of silymarin, suppresses cellular metabolism by reducing glycolysis and mitochondrial function [81][82][83][84]. We recently discovered that silymarin treatment also alters cellular metabolism by reducing glycolysis and mitochondrial respiration [81], and metabolomics analyses demonstrate that silymarin suppresses glycolytic, tricarboxylic acid (TCA) cycle, and amino acid metabolism [57]. Moreover, modulation of cellular metabolism by silymarin involves regulation of multiple metabolic pathways including activation of adenosine monophosphate kinase (AMPK) and suppression of mammalian target of rapamycin (mTOR) ( [57]; Figure 2). The activation of AMPK by silymarin may be indirect via increased AMP:ATP ratios that arise through suppression of glycolysis and mitochondrial function. However, since relatively high doses of silymarin were required to show AMPK activation, it is possible that other mechanisms are involved in AMPK activation by silymarin. In terms of silymarin suppression of mTOR, we showed that silymarin induces DDIT4/Redd1, a novel inhibitor of mTOR that is normally induced during hypoxia [85]. Using mouse knock out cells, suppression of mTOR by silymarin was found to be partially dependent on DDIT4 and AMPK [57] (Figure 2). Thus, in contrast to the direct suppression of mTORC1 by rapamycin binding to mTOR, silymarin may inhibit mTOR via at least 2 pathways: suppression of mitochondrial function to activate AMPK to suppress mTOR and direct induction of DDIT4 to suppress mTOR.
Energy sensing pathways like AMPK and mTOR communicate to inflammatory pathways.
For example, activation of AMPK by metformin and the synthetic compound 5-Aminoimidazole-4-carboxamide riboside (AICAR) inhibits TNF-induced activation of NF-κB dependent pro-inflammatory cytokine production [86] (Figure 2). As described above, silymarin activates AMPK and also inhibits NF-κB-dependent transcription. In murine cells expressing wild type AMPK, silymarin inhibited TNF-α induced NF-κB transcription, as expected. However, in cells with a double knockout of AMPKα1 and α2 subunits, silymarin inhibition of NF-κB transcription was attenuated [57]. Thus, silymarin suppression of inflammatory signaling through the NF-κB pathway was dependent on AMPK. Similar correlations (i.e., without causal connections) between pathways have been shown for curcumin and resveratrol, where natural product activation of AMPK was associated with inhibition of NF-κB phosphorylation [87,88]. Additional research is required to define the steps from AMPK activation to suppression of NF-κB transcriptional activity. mTOR and NF-κB pathways are also similarly connected. For example, activation of mTOR signaling by AKT leads to activation of NF-κB, and this appears to involve an association of IKK with Raptor within the mTORC1 complex [89]. Similarly, suppression of mTOR by curcumin, silymarin, and resveratrol correlates with suppression of NF-κB activity [57,90,91]. Thus, multiple natural products alter cellular metabolism and metabolic signaling pathways like AMPK and mTOR, and we propose that the modulation of these and likely other pathways culminate in the anti-inflammatory cellular phenotype observed with silymarin treatment.
Where do we go from here? There remain several pressing questions that are common to most if not all studies on natural products that have anti-inflammatory action.
What about the metabolism of natural products? Most herbal natural products are consumed orally as extracts, which, after metabolism and uptake in the intestine, get transported via the portal vein into the liver. Upon uptake by hepatocytes (the predominant cell type in the liver), herbal extracts are first modified by Phase I reactions that involve oxidation, reduction, and hydrolysis by cytochrome P450 enzymes. Phase II metabolic enzymes that perform conjugation reactions further alter natural products: transfer of methyl, sulfur, acetyl, or glucuronide moieties. Finally, in Phase III metabolism, compounds are further modified and excreted from the cell for eventual elimination from the body via the kidneys or gastrointestinal system. Thus, the adsorption, distribution, metabolism, and excretion (ADME) define the extent and duration of exposure of cells to natural products. While many herbal natural products are metabolized and excreted from the body within 2-8 h [42,92], there are many examples of beneficial drugs that have similarly short half-lives in vivo including ibuprofen, benadryl, and acetaminophen.
Considerations: Natural products like flavonoids possess biological activity that arises due to interactions with other, non-protein cellular components and/or inherent chemical properties. For example, natural products like curcumin and resveratrol were recently shown known to cause similar effects on membrane protein functions based on their interaction with the bilayer/solution interface of cell membranes [93]. Some natural products can also exhibit the phenomenon known as PAINS (pan assay interference compounds [94]), which typically involve reactive compounds that produce false readouts in in vitro assays, the majority of which are biochemical and non-cell based. For example, resveratrol was originally reported as an activator of Sirtuin 1, a protein involved in prolonging lifespan [95]. However, subsequent studies found that the activation in the screening assay was due to interaction between resveratrol and the fluorescent reporter substrate [96][97][98]. On the other hand, a recent study shows that the Rodgersinine Family of 1,4-benzodioxane neolignan polyphenolic natural products display anti-HCV activity [99]. This result is important since the rodgersinines lack the chromanone (i.e., flavonoid core), indicating that biological activity is not solely dependent on flavone moieties, which have been suggested to be responsible for PAINS activity. Thus, while PAINS activity can occur in non-cellular systems, many natural products reproducibly evoke metabolic and anti-inflammatory effects in biological systems. Regarding chemical properties, both synthetic drugs and natural products have been shown to produce aggregates or colloids, which often form at or below the concentration of compound which shows biological activity [100]. Colloid formation promotes the adsorption of soluble proteins to the surface of the colloid, which can lead to denaturation and nonspecific effects on cellular proteins. For in vitro enzymatic reactions with purified proteins, aggregate activity can be neutralized by addition of detergent [101]. Furthermore, non-toxic concentrations of detergent can be added to cells in culture, which has shown to effectively disperse colloids into monomers in solution [102]. Thus, considerations of PAINS activity, aggregate formation, and assay context (non-cell based versus bioassay) must be incorporated into any natural product research program that seeks to use natural products as tools to define the biology of inflammation. While many natural product studies have been focused on the protein interaction/signaling hypothesis, the ideal approach should also consider the role of natural product-membrane and natural product chemical properties in mediating these diverse biological effects. Collectively, the data described above suggest that modulating cellular metabolism via key checkpoints such as AMPK and mTOR provide novel opportunities for altering immune and inflammatory responses. While both AMPK and mTOR are highlighted in this review, many compounds derived from natural products perturb many pathways to achieve similar anti-inflammatory phenotypes. Therefore, as research continues to delve into controlling deregulated immune states, whether they be in the context of an ongoing, uncontrolled, chronic infection or when an infection has been cleared or suppressed but the immune system has not recovered, it is clear that one possible avenue to reigning in and quenching this deregulation is through modulating the metabolism of the target cells. When the diverse chemical properties of natural products are properly considered, these compounds represent a diverse class of novel and powerful tools for defining how to suppress inflammation in hepatocytes or non-parenchymal cells (in the context of HCV-induced liver disease) and in immune cells (that contribute to chronic inflammation during HIV infection).


extfieldsofstudy: Medicine Biology

text:
The frequency of CCR5 promoter polymorphisms and CCR5 Δ 32 mutation in Iranian populations.

Evidence showed that chemokines serve as pro-migratory factors for immune cells. CCL3, CCL4 and CCL5, as the main CC chemokines subfamily members, activate immune cells through binding to CC chemokine receptor 5 or CCR5. Macrophages, NK cells and T lymphocytes express CCR5 and thus, affected CCR5 expression or functions could be associated with altered immune responses. Deletion of 32 base pairs (Δ 32) in the exon 1 of the CCR5 gene, which is known as CCR5 Δ 32 mutation causes down regulation and malfunction of the molecule. Furthermore, it has been evidenced that three polymorphisms in the promoter region of CCR5 modulate its expression. Altered CCR5 expression in microbial infection and immune related diseases have been reported by several researchers but the role of CCR5 promoter polymorphisms and CCR5 Δ 32 mutation in Iranian patients suffering from these diseases are controversial. Due to the fact that Iranian people have different genetic backgrounds compared to other ethnics, hence, CCR5 promoter polymorphisms and CCR5 32 mutation association with the diseases may be different in Iranian patients. Therefore, this review addresses the most recent information regarding the prevalence as well as association of the mutation and polymorphisms in Iranian patients with microbial infection and immune related diseases as along with normal population.

Introduction
Cytotoxic T lymphocytes, NK cells, DCs and macrophages play important roles in immune responses against foreign antigens, PAMPs and DAMPs (1). Previous investigations evidenced that chemokines serve as recruiters of the abovementioned parts of immune system, through interaction with their receptors (2). Furthermore, CCR5 is a specific receptor for MIP-1α/CCL3, MIP-1β/CCL4 and RANTES/CCL5. It has also been reported that MCP-3/CCL7 bind CCR5 without producing any signal (3). Based on the potential intracellular signaling of CCR5/ligands axis which leads to activation of several transcription factors (see the next section), it seems that CCR5/ligands axis plays important roles in immune cell activation and migration response to microbes, microbial particles and self-antigens ( Figure 1) (4). It has been documented that deletion of 32 nucleotides from exon 1 of the CCR5 gene (known as the  32 mutation) causes a frame shift mutation at position 185 and in turn leads to dysfunction or declined expression of CCR5 receptor (4)(5)(6)(7)(8). The CCR5 Δ 32 mutation is localized on the second extracellular loop of the receptor, which is shown in Figure 1. Previous studies demonstrated that this mutation is prevalent and polymorphic in various ethnic populations (9,10). Therefore, evaluation of CCR5  32 mutation could lead us to a better understanding of the main mechanisms responsible for progression of chronic microbial infections and also immune related diseases including autoimmune diseases. Further-more, previous investigations revealed that the functional polymorphisms within promoter region of CCR5 gene result in alteration of CCR5 expression (11). Moreover, in comparison with other countries, Iran is a vast country with multiple ethnic populations with different genetic backgrounds, hence, CCR5 promoter polymorphisms and especially CCR5  32 mutation associations with the chronic microbial infections and immune related diseases may vary in Iranian patients. On the other side, several investigations identified that CCR5 serves as a co-receptor for HIV; hence, the genetic factors including CCR5 promoter polymorphisms and CCR5  32 mutations that affect CCR5 expression can be significantly associated with HIV restriction. Therefore, Iranian researchers have evaluated genetic variations in general population and specific patients. Therefore, the main aim of this review was to collect the available information on the status of CCR5  32 mutation and the polymorphisms within promoter region of CCR5 gene in Iranian general population and in patients suffering from some chronic microbial infections and immune related diseases.

Evidence acquisition
A literature search in various international and Iranian scientific databases namely Medline, PubMed, Web of Science, Web of Knowledge, Scopus, ProQuest, Google Scholar, Iranian Scientific Information Database (www.sid.ir) and Iranian Magazines Database (www.magiran.com) was conducted using appropriate combinations of medical terms for CCR5, mutation, polymorphisms and Iran, within the maximal date range until 2013. To retrieve more relevant and updated results, some advanced search measures such as Controlled Vocabulary (MeSH), Boolean operators, Truncations, limits and field searching were used.

Introduction to CCR5
CCR5 is defined as a seven transmembranespanning α-helix architecture protein belonging to GPCRs family and its gene is located on the short arm of chromosome 3 (3p21.31) (12). Evidence demonstrated that several pro-inflammatory cytokines (13,14) upregulate CCR5 expression in the immune cells via action of NF-κB transcription factor (15) and CREB pathways (16). CCR5 as a member of the heterotrimeric GPCRs family increases cytoplasmic Ca 2+ , in one side while, inhibits cAMP production in the other side and thus results in activation of crucial enzymes, including PI3-kinase, MAP kinases and other tyrosine kinase cascades such as FAK and Pyk2 (17). These molecules play important roles in immune cell migration and activation (17). Additionally, it has been  (18) suggested that CCR5/ligands axis is crucial for immune cells proliferation and expression of inflammatory cytokines through activating PKB, MAPK family, Rho GTPase and JAK/STAT (ERK1/2, p38, and SAPK/JNK) pathways ( Figure 1) (18,19).

The prevalence of CCR5  32 mutation among Iranian populations
Since CCR5 plays important roles in the induction of appropriate immune responses as well as HIV infection, several investigators have evaluated CCR5  32 mutation in Iranian populations (Table 1).  (20). To the best of our knowledge, the study by Gharagozloo and colleagues is the unique investigation assessing CCR5  32 mutation in Iranian general population, but several researchers have evaluated this mutation among Iranian individuals with specific diseases or conditions. Azmandian et al have evaluated the role of CCR5  32 mutation in both acute (AR) and DGF kidney transplant rejection in 100 donor/recipient pairs. Their results showed that CCR5  32 mutation was neither associated with AR nor DGF in Iranian donor and recipient kidney transplantation (21). In another study, Khademi and colleagues identified the frequency of CCR5  32 mutation in 156, 125 and 31 Iranian patients with malignant head and neck cancer, squamous cell carcinoma and salivary gland tumors, respectively, in comparison to 262 healthy controls (22). Interestingly, their results also revealed that CCR5  32 mutation was not prevalent in Iranian patients with malignant head and neck cancer, squamous cell carcinoma and salivary gland tumors as well as healthy controls (22). Following evaluation of 156 patients with late-onset Alzheimer's disease (AD) and 161 control subjects, Khoram et al reported that CCR5  32 mutation was uncommon in both AD patients and healthy controls (23). Another study on the Persian race (Shiraz, Iran) showed that CCR5  32 mutation was not associated with Behcet's disease (BD) compared to a large healthy control population (24). This study reported that CCR5  32 (380 cases) mutant allele rate was significantly higher in female patients than female control individuals (24); hence, the authors suggested that CCR5  32 mutation may be a potential risk factor for BD in Iranian women. Ardabili et al have assessed the mutation in 160 patients with AD and 163 healthy controls from north-west of Iran (province of Eastern Azerbaijan) and reported that CCR5  32 mutation was not associated with the disease in the Iranian population of the region (25). Our research team has also done several studies on Iranian patients. Accordingly, our previous studies on the 57 Iranian patients with occult HBV infection (OBI) revealed that none of patients carried the mutation, while 3% of healthy controls had heterozygotic form of CCR5  32 mutation (26). Our results from the Iranian patients with chronic HBV infection (CHI) have also demonstrated that neither CHI patients nor healthy controls had homozigotic form of CCR5  32 mutation (27). Interestingly, the study revealed that homozigotic form of CCR5  32 mutation was observed in 3 control subjects out of 300 (1%) Iranian healthy controls (27). Our study in the Iranian asthmatic patients also showed that CCR5  32 mutation was not prevalent in patients suffering from this disease (10). The results were also repeatedly reported when the mutation was assessed in the Iranian patients with type-2 diabetes with and without nephropathy (28) and multiple sclerosis (29). Interestingly, our results revealed that the mutation was rarely prevalent in Iranian healthy controls e.g. general population (10,28,29). In contrast to our and other studies, Shahbazi and colleagues reported that 37 out of 254 (15%) patients with multiple sclerosis (MS) and 8 out of 380 (2%) healthy controls were homozygous for the CCR5  32 mutation (30). Their results also demonstrated that the prevalence of heterozygotic forms of CCR5  32 mutation in MS patients and healthy controls were 6% and 13%, respectively (30). To explain this discrepancy between our result and that reported by Shahbazi et al, several background reasons could be proposed as follow: Iran is a vast country with different ethnic groups and the patients included in Shahbazi et al study were genetically different, since most of them were Turkmen people from northern Iran.
Based on the above studies done by Iranian researchers, except for Shahbazi et al (30), it appears that the rate of CCR5  32 mutation is low and irrelevant to infectious and immune-related diseases in Iranian populations.

Status of CCR5 polymorphisms in Iranians
Previous studies confirmed that variations in promoter region (-59029, -59353 and -2459) of CCR5 gene could influence the expression of this chemokine receptor. Therefore, probably a significant association between this polymorphism and immune-related and infectious diseases could be speculated. In contrast to CCR5  32 mutations; there is not enough information on the other polymorphisms in Iranian populations. To the best of our knowledge, there are only two published articles regarding the polymorphisms in Iranian populations.
First, Abdi et al evaluated 163 renal transplant recipients regarding outcome of renal transplantation and the polymorphism within -59029 region (31). Their results demonstrated that polymorphism was not associated with the outcome of the transplantation (31). Second, Omrani et al conversely reported that the polymorphic region of -59029 within CCR5 was significantly associated with the survival rate of renal allografts (32).
According to the results of the most recent reports which are considered here, it seems that the potential effects of polymorphisms within promoter region of CCR5 on the outcome of immune-related conditions in Iranians is controversial, because the data of the two studies was inconsistent and their sample sizes were too low to give a definite conclusion. Additionally, according to the facts that polymorphisms within promoter region of CCR5 may be associated with its expression and that the ethnicity is an important factor, which alters the effects of genetic variations, it seems that the effects of genetic variations on the expression of CCR5 in Iranian populations need further evaluation. Moreover, due to the fact that CCR5 plays important roles in migration and activation of immune cells in the fight against microbes, it may be hypothesized that the polymorphisms within CCR5 gene may be associated with several immune-related conditions in Iranian patients. More investigations would improve our knowledge in this field.

Conclusion
Based on the present data it is likely that CCR5  32 mutations is not prevalent in Iranian normal population, and accordingly, studies were unable to find out its relationship with infectious and immunerelated diseases the population. Therefore, it is reasonable to conclude that impaired immune responses during chronic infectious diseases in Iranian patients are not related to CCR5  32 mutation. It could also be concluded that uncontrolled immune responses during immune-related diseases including autoimmune responses and inflammation during diseases (e.g. graft rejection and asthma) are not related to CCR5  32 mutation in Iranians. Additionally, because CCR5 is a HIV co-receptor, high prevalence of CCR5  32 mutations in the populations can lead to decreased HIV infections. Therefore, according to the reviewed studies, it may be concluded that Iranian populations are at a high risk for HIV infection and susceptible to be affected by a serious course of the disease. Due to the paucity of studies on the polymorphisms of promoter region of CCR5 within Iranian populations, it seems to be impossible to reach a clear conclusion concerning the relationship between polymorphisms and infectious or immune related diseases in Iranian populations and more studies are required.


s2fieldsofstudy: Biology Medicine

text:
α-Smooth Muscle Actin and ACTA2 Gene Expressions in Vasculopathies

α-smooth muscle actin, encoded by ACTA2 gene, is an isoform of the vascular smooth muscle actins, typically expressed in the vascular smooth muscle cells contributing to vascular motility and contraction. ACTA2 gene mutations cause a diversity of diffuse vasculopathies such as thoracic aortic aneurysms and dissections as well as occlusive vascular diseases, including premature coronary artery disease and ischemic stroke. Dynamics of differentiation-specific α-smooth muscle actin in arterial smooth muscle cells and proliferation of the proteins have been well described. Although a variety of research works have been undertaken in terms of modifications of α-smooth muscle actin and mutations of ACTA2 gene and myosin, the underlying mechanisms towards the pathological processes by way of gene mutations are yet to be clarified. The purpose of the present article is to describe the phenotypes of α-smooth muscle actin and implications of ACTA2 mutations in vasculopathies in order to enhance the understanding of potential mechanisms of aortic and coronary disorders.

INTRODUCTION
The pathogenesis of vasculopathies, including aortic and coronary disorders, are uncertain, even though they have been under constant investigation. The potential roles that α-smooth muscle actin (SMA) might play in the development of vasculopathies have drawn much attention. There are four distinct variants of actins: two vascular SMC (α-SM and γ-SM)-specific, and two cytoplasmic actins (β-NM and γ-NM actins) in eukaryotic cells. α-SMA is located primarily in the microfilament bundles of vascular SMCs and exerts contractile functions. It shows a close relationship with arterial tones and the activation of myofibroblasts [4] . The forceful contractile property of myofibroblasts is attributable to the stress fibers dependant on α-SMA [5] . SMC components and extracellular matrix (collagen and elastin) are determinants of the contractility, distensibility and elasticity of the aortic wall. Extracellular matrix disruption or imbalance of the extracellular matrix components leads to stiffness of the aortic wall and further development of TAAD [6] . In experimental homozygous α-SMA knock-out mice, deficits of vascular contractility and reduced basal blood pressure were noted, implicating the important role of α-SMA in maintaining vascular tone [7] .

Clinical investigations
Normal aortic media predominantly contains α-SMApositive SMCs with homogeneous elastic laminae [8] whereas degeneration is marked by disruptions of α-SMA-positive SMCs [9] . Vascular occlusions are pathologically characterized by medial proliferation of SMCs or enhanced proteoglycan accumulation along with fragmentation and loss of elastic fibers [10] . The α-SMA mutations in TAAD hinder the binding affinity between the monomer and the ligands, thereby generating more unpolymerized monomers [10] . The aortic media of abdominal aortic aneurysm displays fewer α-SMApositive cells, which are secondary to extensive aortic wall structural damages. Medial SMC density may decrease by 79.1% in abdominal aortic aneurysmal tissue in comparison with that of normal control [8] . Aortic aneurysmal wall shows chronic infiltration of the inflammatory cells, degradation of the extracellular matrix, and apoptosis of SMCs [11] . In addition, macrophages express chemoattractant proteins and matrix metalloproteinases in the atherosclerotic plaques [12] similar to aortic aneurysm, in particular in the aneurysm necks for the stent graft anchoring.
An immunohistological study has shown more α-SMA expressions in coronary erosion lesions than in stable plaques or mildly stenotic plaques [13] . Acute arterial injury was found to be associated with changes in glycosaminoglycans of the extracellular matrix ahead of those of SMC phenotype, whereby matrix glycosaminoglycans are substituted by SMCs rapidly during the modification process where neointima does not generate enough heparin sulphates [14] .
Coronary stenting may cause concentric narrowing of the lumen due to neointimal formation, in which the extracellular matrix increases and number of SMCs decreases over time. The neointima is composed of three-layered and circular-arranged α-SMA-positive cells. Immunohistochemical studies of the common carotid artery with stent deployment showed in-stent thrombus around the stent struts with the neointima composed mainly of α-SMA-positive cells within type I collagen-positive matrix [15] . Comparative studies on the abdominal aortopathies revealed that α-SMA-positive SMCs were predominant in the aortic media of the occlusive abdominal aorta and the aneurysmal neck; however, SMCs were disrupted and disarrayed in the aneurysmal body, in which much fewer SMCs were present than in the occlusive aorta and the aneurysmal neck [16] .
Transforming growth factor (TGF)-β-antagonist reduces α-SMA expression, and treatment with TGF-β may upregulate α-SMA expression. TGF-β 1 -stimulated adventitial fibroblasts in TAAD patients attenuated extracellular matrix production and SMC transformation [6] . In the aneurysmal neck, there was increased TGF-β 1 expression and reduced SMC density, and even more upregulated TGF-β 1 expression and much more reduced SMC density were found in the aneurysmal body in comparison with those of the occlusive aorta. Therefore, a link between TGF-β 1 overexpression and reduced SMC density as a result of SMC apoptosis and attenuated SMC proliferative capability may characterize the aneurysmal formation [16] .

Genetic aspects of ACTA2 mutations
ACTA2, the encoding gene of α-SMA, located at 10q22-q24 and composed of eight coding exons, is a major contractile component of the arterial SMCs [2] . ACTA2 gene defects that cause familial TAAD type 6 have been reported [17] . So far, more than 20 different missense mutations of ACTA2 have been found to be associated with aortopathies [18] . ACTA2 mutations may show reduced penetrance and variable expressivity due to a dominant negative mechanism [19] . Heterozygous ACTA2 mutations may lead to impaired SMCs, showing pathological hyperplasia and disarray due to medial degeneration, vascular wall remodeling, increased wall stiffness, and aortic dilation [20,21] . It may indicate a possible increased risk of stroke and CAD rather than hypertrophy [2] , and it may be a causative etiology of multiple tiny aneurysms of the cerebral arteries, responsible for pediatric acute ischemic stroke [22] . The relationship among SMA, ACTA2 gene, and contractile property in vasculopathies is listed in Figure 1.

Basic Research for Possible Mechanisms
α-SMA is important for contractile functions of the developing heart at an early embryonic stage. Research on avian epimyocardial cells demonstrated α-SMA mRNAs were mainly detected in conus arteriosus and the ventral aorta before Hamburger-Hamilton stage (HH) 12. In subsequent stages, the α-SMA mRNA was apparently confined to SMCs of the vascular system. The contractive properties of SMCs can be suspended while secretory functions prevail by exertions of mitogens, including platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), and extracellular matrix components such as collagen, elastin and proteoglycans that contribute to SMC differentiation and proliferation [3] . Hence, α-SMA expression inversely correlates with cell proliferation, but directly correlates with contractile efficiency of SMCs [46] .
Fibronectin and laminin are expressed typically in the arterial media during fetal and adult life in different forms, and a paradoxical transformation of the proteins from adult to fetal variants takes place during the vascular pathological processes. Vascular SMCs are also implicated in vessel remodeling, in either physiological or pathological conditions, such as pregnancy, exercise, or vascular injury [47] . During the remodeling process after vascular injury, the SMCs resume a contractile phenotype, with a cytoplasm dominated by α-actin filaments until the final extent of neointimal formation. The phenotype of cultured rabbit SMCs can be modified by PDGF-BB, resulting in faster growth, but SMCs preincubated with conditioned medium of macrophages plus anti-PDGF antibody did not grow faster. Human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Proliferation of arterial  p.Arg179His TAAD [23] G304R (in the vicinity of the ATP-binding site) TAAD, isolated [20] p.G152_T205del (c.616+1G>T), p.R212Q & p.R149C TAAD, familial [17] p.Y145C TAAD, sporadic & young-onset [17] c.76G>T; p.Asp26Tyr TAAD, non-syndromic familial [19] R39C (the DNAse-I-binding loop within subdomain 2 of α-smooth muscle actin) Aortic aneurysm, recurrent [20] M49V SMCs was strongly stimulated by PDGF-AA, but venous SMCs showed no proliferative response to PDGF-AA, demonstrating instead a significantly greater proliferative response to PDGF-BB than arterial SMCs [48] . In cultured rat aortic SMCs, enhanced proliferation, elongated cellular deformity as well as a fluctuation pattern of growth under the exertion of TGF-β 1 were present [49] .
During angiogenesis and vascular remodeling due to restenosis and atherosclerosis, SMCs regulate endothelial cell quiescence and angiogenic responsiveness to cytokines. A series of studies revealed TGF-β stimulates the matrix production of vascular SMCs, including type I collagen. The enhanced α-SMA expression induced by TGF-β 1 increases the contraction of collagen gel mediated by bovine corneal fibroblasts in a dosedependent manner. Integrin α 2 β 1 , one of the collagen-binding receptors, binds fibril-forming collagens, collagens I, II, III and X. It joins the process of TGF-β 1 -induced α-SMA production and regulation, capable of generating intracellular tension.
α-SMA disruption results in SMC hyperplasia via certain cellular pathways involving FAK, p53 and PDGF receptor-β, contributing to vascular occlusive disorders in patients with ACTA2 missense mutations. This is supported by the experiments in ACTA2 knockout mice, in which decreased aortic contractility was displayed [50] . The structural heterogeneity of genomic DNA encoding the chicken α-SMA gene in 3' untranslated regions is of alternative polyadenylation signals, but with uncertain functional implications. Moreover, actin isoform synthesis after balloon induced endothelial denudation warrants further investigations in order to be employed early in clinical practice.

CONCLUSION
ACTA2 mutations are associated with structural disruption and functional impairment of contractile proteins, and predispose to a variety of diffuse vascular diseases including TAAD, CAD, ischemic strokes, and Moyamoya disease. Vascular SMCs are also implicated in vascular remodeling in both physiological and pathological conditions. Regulation of differentiation and proliferation of SMCs may control the expression of genes encoding for proteins responsible for the contractile function of the SMCs. In the process of vascular remodeling, contractive properties of SMCs can be suspended while secretory functions prevail by mitogen impacts. Research on α-SMA and ACTA2 mutations is imperative for understanding the pathogenesis and determining the pertinent management strategies of vasculopathies. Further in-depth studies of causative genes of ACTA2 mutations may largely facilitate the diagnosis and treatment of the underlying disorders.