Research ArticleModulation of Corneal FAKPI3KAkt SignalingExpression and of Metalloproteinase-2 and Metalloproteinase-9during the Development of Herpes Simplex Keratitis

Lan Ke Yanning Yang Jing wei Li BoWang YujingWang Wanju Yang and Jiangbo Yan

Department of Ophthalmology Renmin Hospital of Wuhan University Wuhan Hubei China

Correspondence should be addressed to Yanning Yang ophyyn163com

Received 15 December 2018 Revised 26 January 2019 Accepted 7 February 2019 Published 2 April 2019

Academic Editor SivagnanamThamilselvan

Copyright copy 2019 Lan Ke et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To observe the expression of MMP-2 and MMP-9 and of the FAKPI3KAkt signaling pathway in HSK Fifty BALBc mice wereinfected to establish the model and killed on days 0 2 7 14 and 28 The cornea samples were prepared respectively Slit lampexamination immunofluorescence staining reverse transcription PCR andWestern blot were used to detect the index After HSV-1 infection different degrees of epithelial or stromal damage and corneal opacity were observed Immunofluorescence stainingshowed that the expressions of MMP-2 and MMP-9 at different levels of corneal tissue were observed on the 0d 2d 7d 14d and28d Compared with 0d the relative expression levels of MMP-2 and MMP-9 mRNA at 2d 7d 14d and 28d were significantlyincreased (all Plt 005) Compared with 14d the relative expression ofMMP-2 andMMP-9mRNA decreased on the 2d 7d and 28d(all Plt 005) Western blot showed that the protein expressions of p-FAK p-PI3K p-Akt MMP-2 and MMP-9 at 2d 14d and 28dwere all significantly higher than 0d (all Plt 005) At 14 d the expressions of p-FAK p-PI3K p-Akt and MMP-2 were significantlyhigher than those at 2d 7d and 28d (all Plt 005) The protein expression of FAK PI3K and Akt in corneal of mice in each timeperiod had no significant (all Pgt 005) These data suggest that FAKPI3KAkt signaling pathway and MMP-2 and MMP-9 may beinvolved in the development of HSK

1 Introduction

Herpes stromal keratitis (HSK) is a disease that developsdue to infection of the cornea with herpes simplex type1 HSK is characterized by recurrence and its repeatedattacks can cause different degrees of damage to the cornealtissue especially to the stroma resulting in severe patho-logical damage such as corneal melting neovasculariza-tion secondary glaucoma ulcer perforation and cornealscarring [1 2] The incidence of HSK is approximately15 million per year of which about 40000 people cancause severe visual impairment or corneal blindness eachyear [3] However the pathogenesis of HSK has not yetbeen fully clarified Our previous study found that matrixmetalloproteinase-2 andmetalloproteinase-9 (MMP-2MMP-9) play an important role in the development of HSK

[4] However studies have not yet clearly defined whichpathway leads to the secretion and expression of MMP-2 andMMP-9 Focal adhesional kinase (FAK)phosphoinositide-3-kinase (PI3K)protein kinase B (AKTPKB) pathway is animportant signal transduction pathway which can regulate avariety of important biological signaling PI3KAkt signalingpathway has biological effects in ocular diseases such asthe occurrence and development of postcataract choroidalneovascularization and retinopathy of prematurity [5ndash7]Recently our group has found that FAKPI3KAkt signalingpathway plays an important role in keratocytes infected withHSV-1 Therefore in this study the HSV-1 KOS strain wasused to induce the mice model of HSK The expression ofrelated factors mRNA and protein in the cornea of HSKwas detected by immunofluorescence staining RT-PCR andWestern blot We aim to study whether the FAKPI3KAkt

HindawiBioMed Research InternationalVolume 2019 Article ID 4143981 8 pageshttpsdoiorg10115520194143981

2 BioMed Research International

signaling pathway and MMP-2 and MMP-9 are involved inthe development of HSK in the cornea to facilitate the studyof the mechanism of HSK in the future

2 Materials and Methods

21 Materials

211 Animals Fifty inbred male BALBc mice of SPF grade6sim8 weeks of age and weighing 20sim25 g were provided bytheExperimentalAnimal ResearchCenter ofHubei ProvinceThe license number was SCXK (E) 2015-0018Mice were bredin a temperature-controlled room at about 22sim25∘Cwith 12 hdaynight cycles The entire study complied with the ARVOstatement on the use of animals in research and was approvedby the Ethics Committee of the Renmin Hospital of WuhanUniversity

212 Main Reagents and Instruments These included rab-bit anti-mouse P-FAK antibody (ab39967 abcam) rab-bit anti-mouse FAK antibody (ab61113 abcam) rabbitanti-mouse P-PI3K antibody (4228 CST) rabbit anti-mouse PI3K antibody (4292 CST) rabbit anti-mouseP-AKT Antibody (4060CST) rabbit anti-mouse AKTantibody (4691CST) rabbit anti-mouse MMP-2 Anti-body (ab92536abcam) mouse anti-mouseMMP-9 Antibody(ab58803 Abcam) FITC labeled goat anti-rabbit IgG (AS-1110 Aspen) CY3 labeled goat anti-mouse IgG (AS-1111Aspen) trizol reagent (Invitrogen USA) PrimeScriptRTKit with gDNA Eraser SYBR Premix Ex Taq kit (TaKaRa)slicer (Shanghai Leica Instruments Co Ltd) ordinaryoptical microscope Inverted microscope imaging system(OLYMPUS) StepOne Real-Time PCR (Life technologies)

22 Methods

221 Establishment of HSK Animal Model at 0d The HSV-1KOS strainwas kindly provided by theWuhanVirusResearchInstitute The virus HSV-1 KOS strain had a titer of 2 x 107pfuml before use The virus-producing cells were Vero cells(African green monkey kidney fibroblasts) Vero cells wereobtained from American Type Culture Collection (ATCC)Mice were anesthetized by intraperitoneally injecting 5chloral hydrate at a dose of 6 mlkg Under the microscopewe scratch the mouse corneal epithelium with the ldquordquo markon the back of the blade of the No 5 surgical blade (thescratching depth needs to break through the cornea elasticlayer) Subsequently 5 120583l of a solution containing HSV-1(KOS strain 105 spot forming units (pfu)) was spotted andretained for 10s on the cornea and the eyelids were closedand massaged for 30s to allow the virus fluid to sufficientlycontact the cornea After surgery 05 gentamicin eye dropswere used to avoid bacterial infection All experiments wereconducted at the Center for Animal Experiment of WuhanUniversity

222 Slit Lamp Examination The severity of epithelial orstromal damage and corneal opacity was assessed under the

microscope at 0d 2d 7d 14d and 28d after infection withHSV-1 The evaluation index was based on the method ofHeiligenhaus A[8] (Table 1)

223 Immunofluorescence Staining According to the ran-dom number table method mice were sacrificed by cervicaldislocation 0d 2d 7d 14d and 28d after infection with HSV-1 (n=2) The tissue was fixed with 4 paraformaldehydeembedded in paraffin and sectioned at 10 120583m thicknessParaffin sections were subjected to baking dewaxing andantigen retrieval After washing with PBS for 3 timesblocking with 5 BSA removal of BSA solution dilutedMMP-2 primary antibody (1150) and MMP-9 primary anti-body (1200) working solution were added and incubatedovernight at 4∘C After washing 3 times with PBS diluteMMP-2 secondary antibody (150) and MMP-9 secondaryantibody (150) working solution were respectively addedand incubated at 37∘C for 50 min After washing with PBS 3times appropriate amount of DAPI dye solution was addedIncubation was done in the dark at room temperature for 5minutes Finally after washing 3 times in PBS an appropriateamount of antifluorescence quencher was placed on the slideand the expression of MMP-2 and MMP-9 was observedunder the fluorescence microscope

224 RT-PCR According to the random number tablemethod mice were sacrificed by cervical dislocation 0d2d 7d 14d and 28d after infection with HSV-1 (n=2) Asmall piece of tissue was placed in a solution containing1 ml TRIzol Reagent and ground in a homogenizer Sub-sequently procedures for extracting RNA measuring RNAconcentration reverse transcription of cDNA and PCRamplification are routinely performed cDNA synthesis wasperformed using the PrimeScriptRT reagent kit with gDNAEraser (TaKaRa) and PCR amplification was performed onthe StepOne Real-Time Cycler (Life technologies) using 3replicate wells per sample using SYBR Premix Ex Taq kit(TaKaRa) performed The molecular primer sequence wasdesigned as MMP-2 upstream primer 51015840- TCAACGGTC-GGGAATACAGC-31015840 downstream primer 51015840- TAGCTG-TTGTAGGAGGTGCCCT-31015840 amplified fragment length136 MMP-9 upstream primer 51015840- TAAGGGTACAGCCTG-TTCCTGGT-31015840 downstream primer 51015840- TCTGGATGC-CGTCTATGTCGTCT-31015840 and amplified fragment length149 The experiment was repeated three times The obtainedCT value was converted by 2-ΔΔCT and represents the relativeexpression level of mRNA

225 Western Blot According to the random number tablemethod mice were sacrificed by cervical dislocation 0d2d 7d 14d and 28d after infection with HSV-1 (n=6) Themouse corneal tissue blocks were placed in 04 ml tissuelysis buffer lysed on ice and subjected to rapid electricalhomogenization Repeat asmany times as possible to crumplethe tissue The centrifuge was adjusted to 12000 rpm at4∘C and centrifuged for approximately 10 minutes Afterthe supernatant was sampled for protein concentration theprotein concentration of the sample was adjusted After

BioMed Research International 3

Table 1 Evaluate different indexes of cornea in HSV-1 infected mice

Score Epithelial or stromal damage Corneal opacity0 - Transparent1+ lt25 Slightly turbid2+ ge25 lt50 Moderately turbid visible iris3+ ge50 lt75 Severe turbidity pupillary margin can be judged4+ ge75 Complete opacity invisible posterior features

Table 2 Evaluate different indexes of cornea in HSV-1 infected mice (Xplusmns)

Indexes 0d 2d 7d 14d 28dEpithelial or stromal damage 000plusmn000 103plusmn016a 100plusmn037a 150plusmn069a 160plusmn084a

Corneal opacity 000plusmn000 103plusmn016a 103plusmn032a 220plusmn041a 250plusmn071a

Note Compared with 0d aP lt 005 (t test)

centrifugation 12 gel electrophoresis was performed Afterelectrotransfer of PVDFmembranes there was blocking with5 skim milk blocking solution (TBST) anti-FAK primaryantibody (1500) p-FAK primary antibody (1500) PI3K pri-mary antibody (14000) p-PI3K primary antibody (11000)Akt primary antibody (12000) p-Akt primary antibody(11000) MMP-2 primary antibody (11000) MMP-9 primaryantibody (11000) ) Incubate overnight at 4∘C thenwash addthe corresponding secondary antibody at 4∘C overnight anddevelop with colorThe experiment was repeated three timesThe results obtained were density scanned and gray-scaleanalysis was performed using Gel-proAnalyzer 45 imageanalysis software

23 StatisticalMethod SPSS 220 statistical softwarewas usedto analyze the experimental data All indicators are expressedas mean plusmn standard deviation (XplusmnS) The t-test was usedto compare the differences in the scores of different cornealindexes at different times One-way analysis of variancewas used to compare the differences in values obtained atdifferent time points Multiple comparisons were performedusing Dunnettrsquos test or LSD-t test Plt 005 was consideredstatistically significant

3 Results

31 Clinical Course ofHSK andEvaluation of Different CornealIndexes We found that after infected with HSV-1 differentdegrees of epithelial or stromal damage corneal opacityandor corneal neovascularization were observed on the 2d7d 14d and 28d First inflammation in corneal epithelialappeared in 1 to 3 days after corneal infection in normalBALBc mice The corneal epithelium showed a punctatedefect or a map-like defect or a dendritic defect in thecornea Secondly stroma gradually developed edema afterthe corneal epithelium healed after 4-7 days A few severecases were even accompanied by corneal neovascularizationcorneal ulcers andor corneal neovascularization Cornealulcers and corneal neovascularization can be seen around14 days and corneal ulcer perforation can occur in severecases Then about 28 days the inflammation of the cornea

Figure 1 Cornea image of typical BALBc mouse infected withHSV-1

was reduced and corneal neovascularization became coarseand dark (Figure 1) In Table 2 we can find that with thetime of corneal infection prolonged the score of cornealopacity gradually increased Compared with 0d the score ofepithelial or stromal damage and corneal opacity increased in2d 7d 14d and 28d after infection and the differences werestatistically significant (Plt 005)

32 Expression and Distribution of MMP-2 and MMP-9Protein in Corneal Tissue at Different Time Points in HSKMouseModel Immunofluorescence staining showed that theexpression of MMP-2 and MMP-9 was observed in thecorneal tissue of HSK at 0d 2d 7d 14d and 28d (Figure 2) Inthe corneal tissue the expression ofMMP-2was located in theepithelium and the expression of MMP-9 was located in thestroma of the cornea After 2 days the expression of MMP-2in the corneal tissuewas located in the corneal epitheliumanddiffused in the corneal stromal layer while the expression ofMMP-9 was located in the corneal stroma Compared with0d the expression of MMP-2 and MMP-9 was stronger in

4 BioMed Research International

MMP-2 MMP-9 DAPI merge

0d

2d

7d

14d

28d

Figure 2 Expression of MMP-2 and MMP-9 protein in corneal tissue of BALBc mice after 0 2 7 14 and 28d of infection with HSV-1 Theexpression of MMP-2 in the epithelium and the expression of MMP-9 in the stroma showed diffuse fluorescence after 0d of infection Thefluorescence intensity of MMP-2 and MMP-9 at 2d was higher than that of 0d and 7d The fluorescence intensity of MMP-2 and MMP-9reached summit at 14d After 28d of infection expressions of MMP-2 and MMP-9 protein were weaker than that of 14d

0d 2d 7d 14d 28d0d 2d 7d 14d 28d

ab

ab

abab

ab

ab

aa

The r

elativ

e exp

ress

ion

of M

MP-

2 m

RNA 4

3

2

1

0 The r

elativ

e exp

ress

ion

of M

MP-

9m

RNA 3

2

1

0

Figure 3 The relative expression of MMP-2 and MMP-9 mRNA in cornea at each time point Note aP lt 005 compared with 0d bP lt 005compared with 14d (One-way ANOVA Dunnettrsquos t test) MMP Matrix Metalloproteinases

2d After 7 days of infection with HSV-1 the expression ofMMP-2 and MMP-9 in corneal tissue was stronger than thatof 0d but lower than that of 2d After 14 days the expressionof MMP-2 and MMP-9 in the corneal tissue was strongerthan that of 0d 2d and 7d and the intensity of fluorescenceexpression peaked At 28d the expression of MMP-2 andMMP-9 in the corneal tissue was lower than that of 14d butit was stronger than that of 0d

33 Expression Changes of MMP-2 and MMP-9 mRNA inCorneal Tissue at Different Time Points in HSK Mouse ModelThe results of RT-PCR showed that the mRNA levels ofMMP-2 and MMP-9 increased at 2 days after HSV infectiondecreased after 7 days then peaked again at 14 days andthen decreased at 28 days (Figure 3) The relative expressionlevels of MMP-2 mRNA at 0 d 2 d 7 d 14 d and 28 d afterinfection in HSK mouse model were 100plusmn000 159plusmn004

BioMed Research International 5

0d 2d 7d 14d 28d

119 kDa

119 kDa

85 kDa

85 kDa

60 kDa

60 kDa

74 kDa

92 kDa

43 kDa

P-FAK

FAK

P-PI3K

PI3K

P-AKT

AKT

MMP-2

MMP-9

-Actin

Figure 4 Western Blot results of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 protein expression in mouse cornea at eachtime point

ab

ab

ab

ab

ab ab

ababab

ab

ab

a

a a

a

a

aa

b

b

0d 2d 7d 14d 28d

p-FAK

FAK

p-PI3K

PI3K

p-Akt

AktMMP-2MMP-9

The r

elativ

e exp

ress

ion

of p

rote

in

10

08

06

04

02

00

Figure 5 Relative expression of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 in cornea at each time point Note aP lt 005compared with 0d bP lt 005 compared with 14d (one-way ANOVA LSD-t test)

202plusmn015 270plusmn026 and 199plusmn186 respectively The relativeexpression levels ofMMP-9mRNAwere 100plusmn000 151plusmn005181plusmn007 230plusmn015 and 194plusmn171 Compared with 0d therelative expression levels of MMP-2 and MMP-9 mRNAwere significantly increased at 2d 7d 14d and 28d andthe differences were statistically significant (all Plt 005)Compared with 14d the relative expression levels of MMP-2 and MMP-9 mRNA decreased at 2d 7d and 28d and thedifferences were statistically significant (all Plt 005) (Table 3)

34 Expression of p-FAK FAK p-PI3K PI3K p-Akt AktMMP-2 and MMP-9 in Corneal Tissue at Different TimePoints in HSK Mouse Model Western blot analysis showedthat the expression of p-FAK p-PI3K p-Akt MMP-2 andMMP-9 was increased in corneal tissue at 2 days afterinfection and decreased from 7d to 2d 14d and 28d Itrose again and peaked at 14d (Figures 4 and 5) The proteinexpressions of p-FAK p-PI3K p-Akt MMP-2 and MMP-9at 2d 14d and 28d were all significantly higher than those at

6 BioMed Research International

Table 3 The relative expression of MMP-2 and MMP-9 mRNA in the cornea at each time point (Xplusmns)

Time point MMP-2 MMP-90d 100plusmn000 100plusmn0002d 202plusmn015ab 181plusmn007ab

7d 159plusmn004ab 151plusmn005ab

14d 270plusmn026a 230plusmn015a

28d 199plusmn015ab 194plusmn002ab

F 5069 12216P lt005 lt005NoteaP lt 005 compared with 0d bP lt 005 compared with 14d (One-way ANOVA Dunnettrsquos t test)MMP matrix metalloproteinases

0d (Plt 005) The expression of p-Akt MMP-2 and MMP-9at 7 days after infection was significantly higher than that at 0days (Plt 005) At 14 days the expression of p-FAK p-PI3Kp-Akt andMMP-2 protein was higher than that at 2d 7d and28d and the difference was statistically significant (Plt 005)The expression ofMMP-9 protein at 7d was lower than that at14d and the difference was statistically significant (Plt 005)There was no significant difference in the expression levelsof FAK PI3K and Akt between tissues at all time points (Pgt005) (Table 4)

4 Discussion

HSK is one of the leading eye diseases in the world leadingto corneal blindness [3] Recurrent HSK will undoubtedlyaggravate the deterioration of the disease and eventually leadto blinding outcomes Although the research on molecularmechanisms related to the formation of HSK is increasingand deepening the pathogenesis of HSK is complex andits pathogenesis is still not fully understood There is noclinically satisfactory drug treatment that can prevent it fromrecurring or prevent it from developing into a bad outcomeTherefore it is imperative to explore drug treatment withmultiple pathways multiple targets and multiple therapeuticeffects

Recent research indicates that FAK not only participatesin the regulation of cell proliferation apoptosis migrationinvasion metastasis and adhesion but also participates in theregulation of the expression and activity ofMMPs and TIMPsthrough multiple signaling pathways [9ndash12] At the sametime our previous study found that there were two peaksin the phosphorylation of FAK in the corneal epithelium ofHSV-1-infected mouse corneal models The phosphorylationlevels of FAK increased on the 2d 14d and 28d afterinfection and decreased on the 7d compared with 2d [13]The expression pattern of MMP-2 andMMP-9 in the cornealmodel of HSV-1-infected mice in this study is very similarto our previous studies [4] In addition we also found thatTNF-120572 can increase the expression and secretion of MMP-2 and MMP-9 through the FAKERK signaling pathway invitro experiments [14] This further suggests the role of theFAK signaling pathway in HSK

PI3KAkt is an important signaling pathway downstreamof FAK that regulates many important biological signaling

pathways It has been reported that on the one handthe herpes virus can enhance its function of replicationtranscription and translation by regulating the PI3KAktsignaling pathwayOn the other hand the PI3KAkt signalingpathway can be used to facilitate virus entry replicationdelay and activation [15ndash17] Activation of PI3KAkt sig-naling can occur at multiple stages of the herpes virus lifecycle [16 17] Cheshenko reported that HSV can activateand release intracellular calcium and phosphorylate FAKvia the integrin signaling pathway which activates the AKTsignaling pathway and provides suitable conditions for virusentry into cells [18] Therefore we hypothesized that theFAKPI3KAkt signaling pathway and MMP-2 and MMP-9 may be involved in the development of HSK in thecornea

In this study we first studied the patterns of FAK p-FAKPI3K p-PI3K Akt and p-Akt in the FAKPI3KAkt signalingpathway and MMP-2 and MMP-9 in the HSK mouse modelBy Western blot we observed that the levels of p-FAK p-PI3K and p-Akt were upregulated on the 2d 7d 14d and28d after infection The upregulated level on the 7th daydecreased compared with that on the 2d day and reachedthe maximum on the 14d day At the same time the changesin the levels of MMP-2 and MMP-9 protein were similar tothose of p-FAK p-PI3K and p-Akt This is similar to ourprevious study [4] suggesting a potential link between theFAKPI3KAkt signaling pathway and theMMP-2 andMMP-9 In addition we have further demonstrated by RT-PCRand Immunofluorescence staining that the expression andactivation of MMP-2 and MMP-9 in the mouse cornea atthe early stage of HSV-1 infection play an active role in thedevelopment of HSK To further elucidate the relationshipbetween the FAKPI3KAkt signaling pathway and the HSKwe plan to use the inhibitor

In summary our results provide in vivo evidence thatFAKPI3KAkt signaling pathway and MMP-2 and MMP-9may be involved in the development of HSK The discoveryof the association between FAKPI3KAkt signaling pathwayMMP-2 and MMP-9 and HSK may facilitate the study ofthe mechanism of HSK and studies of the FAKPI3KAKTsignaling pathway may provide new targets for the treatmentofHSKHowever in order to show the regulatory relationshipbetween FAKPI3KAkt signaling pathway and MMP-2 andMMP-9 further research is necessary

BioMed Research International 7

Table4Com

paris

onof

relativee

xpressionlevelsof

p-FA

KFA

Kp-PI3K

PI3K

p-AktA

ktM

MP-2andMMP-9in

cornea

ateach

timep

oint

(Xplusmns)

Timep

oint

p-FA

KFA

Kp-PI3K

PI3K

p-Akt

Akt

MMP-2

MMP-9

0d004plusmn001

054plusmn002

004plusmn001

063plusmn002

004plusmn001

063plusmn000

004plusmn000

006plusmn004

2d026plusmn010

ab056plusmn002

024plusmn001

ab064plusmn001

029plusmn004

ab062plusmn001

045plusmn013

ab033plusmn016

a

7d019plusmn010

b055plusmn002

009plusmn003

b064plusmn001

014plusmn002

ab061plusmn000

024plusmn011

ab020plusmn015

ab

14d

054plusmn005

a057plusmn002

053plusmn003

a067plusmn000

047plusmn001

a064plusmn001

074plusmn009

a050plusmn013

a

28d

035plusmn010

ab057plusmn001

039plusmn005

ab066plusmn001

035plusmn004

ab063plusmn000

056plusmn008

ab042plusmn010

a

F1551

151

12338

873

11907

1014

2531

584

Plt005

gt005

lt005

gt005

lt005

gt005

lt005

lt005

Notea Plt005comparedwith

0dbPlt005comparedwith

14d(one-w

ayANOVA

LSD

-ttest)

8 BioMed Research International

Data Availability

The data used to support the findings of this study arecurrently kept under raps while the research findings aresignificant Requests for data 12 months after publication ofthis article will be considered by the corresponding authoron reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Project approval number 81370986)

References

[1] A J Lepisto G M Frank and R L Hendricks ldquoHow herpessimplex virus type 1 rescinds corneal privilegerdquo ChemicalImmunology and Allergy vol 92 pp 203ndash212 2007

[2] L Xie H Zhai X Dong et al ldquoPrimary diseases of corneal per-foration in Shandong Province China a 10-year retrospectivestudyrdquo American Journal of Ophthalmology vol 145 no 4 pp662ndash666 2008

[3] A V Farooq and D Shukla ldquoHerpes simplex epithelial andstromal keratitis an epidemiologic updaterdquo Survey of Ophthal-mology vol 57 no 5 pp 448ndash462 2012

[4] Y-N Yang D Bauer S Wasmuth K-P Steuhl and A Heili-genhaus ldquoMatrix metalloproteinases (MMP-2 and 9) and tissueinhibitors of matrix metalloproteinases (TIMP-1 and 2) dur-ing the course of experimental necrotizing herpetic keratitisrdquoExperimental Eye Research vol 77 no 2 pp 227ndash237 2003

[5] Z L Teo L McQueen-Miscamble K Turner et al ldquoIntegrinlinked kinase (ILK) is required for lens epithelial cell survivalproliferation and differentiationrdquo Experimental Eye Researchvol 121 no 3 pp 130ndash142 2014

[6] X-M Yang Y-S Wang J Zhang et al ldquoRole of PI3KAkt andMEKERK inmediating hypoxia-induced expression of HIF-1120572and VEGF in laser-induced rat choroidal neovascularizationrdquoInvestigative Ophthalmology amp Visual Science vol 50 no 4 pp1873ndash1879 2009

[7] S P Narayanan J Suwanpradid W Zhang et al ldquoArginasedepletion modulates Akt and iNOS to reduce retinal degenera-tion in a mouse model of retinopathy of prematurityrdquo Gut vol58 no 1 p 147 2010 author reply 147-8

[8] A Heiligenhaus D Bauer D Meller K-P Steuhl and S C GTseng ldquoImprovement of HSV-1 necrotizing keratitis with amni-otic membrane transplantationrdquo Investigative Ophthalmology ampVisual Science vol 42 no 9 pp 1969ndash1974 2001

[9] N A Chatzizacharias G P Kouraklis and S E TheocharisldquoClinical significance of FAK expression in human neoplasiardquoHistology and Histopathology vol 23 no 5 pp 629ndash650 2008

[10] B Hu M J Jarzynka P Guo Y Imanishi D D Schlaepfer andS-Y Cheng ldquoAngiopoietin 2 induces glioma cell invasion bystimulating matrix metalloprotease 2 expression through the120572v1205731 integrin and focal adhesion kinase signaling pathwayrdquoCancer Research vol 66 no 2 pp 775ndash783 2006

[11] M Canel P Secades M Garzon-Arango et al ldquoInvolvementof focal adhesion kinase in cellular invasion of head and neck

squamous cell carcinomas via regulation ofMMP-2 expressionrdquoBritish Journal of Cancer vol 98 no 7 pp 1274ndash1284 2008

[12] L-C Chang C-H Huang C-H Cheng B-H Chen and H-C Chen ldquoDifferential effect of the focal adhesion kinase Y397Fmutant on v-Src-stimulated cell invasion and tumor growthrdquoJournal of Biomedical Science vol 12 no 4 pp 571ndash585 2005

[13] Q Z Wu L Z Zhang W S Nie et al ldquoActivity of matrixmetalloproteinases 2 and 9 in cultured rabbit corneal epitheliumcells stimulated by tumor necrosis factor alphardquo Genetics andMolecular Research GMR vol 14 no 2 p 6360 2015

[14] Y-N Yang F Wang W Zhou Z-Q Wu and Y-Q XingldquoTNF-120572 stimulates MMP-2 and MMP-9 activities in humancorneal epithelial cells via the activation of FAKERK signalingrdquoOphthalmic Research vol 48 no 4 pp 165ndash170 2012

[15] A P Bhatt and B Damania ldquoAKTivation of PI3KAKTmTORsignaling pathway by KSHVrdquo Frontiers in Immunology vol 3 p401 2012

[16] N J Buchkovich Y Yu C A Zampieri and J C Alwine ldquoTheTORrid affairs of viruses Effects of mammalian DNA viruseson the PI3K-Akt-mTOR signalling pathwayrdquo Nature ReviewsMicrobiology vol 6 no 4 pp 265ndash275 2008

[17] N Diehl and H Schaal ldquoMake yourself at home viral hijackingof the PI3KAkt signaling pathwayrdquo Viruses vol 5 no 12 pp3192ndash3212 2013

[18] N Cheshenko J B Trepanier P A Gonzalez E A EugeninW R Jacobs and B C Herold ldquoHerpes simplex virus type 2glycoprotein h interacts with integrin 120572v1205733 to facilitate viralentry and calcium signaling in human genital tract epithelialcellsrdquo Journal of Virology vol 88 no 17 pp 10026ndash10038 2014

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BioMed Research International 3

Table 1 Evaluate different indexes of cornea in HSV-1 infected mice

Score Epithelial or stromal damage Corneal opacity0 - Transparent1+ lt25 Slightly turbid2+ ge25 lt50 Moderately turbid visible iris3+ ge50 lt75 Severe turbidity pupillary margin can be judged4+ ge75 Complete opacity invisible posterior features

Table 2 Evaluate different indexes of cornea in HSV-1 infected mice (Xplusmns)

Indexes 0d 2d 7d 14d 28dEpithelial or stromal damage 000plusmn000 103plusmn016a 100plusmn037a 150plusmn069a 160plusmn084a

Corneal opacity 000plusmn000 103plusmn016a 103plusmn032a 220plusmn041a 250plusmn071a

Note Compared with 0d aP lt 005 (t test)

centrifugation 12 gel electrophoresis was performed Afterelectrotransfer of PVDFmembranes there was blocking with5 skim milk blocking solution (TBST) anti-FAK primaryantibody (1500) p-FAK primary antibody (1500) PI3K pri-mary antibody (14000) p-PI3K primary antibody (11000)Akt primary antibody (12000) p-Akt primary antibody(11000) MMP-2 primary antibody (11000) MMP-9 primaryantibody (11000) ) Incubate overnight at 4∘C thenwash addthe corresponding secondary antibody at 4∘C overnight anddevelop with colorThe experiment was repeated three timesThe results obtained were density scanned and gray-scaleanalysis was performed using Gel-proAnalyzer 45 imageanalysis software

23 StatisticalMethod SPSS 220 statistical softwarewas usedto analyze the experimental data All indicators are expressedas mean plusmn standard deviation (XplusmnS) The t-test was usedto compare the differences in the scores of different cornealindexes at different times One-way analysis of variancewas used to compare the differences in values obtained atdifferent time points Multiple comparisons were performedusing Dunnettrsquos test or LSD-t test Plt 005 was consideredstatistically significant

3 Results

31 Clinical Course ofHSK andEvaluation of Different CornealIndexes We found that after infected with HSV-1 differentdegrees of epithelial or stromal damage corneal opacityandor corneal neovascularization were observed on the 2d7d 14d and 28d First inflammation in corneal epithelialappeared in 1 to 3 days after corneal infection in normalBALBc mice The corneal epithelium showed a punctatedefect or a map-like defect or a dendritic defect in thecornea Secondly stroma gradually developed edema afterthe corneal epithelium healed after 4-7 days A few severecases were even accompanied by corneal neovascularizationcorneal ulcers andor corneal neovascularization Cornealulcers and corneal neovascularization can be seen around14 days and corneal ulcer perforation can occur in severecases Then about 28 days the inflammation of the cornea

Figure 1 Cornea image of typical BALBc mouse infected withHSV-1

was reduced and corneal neovascularization became coarseand dark (Figure 1) In Table 2 we can find that with thetime of corneal infection prolonged the score of cornealopacity gradually increased Compared with 0d the score ofepithelial or stromal damage and corneal opacity increased in2d 7d 14d and 28d after infection and the differences werestatistically significant (Plt 005)

32 Expression and Distribution of MMP-2 and MMP-9Protein in Corneal Tissue at Different Time Points in HSKMouseModel Immunofluorescence staining showed that theexpression of MMP-2 and MMP-9 was observed in thecorneal tissue of HSK at 0d 2d 7d 14d and 28d (Figure 2) Inthe corneal tissue the expression ofMMP-2was located in theepithelium and the expression of MMP-9 was located in thestroma of the cornea After 2 days the expression of MMP-2in the corneal tissuewas located in the corneal epitheliumanddiffused in the corneal stromal layer while the expression ofMMP-9 was located in the corneal stroma Compared with0d the expression of MMP-2 and MMP-9 was stronger in

4 BioMed Research International

MMP-2 MMP-9 DAPI merge

0d

2d

7d

14d

28d

Figure 2 Expression of MMP-2 and MMP-9 protein in corneal tissue of BALBc mice after 0 2 7 14 and 28d of infection with HSV-1 Theexpression of MMP-2 in the epithelium and the expression of MMP-9 in the stroma showed diffuse fluorescence after 0d of infection Thefluorescence intensity of MMP-2 and MMP-9 at 2d was higher than that of 0d and 7d The fluorescence intensity of MMP-2 and MMP-9reached summit at 14d After 28d of infection expressions of MMP-2 and MMP-9 protein were weaker than that of 14d

0d 2d 7d 14d 28d0d 2d 7d 14d 28d

ab

ab

abab

ab

ab

aa

The r

elativ

e exp

ress

ion

of M

MP-

2 m

RNA 4

3

2

1

0 The r

elativ

e exp

ress

ion

of M

MP-

9m

RNA 3

2

1

0

Figure 3 The relative expression of MMP-2 and MMP-9 mRNA in cornea at each time point Note aP lt 005 compared with 0d bP lt 005compared with 14d (One-way ANOVA Dunnettrsquos t test) MMP Matrix Metalloproteinases

2d After 7 days of infection with HSV-1 the expression ofMMP-2 and MMP-9 in corneal tissue was stronger than thatof 0d but lower than that of 2d After 14 days the expressionof MMP-2 and MMP-9 in the corneal tissue was strongerthan that of 0d 2d and 7d and the intensity of fluorescenceexpression peaked At 28d the expression of MMP-2 andMMP-9 in the corneal tissue was lower than that of 14d butit was stronger than that of 0d

33 Expression Changes of MMP-2 and MMP-9 mRNA inCorneal Tissue at Different Time Points in HSK Mouse ModelThe results of RT-PCR showed that the mRNA levels ofMMP-2 and MMP-9 increased at 2 days after HSV infectiondecreased after 7 days then peaked again at 14 days andthen decreased at 28 days (Figure 3) The relative expressionlevels of MMP-2 mRNA at 0 d 2 d 7 d 14 d and 28 d afterinfection in HSK mouse model were 100plusmn000 159plusmn004

BioMed Research International 5

0d 2d 7d 14d 28d

119 kDa

119 kDa

85 kDa

85 kDa

60 kDa

60 kDa

74 kDa

92 kDa

43 kDa

P-FAK

FAK

P-PI3K

PI3K

P-AKT

AKT

MMP-2

MMP-9

-Actin

Figure 4 Western Blot results of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 protein expression in mouse cornea at eachtime point

ab

ab

ab

ab

ab ab

ababab

ab

ab

a

a a

a

a

aa

b

b

0d 2d 7d 14d 28d

p-FAK

FAK

p-PI3K

PI3K

p-Akt

AktMMP-2MMP-9

The r

elativ

e exp

ress

ion

of p

rote

in

10

08

06

04

02

00

Figure 5 Relative expression of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 in cornea at each time point Note aP lt 005compared with 0d bP lt 005 compared with 14d (one-way ANOVA LSD-t test)

202plusmn015 270plusmn026 and 199plusmn186 respectively The relativeexpression levels ofMMP-9mRNAwere 100plusmn000 151plusmn005181plusmn007 230plusmn015 and 194plusmn171 Compared with 0d therelative expression levels of MMP-2 and MMP-9 mRNAwere significantly increased at 2d 7d 14d and 28d andthe differences were statistically significant (all Plt 005)Compared with 14d the relative expression levels of MMP-2 and MMP-9 mRNA decreased at 2d 7d and 28d and thedifferences were statistically significant (all Plt 005) (Table 3)

34 Expression of p-FAK FAK p-PI3K PI3K p-Akt AktMMP-2 and MMP-9 in Corneal Tissue at Different TimePoints in HSK Mouse Model Western blot analysis showedthat the expression of p-FAK p-PI3K p-Akt MMP-2 andMMP-9 was increased in corneal tissue at 2 days afterinfection and decreased from 7d to 2d 14d and 28d Itrose again and peaked at 14d (Figures 4 and 5) The proteinexpressions of p-FAK p-PI3K p-Akt MMP-2 and MMP-9at 2d 14d and 28d were all significantly higher than those at

6 BioMed Research International

Table 3 The relative expression of MMP-2 and MMP-9 mRNA in the cornea at each time point (Xplusmns)

Time point MMP-2 MMP-90d 100plusmn000 100plusmn0002d 202plusmn015ab 181plusmn007ab

7d 159plusmn004ab 151plusmn005ab

14d 270plusmn026a 230plusmn015a

28d 199plusmn015ab 194plusmn002ab

F 5069 12216P lt005 lt005NoteaP lt 005 compared with 0d bP lt 005 compared with 14d (One-way ANOVA Dunnettrsquos t test)MMP matrix metalloproteinases

0d (Plt 005) The expression of p-Akt MMP-2 and MMP-9at 7 days after infection was significantly higher than that at 0days (Plt 005) At 14 days the expression of p-FAK p-PI3Kp-Akt andMMP-2 protein was higher than that at 2d 7d and28d and the difference was statistically significant (Plt 005)The expression ofMMP-9 protein at 7d was lower than that at14d and the difference was statistically significant (Plt 005)There was no significant difference in the expression levelsof FAK PI3K and Akt between tissues at all time points (Pgt005) (Table 4)

4 Discussion

HSK is one of the leading eye diseases in the world leadingto corneal blindness [3] Recurrent HSK will undoubtedlyaggravate the deterioration of the disease and eventually leadto blinding outcomes Although the research on molecularmechanisms related to the formation of HSK is increasingand deepening the pathogenesis of HSK is complex andits pathogenesis is still not fully understood There is noclinically satisfactory drug treatment that can prevent it fromrecurring or prevent it from developing into a bad outcomeTherefore it is imperative to explore drug treatment withmultiple pathways multiple targets and multiple therapeuticeffects

Recent research indicates that FAK not only participatesin the regulation of cell proliferation apoptosis migrationinvasion metastasis and adhesion but also participates in theregulation of the expression and activity ofMMPs and TIMPsthrough multiple signaling pathways [9ndash12] At the sametime our previous study found that there were two peaksin the phosphorylation of FAK in the corneal epithelium ofHSV-1-infected mouse corneal models The phosphorylationlevels of FAK increased on the 2d 14d and 28d afterinfection and decreased on the 7d compared with 2d [13]The expression pattern of MMP-2 andMMP-9 in the cornealmodel of HSV-1-infected mice in this study is very similarto our previous studies [4] In addition we also found thatTNF-120572 can increase the expression and secretion of MMP-2 and MMP-9 through the FAKERK signaling pathway invitro experiments [14] This further suggests the role of theFAK signaling pathway in HSK

PI3KAkt is an important signaling pathway downstreamof FAK that regulates many important biological signaling

pathways It has been reported that on the one handthe herpes virus can enhance its function of replicationtranscription and translation by regulating the PI3KAktsignaling pathwayOn the other hand the PI3KAkt signalingpathway can be used to facilitate virus entry replicationdelay and activation [15ndash17] Activation of PI3KAkt sig-naling can occur at multiple stages of the herpes virus lifecycle [16 17] Cheshenko reported that HSV can activateand release intracellular calcium and phosphorylate FAKvia the integrin signaling pathway which activates the AKTsignaling pathway and provides suitable conditions for virusentry into cells [18] Therefore we hypothesized that theFAKPI3KAkt signaling pathway and MMP-2 and MMP-9 may be involved in the development of HSK in thecornea

In this study we first studied the patterns of FAK p-FAKPI3K p-PI3K Akt and p-Akt in the FAKPI3KAkt signalingpathway and MMP-2 and MMP-9 in the HSK mouse modelBy Western blot we observed that the levels of p-FAK p-PI3K and p-Akt were upregulated on the 2d 7d 14d and28d after infection The upregulated level on the 7th daydecreased compared with that on the 2d day and reachedthe maximum on the 14d day At the same time the changesin the levels of MMP-2 and MMP-9 protein were similar tothose of p-FAK p-PI3K and p-Akt This is similar to ourprevious study [4] suggesting a potential link between theFAKPI3KAkt signaling pathway and theMMP-2 andMMP-9 In addition we have further demonstrated by RT-PCRand Immunofluorescence staining that the expression andactivation of MMP-2 and MMP-9 in the mouse cornea atthe early stage of HSV-1 infection play an active role in thedevelopment of HSK To further elucidate the relationshipbetween the FAKPI3KAkt signaling pathway and the HSKwe plan to use the inhibitor

In summary our results provide in vivo evidence thatFAKPI3KAkt signaling pathway and MMP-2 and MMP-9may be involved in the development of HSK The discoveryof the association between FAKPI3KAkt signaling pathwayMMP-2 and MMP-9 and HSK may facilitate the study ofthe mechanism of HSK and studies of the FAKPI3KAKTsignaling pathway may provide new targets for the treatmentofHSKHowever in order to show the regulatory relationshipbetween FAKPI3KAkt signaling pathway and MMP-2 andMMP-9 further research is necessary

BioMed Research International 7

Table4Com

paris

onof

relativee

xpressionlevelsof

p-FA

KFA

Kp-PI3K

PI3K

p-AktA

ktM

MP-2andMMP-9in

cornea

ateach

timep

oint

(Xplusmns)

Timep

oint

p-FA

KFA

Kp-PI3K

PI3K

p-Akt

Akt

MMP-2

MMP-9

0d004plusmn001

054plusmn002

004plusmn001

063plusmn002

004plusmn001

063plusmn000

004plusmn000

006plusmn004

2d026plusmn010

ab056plusmn002

024plusmn001

ab064plusmn001

029plusmn004

ab062plusmn001

045plusmn013

ab033plusmn016

a

7d019plusmn010

b055plusmn002

009plusmn003

b064plusmn001

014plusmn002

ab061plusmn000

024plusmn011

ab020plusmn015

ab

14d

054plusmn005

a057plusmn002

053plusmn003

a067plusmn000

047plusmn001

a064plusmn001

074plusmn009

a050plusmn013

a

28d

035plusmn010

ab057plusmn001

039plusmn005

ab066plusmn001

035plusmn004

ab063plusmn000

056plusmn008

ab042plusmn010

a

F1551

151

12338

873

11907

1014

2531

584

Plt005

gt005

lt005

gt005

lt005

gt005

lt005

lt005

Notea Plt005comparedwith

0dbPlt005comparedwith

14d(one-w

ayANOVA

LSD

-ttest)

8 BioMed Research International

Data Availability

The data used to support the findings of this study arecurrently kept under raps while the research findings aresignificant Requests for data 12 months after publication ofthis article will be considered by the corresponding authoron reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Project approval number 81370986)

References

[1] A J Lepisto G M Frank and R L Hendricks ldquoHow herpessimplex virus type 1 rescinds corneal privilegerdquo ChemicalImmunology and Allergy vol 92 pp 203ndash212 2007

[2] L Xie H Zhai X Dong et al ldquoPrimary diseases of corneal per-foration in Shandong Province China a 10-year retrospectivestudyrdquo American Journal of Ophthalmology vol 145 no 4 pp662ndash666 2008

[3] A V Farooq and D Shukla ldquoHerpes simplex epithelial andstromal keratitis an epidemiologic updaterdquo Survey of Ophthal-mology vol 57 no 5 pp 448ndash462 2012

[4] Y-N Yang D Bauer S Wasmuth K-P Steuhl and A Heili-genhaus ldquoMatrix metalloproteinases (MMP-2 and 9) and tissueinhibitors of matrix metalloproteinases (TIMP-1 and 2) dur-ing the course of experimental necrotizing herpetic keratitisrdquoExperimental Eye Research vol 77 no 2 pp 227ndash237 2003

[5] Z L Teo L McQueen-Miscamble K Turner et al ldquoIntegrinlinked kinase (ILK) is required for lens epithelial cell survivalproliferation and differentiationrdquo Experimental Eye Researchvol 121 no 3 pp 130ndash142 2014

[6] X-M Yang Y-S Wang J Zhang et al ldquoRole of PI3KAkt andMEKERK inmediating hypoxia-induced expression of HIF-1120572and VEGF in laser-induced rat choroidal neovascularizationrdquoInvestigative Ophthalmology amp Visual Science vol 50 no 4 pp1873ndash1879 2009

[7] S P Narayanan J Suwanpradid W Zhang et al ldquoArginasedepletion modulates Akt and iNOS to reduce retinal degenera-tion in a mouse model of retinopathy of prematurityrdquo Gut vol58 no 1 p 147 2010 author reply 147-8

[8] A Heiligenhaus D Bauer D Meller K-P Steuhl and S C GTseng ldquoImprovement of HSV-1 necrotizing keratitis with amni-otic membrane transplantationrdquo Investigative Ophthalmology ampVisual Science vol 42 no 9 pp 1969ndash1974 2001

[9] N A Chatzizacharias G P Kouraklis and S E TheocharisldquoClinical significance of FAK expression in human neoplasiardquoHistology and Histopathology vol 23 no 5 pp 629ndash650 2008

[10] B Hu M J Jarzynka P Guo Y Imanishi D D Schlaepfer andS-Y Cheng ldquoAngiopoietin 2 induces glioma cell invasion bystimulating matrix metalloprotease 2 expression through the120572v1205731 integrin and focal adhesion kinase signaling pathwayrdquoCancer Research vol 66 no 2 pp 775ndash783 2006

[11] M Canel P Secades M Garzon-Arango et al ldquoInvolvementof focal adhesion kinase in cellular invasion of head and neck

squamous cell carcinomas via regulation ofMMP-2 expressionrdquoBritish Journal of Cancer vol 98 no 7 pp 1274ndash1284 2008

[12] L-C Chang C-H Huang C-H Cheng B-H Chen and H-C Chen ldquoDifferential effect of the focal adhesion kinase Y397Fmutant on v-Src-stimulated cell invasion and tumor growthrdquoJournal of Biomedical Science vol 12 no 4 pp 571ndash585 2005

[13] Q Z Wu L Z Zhang W S Nie et al ldquoActivity of matrixmetalloproteinases 2 and 9 in cultured rabbit corneal epitheliumcells stimulated by tumor necrosis factor alphardquo Genetics andMolecular Research GMR vol 14 no 2 p 6360 2015

[14] Y-N Yang F Wang W Zhou Z-Q Wu and Y-Q XingldquoTNF-120572 stimulates MMP-2 and MMP-9 activities in humancorneal epithelial cells via the activation of FAKERK signalingrdquoOphthalmic Research vol 48 no 4 pp 165ndash170 2012

[15] A P Bhatt and B Damania ldquoAKTivation of PI3KAKTmTORsignaling pathway by KSHVrdquo Frontiers in Immunology vol 3 p401 2012

[16] N J Buchkovich Y Yu C A Zampieri and J C Alwine ldquoTheTORrid affairs of viruses Effects of mammalian DNA viruseson the PI3K-Akt-mTOR signalling pathwayrdquo Nature ReviewsMicrobiology vol 6 no 4 pp 265ndash275 2008

[17] N Diehl and H Schaal ldquoMake yourself at home viral hijackingof the PI3KAkt signaling pathwayrdquo Viruses vol 5 no 12 pp3192ndash3212 2013

[18] N Cheshenko J B Trepanier P A Gonzalez E A EugeninW R Jacobs and B C Herold ldquoHerpes simplex virus type 2glycoprotein h interacts with integrin 120572v1205733 to facilitate viralentry and calcium signaling in human genital tract epithelialcellsrdquo Journal of Virology vol 88 no 17 pp 10026ndash10038 2014

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of

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Disease Markers

Hindawiwwwhindawicom Volume 2018

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OncologyJournal of

Hindawiwwwhindawicom Volume 2013

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Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

4 BioMed Research International

MMP-2 MMP-9 DAPI merge

0d

2d

7d

14d

28d

Figure 2 Expression of MMP-2 and MMP-9 protein in corneal tissue of BALBc mice after 0 2 7 14 and 28d of infection with HSV-1 Theexpression of MMP-2 in the epithelium and the expression of MMP-9 in the stroma showed diffuse fluorescence after 0d of infection Thefluorescence intensity of MMP-2 and MMP-9 at 2d was higher than that of 0d and 7d The fluorescence intensity of MMP-2 and MMP-9reached summit at 14d After 28d of infection expressions of MMP-2 and MMP-9 protein were weaker than that of 14d

0d 2d 7d 14d 28d0d 2d 7d 14d 28d

ab

ab

abab

ab

ab

aa

The r

elativ

e exp

ress

ion

of M

MP-

2 m

RNA 4

3

2

1

0 The r

elativ

e exp

ress

ion

of M

MP-

9m

RNA 3

2

1

0

Figure 3 The relative expression of MMP-2 and MMP-9 mRNA in cornea at each time point Note aP lt 005 compared with 0d bP lt 005compared with 14d (One-way ANOVA Dunnettrsquos t test) MMP Matrix Metalloproteinases

2d After 7 days of infection with HSV-1 the expression ofMMP-2 and MMP-9 in corneal tissue was stronger than thatof 0d but lower than that of 2d After 14 days the expressionof MMP-2 and MMP-9 in the corneal tissue was strongerthan that of 0d 2d and 7d and the intensity of fluorescenceexpression peaked At 28d the expression of MMP-2 andMMP-9 in the corneal tissue was lower than that of 14d butit was stronger than that of 0d

33 Expression Changes of MMP-2 and MMP-9 mRNA inCorneal Tissue at Different Time Points in HSK Mouse ModelThe results of RT-PCR showed that the mRNA levels ofMMP-2 and MMP-9 increased at 2 days after HSV infectiondecreased after 7 days then peaked again at 14 days andthen decreased at 28 days (Figure 3) The relative expressionlevels of MMP-2 mRNA at 0 d 2 d 7 d 14 d and 28 d afterinfection in HSK mouse model were 100plusmn000 159plusmn004

BioMed Research International 5

0d 2d 7d 14d 28d

119 kDa

119 kDa

85 kDa

85 kDa

60 kDa

60 kDa

74 kDa

92 kDa

43 kDa

P-FAK

FAK

P-PI3K

PI3K

P-AKT

AKT

MMP-2

MMP-9

-Actin

Figure 4 Western Blot results of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 protein expression in mouse cornea at eachtime point

ab

ab

ab

ab

ab ab

ababab

ab

ab

a

a a

a

a

aa

b

b

0d 2d 7d 14d 28d

p-FAK

FAK

p-PI3K

PI3K

p-Akt

AktMMP-2MMP-9

The r

elativ

e exp

ress

ion

of p

rote

in

10

08

06

04

02

00

Figure 5 Relative expression of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 in cornea at each time point Note aP lt 005compared with 0d bP lt 005 compared with 14d (one-way ANOVA LSD-t test)

202plusmn015 270plusmn026 and 199plusmn186 respectively The relativeexpression levels ofMMP-9mRNAwere 100plusmn000 151plusmn005181plusmn007 230plusmn015 and 194plusmn171 Compared with 0d therelative expression levels of MMP-2 and MMP-9 mRNAwere significantly increased at 2d 7d 14d and 28d andthe differences were statistically significant (all Plt 005)Compared with 14d the relative expression levels of MMP-2 and MMP-9 mRNA decreased at 2d 7d and 28d and thedifferences were statistically significant (all Plt 005) (Table 3)

34 Expression of p-FAK FAK p-PI3K PI3K p-Akt AktMMP-2 and MMP-9 in Corneal Tissue at Different TimePoints in HSK Mouse Model Western blot analysis showedthat the expression of p-FAK p-PI3K p-Akt MMP-2 andMMP-9 was increased in corneal tissue at 2 days afterinfection and decreased from 7d to 2d 14d and 28d Itrose again and peaked at 14d (Figures 4 and 5) The proteinexpressions of p-FAK p-PI3K p-Akt MMP-2 and MMP-9at 2d 14d and 28d were all significantly higher than those at

6 BioMed Research International

Table 3 The relative expression of MMP-2 and MMP-9 mRNA in the cornea at each time point (Xplusmns)

Time point MMP-2 MMP-90d 100plusmn000 100plusmn0002d 202plusmn015ab 181plusmn007ab

7d 159plusmn004ab 151plusmn005ab

14d 270plusmn026a 230plusmn015a

28d 199plusmn015ab 194plusmn002ab

F 5069 12216P lt005 lt005NoteaP lt 005 compared with 0d bP lt 005 compared with 14d (One-way ANOVA Dunnettrsquos t test)MMP matrix metalloproteinases

0d (Plt 005) The expression of p-Akt MMP-2 and MMP-9at 7 days after infection was significantly higher than that at 0days (Plt 005) At 14 days the expression of p-FAK p-PI3Kp-Akt andMMP-2 protein was higher than that at 2d 7d and28d and the difference was statistically significant (Plt 005)The expression ofMMP-9 protein at 7d was lower than that at14d and the difference was statistically significant (Plt 005)There was no significant difference in the expression levelsof FAK PI3K and Akt between tissues at all time points (Pgt005) (Table 4)

4 Discussion

HSK is one of the leading eye diseases in the world leadingto corneal blindness [3] Recurrent HSK will undoubtedlyaggravate the deterioration of the disease and eventually leadto blinding outcomes Although the research on molecularmechanisms related to the formation of HSK is increasingand deepening the pathogenesis of HSK is complex andits pathogenesis is still not fully understood There is noclinically satisfactory drug treatment that can prevent it fromrecurring or prevent it from developing into a bad outcomeTherefore it is imperative to explore drug treatment withmultiple pathways multiple targets and multiple therapeuticeffects

Recent research indicates that FAK not only participatesin the regulation of cell proliferation apoptosis migrationinvasion metastasis and adhesion but also participates in theregulation of the expression and activity ofMMPs and TIMPsthrough multiple signaling pathways [9ndash12] At the sametime our previous study found that there were two peaksin the phosphorylation of FAK in the corneal epithelium ofHSV-1-infected mouse corneal models The phosphorylationlevels of FAK increased on the 2d 14d and 28d afterinfection and decreased on the 7d compared with 2d [13]The expression pattern of MMP-2 andMMP-9 in the cornealmodel of HSV-1-infected mice in this study is very similarto our previous studies [4] In addition we also found thatTNF-120572 can increase the expression and secretion of MMP-2 and MMP-9 through the FAKERK signaling pathway invitro experiments [14] This further suggests the role of theFAK signaling pathway in HSK

PI3KAkt is an important signaling pathway downstreamof FAK that regulates many important biological signaling

pathways It has been reported that on the one handthe herpes virus can enhance its function of replicationtranscription and translation by regulating the PI3KAktsignaling pathwayOn the other hand the PI3KAkt signalingpathway can be used to facilitate virus entry replicationdelay and activation [15ndash17] Activation of PI3KAkt sig-naling can occur at multiple stages of the herpes virus lifecycle [16 17] Cheshenko reported that HSV can activateand release intracellular calcium and phosphorylate FAKvia the integrin signaling pathway which activates the AKTsignaling pathway and provides suitable conditions for virusentry into cells [18] Therefore we hypothesized that theFAKPI3KAkt signaling pathway and MMP-2 and MMP-9 may be involved in the development of HSK in thecornea

In this study we first studied the patterns of FAK p-FAKPI3K p-PI3K Akt and p-Akt in the FAKPI3KAkt signalingpathway and MMP-2 and MMP-9 in the HSK mouse modelBy Western blot we observed that the levels of p-FAK p-PI3K and p-Akt were upregulated on the 2d 7d 14d and28d after infection The upregulated level on the 7th daydecreased compared with that on the 2d day and reachedthe maximum on the 14d day At the same time the changesin the levels of MMP-2 and MMP-9 protein were similar tothose of p-FAK p-PI3K and p-Akt This is similar to ourprevious study [4] suggesting a potential link between theFAKPI3KAkt signaling pathway and theMMP-2 andMMP-9 In addition we have further demonstrated by RT-PCRand Immunofluorescence staining that the expression andactivation of MMP-2 and MMP-9 in the mouse cornea atthe early stage of HSV-1 infection play an active role in thedevelopment of HSK To further elucidate the relationshipbetween the FAKPI3KAkt signaling pathway and the HSKwe plan to use the inhibitor

In summary our results provide in vivo evidence thatFAKPI3KAkt signaling pathway and MMP-2 and MMP-9may be involved in the development of HSK The discoveryof the association between FAKPI3KAkt signaling pathwayMMP-2 and MMP-9 and HSK may facilitate the study ofthe mechanism of HSK and studies of the FAKPI3KAKTsignaling pathway may provide new targets for the treatmentofHSKHowever in order to show the regulatory relationshipbetween FAKPI3KAkt signaling pathway and MMP-2 andMMP-9 further research is necessary

BioMed Research International 7

Table4Com

paris

onof

relativee

xpressionlevelsof

p-FA

KFA

Kp-PI3K

PI3K

p-AktA

ktM

MP-2andMMP-9in

cornea

ateach

timep

oint

(Xplusmns)

Timep

oint

p-FA

KFA

Kp-PI3K

PI3K

p-Akt

Akt

MMP-2

MMP-9

0d004plusmn001

054plusmn002

004plusmn001

063plusmn002

004plusmn001

063plusmn000

004plusmn000

006plusmn004

2d026plusmn010

ab056plusmn002

024plusmn001

ab064plusmn001

029plusmn004

ab062plusmn001

045plusmn013

ab033plusmn016

a

7d019plusmn010

b055plusmn002

009plusmn003

b064plusmn001

014plusmn002

ab061plusmn000

024plusmn011

ab020plusmn015

ab

14d

054plusmn005

a057plusmn002

053plusmn003

a067plusmn000

047plusmn001

a064plusmn001

074plusmn009

a050plusmn013

a

28d

035plusmn010

ab057plusmn001

039plusmn005

ab066plusmn001

035plusmn004

ab063plusmn000

056plusmn008

ab042plusmn010

a

F1551

151

12338

873

11907

1014

2531

584

Plt005

gt005

lt005

gt005

lt005

gt005

lt005

lt005

Notea Plt005comparedwith

0dbPlt005comparedwith

14d(one-w

ayANOVA

LSD

-ttest)

8 BioMed Research International

Data Availability

The data used to support the findings of this study arecurrently kept under raps while the research findings aresignificant Requests for data 12 months after publication ofthis article will be considered by the corresponding authoron reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Project approval number 81370986)

References

[1] A J Lepisto G M Frank and R L Hendricks ldquoHow herpessimplex virus type 1 rescinds corneal privilegerdquo ChemicalImmunology and Allergy vol 92 pp 203ndash212 2007

[2] L Xie H Zhai X Dong et al ldquoPrimary diseases of corneal per-foration in Shandong Province China a 10-year retrospectivestudyrdquo American Journal of Ophthalmology vol 145 no 4 pp662ndash666 2008

[3] A V Farooq and D Shukla ldquoHerpes simplex epithelial andstromal keratitis an epidemiologic updaterdquo Survey of Ophthal-mology vol 57 no 5 pp 448ndash462 2012

[4] Y-N Yang D Bauer S Wasmuth K-P Steuhl and A Heili-genhaus ldquoMatrix metalloproteinases (MMP-2 and 9) and tissueinhibitors of matrix metalloproteinases (TIMP-1 and 2) dur-ing the course of experimental necrotizing herpetic keratitisrdquoExperimental Eye Research vol 77 no 2 pp 227ndash237 2003

[5] Z L Teo L McQueen-Miscamble K Turner et al ldquoIntegrinlinked kinase (ILK) is required for lens epithelial cell survivalproliferation and differentiationrdquo Experimental Eye Researchvol 121 no 3 pp 130ndash142 2014

[6] X-M Yang Y-S Wang J Zhang et al ldquoRole of PI3KAkt andMEKERK inmediating hypoxia-induced expression of HIF-1120572and VEGF in laser-induced rat choroidal neovascularizationrdquoInvestigative Ophthalmology amp Visual Science vol 50 no 4 pp1873ndash1879 2009

[7] S P Narayanan J Suwanpradid W Zhang et al ldquoArginasedepletion modulates Akt and iNOS to reduce retinal degenera-tion in a mouse model of retinopathy of prematurityrdquo Gut vol58 no 1 p 147 2010 author reply 147-8

[8] A Heiligenhaus D Bauer D Meller K-P Steuhl and S C GTseng ldquoImprovement of HSV-1 necrotizing keratitis with amni-otic membrane transplantationrdquo Investigative Ophthalmology ampVisual Science vol 42 no 9 pp 1969ndash1974 2001

[9] N A Chatzizacharias G P Kouraklis and S E TheocharisldquoClinical significance of FAK expression in human neoplasiardquoHistology and Histopathology vol 23 no 5 pp 629ndash650 2008

[10] B Hu M J Jarzynka P Guo Y Imanishi D D Schlaepfer andS-Y Cheng ldquoAngiopoietin 2 induces glioma cell invasion bystimulating matrix metalloprotease 2 expression through the120572v1205731 integrin and focal adhesion kinase signaling pathwayrdquoCancer Research vol 66 no 2 pp 775ndash783 2006

[11] M Canel P Secades M Garzon-Arango et al ldquoInvolvementof focal adhesion kinase in cellular invasion of head and neck

squamous cell carcinomas via regulation ofMMP-2 expressionrdquoBritish Journal of Cancer vol 98 no 7 pp 1274ndash1284 2008

[12] L-C Chang C-H Huang C-H Cheng B-H Chen and H-C Chen ldquoDifferential effect of the focal adhesion kinase Y397Fmutant on v-Src-stimulated cell invasion and tumor growthrdquoJournal of Biomedical Science vol 12 no 4 pp 571ndash585 2005

[13] Q Z Wu L Z Zhang W S Nie et al ldquoActivity of matrixmetalloproteinases 2 and 9 in cultured rabbit corneal epitheliumcells stimulated by tumor necrosis factor alphardquo Genetics andMolecular Research GMR vol 14 no 2 p 6360 2015

[14] Y-N Yang F Wang W Zhou Z-Q Wu and Y-Q XingldquoTNF-120572 stimulates MMP-2 and MMP-9 activities in humancorneal epithelial cells via the activation of FAKERK signalingrdquoOphthalmic Research vol 48 no 4 pp 165ndash170 2012

[15] A P Bhatt and B Damania ldquoAKTivation of PI3KAKTmTORsignaling pathway by KSHVrdquo Frontiers in Immunology vol 3 p401 2012

[16] N J Buchkovich Y Yu C A Zampieri and J C Alwine ldquoTheTORrid affairs of viruses Effects of mammalian DNA viruseson the PI3K-Akt-mTOR signalling pathwayrdquo Nature ReviewsMicrobiology vol 6 no 4 pp 265ndash275 2008

[17] N Diehl and H Schaal ldquoMake yourself at home viral hijackingof the PI3KAkt signaling pathwayrdquo Viruses vol 5 no 12 pp3192ndash3212 2013

[18] N Cheshenko J B Trepanier P A Gonzalez E A EugeninW R Jacobs and B C Herold ldquoHerpes simplex virus type 2glycoprotein h interacts with integrin 120572v1205733 to facilitate viralentry and calcium signaling in human genital tract epithelialcellsrdquo Journal of Virology vol 88 no 17 pp 10026ndash10038 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 5

0d 2d 7d 14d 28d

119 kDa

119 kDa

85 kDa

85 kDa

60 kDa

60 kDa

74 kDa

92 kDa

43 kDa

P-FAK

FAK

P-PI3K

PI3K

P-AKT

AKT

MMP-2

MMP-9

-Actin

Figure 4 Western Blot results of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 protein expression in mouse cornea at eachtime point

ab

ab

ab

ab

ab ab

ababab

ab

ab

a

a a

a

a

aa

b

b

0d 2d 7d 14d 28d

p-FAK

FAK

p-PI3K

PI3K

p-Akt

AktMMP-2MMP-9

The r

elativ

e exp

ress

ion

of p

rote

in

10

08

06

04

02

00

Figure 5 Relative expression of p-FAK FAK p-PI3K PI3K p-Akt Akt MMP-2 and MMP-9 in cornea at each time point Note aP lt 005compared with 0d bP lt 005 compared with 14d (one-way ANOVA LSD-t test)

202plusmn015 270plusmn026 and 199plusmn186 respectively The relativeexpression levels ofMMP-9mRNAwere 100plusmn000 151plusmn005181plusmn007 230plusmn015 and 194plusmn171 Compared with 0d therelative expression levels of MMP-2 and MMP-9 mRNAwere significantly increased at 2d 7d 14d and 28d andthe differences were statistically significant (all Plt 005)Compared with 14d the relative expression levels of MMP-2 and MMP-9 mRNA decreased at 2d 7d and 28d and thedifferences were statistically significant (all Plt 005) (Table 3)

34 Expression of p-FAK FAK p-PI3K PI3K p-Akt AktMMP-2 and MMP-9 in Corneal Tissue at Different TimePoints in HSK Mouse Model Western blot analysis showedthat the expression of p-FAK p-PI3K p-Akt MMP-2 andMMP-9 was increased in corneal tissue at 2 days afterinfection and decreased from 7d to 2d 14d and 28d Itrose again and peaked at 14d (Figures 4 and 5) The proteinexpressions of p-FAK p-PI3K p-Akt MMP-2 and MMP-9at 2d 14d and 28d were all significantly higher than those at

6 BioMed Research International

Table 3 The relative expression of MMP-2 and MMP-9 mRNA in the cornea at each time point (Xplusmns)

Time point MMP-2 MMP-90d 100plusmn000 100plusmn0002d 202plusmn015ab 181plusmn007ab

7d 159plusmn004ab 151plusmn005ab

14d 270plusmn026a 230plusmn015a

28d 199plusmn015ab 194plusmn002ab

F 5069 12216P lt005 lt005NoteaP lt 005 compared with 0d bP lt 005 compared with 14d (One-way ANOVA Dunnettrsquos t test)MMP matrix metalloproteinases

0d (Plt 005) The expression of p-Akt MMP-2 and MMP-9at 7 days after infection was significantly higher than that at 0days (Plt 005) At 14 days the expression of p-FAK p-PI3Kp-Akt andMMP-2 protein was higher than that at 2d 7d and28d and the difference was statistically significant (Plt 005)The expression ofMMP-9 protein at 7d was lower than that at14d and the difference was statistically significant (Plt 005)There was no significant difference in the expression levelsof FAK PI3K and Akt between tissues at all time points (Pgt005) (Table 4)

4 Discussion

HSK is one of the leading eye diseases in the world leadingto corneal blindness [3] Recurrent HSK will undoubtedlyaggravate the deterioration of the disease and eventually leadto blinding outcomes Although the research on molecularmechanisms related to the formation of HSK is increasingand deepening the pathogenesis of HSK is complex andits pathogenesis is still not fully understood There is noclinically satisfactory drug treatment that can prevent it fromrecurring or prevent it from developing into a bad outcomeTherefore it is imperative to explore drug treatment withmultiple pathways multiple targets and multiple therapeuticeffects

Recent research indicates that FAK not only participatesin the regulation of cell proliferation apoptosis migrationinvasion metastasis and adhesion but also participates in theregulation of the expression and activity ofMMPs and TIMPsthrough multiple signaling pathways [9ndash12] At the sametime our previous study found that there were two peaksin the phosphorylation of FAK in the corneal epithelium ofHSV-1-infected mouse corneal models The phosphorylationlevels of FAK increased on the 2d 14d and 28d afterinfection and decreased on the 7d compared with 2d [13]The expression pattern of MMP-2 andMMP-9 in the cornealmodel of HSV-1-infected mice in this study is very similarto our previous studies [4] In addition we also found thatTNF-120572 can increase the expression and secretion of MMP-2 and MMP-9 through the FAKERK signaling pathway invitro experiments [14] This further suggests the role of theFAK signaling pathway in HSK

PI3KAkt is an important signaling pathway downstreamof FAK that regulates many important biological signaling

pathways It has been reported that on the one handthe herpes virus can enhance its function of replicationtranscription and translation by regulating the PI3KAktsignaling pathwayOn the other hand the PI3KAkt signalingpathway can be used to facilitate virus entry replicationdelay and activation [15ndash17] Activation of PI3KAkt sig-naling can occur at multiple stages of the herpes virus lifecycle [16 17] Cheshenko reported that HSV can activateand release intracellular calcium and phosphorylate FAKvia the integrin signaling pathway which activates the AKTsignaling pathway and provides suitable conditions for virusentry into cells [18] Therefore we hypothesized that theFAKPI3KAkt signaling pathway and MMP-2 and MMP-9 may be involved in the development of HSK in thecornea

In this study we first studied the patterns of FAK p-FAKPI3K p-PI3K Akt and p-Akt in the FAKPI3KAkt signalingpathway and MMP-2 and MMP-9 in the HSK mouse modelBy Western blot we observed that the levels of p-FAK p-PI3K and p-Akt were upregulated on the 2d 7d 14d and28d after infection The upregulated level on the 7th daydecreased compared with that on the 2d day and reachedthe maximum on the 14d day At the same time the changesin the levels of MMP-2 and MMP-9 protein were similar tothose of p-FAK p-PI3K and p-Akt This is similar to ourprevious study [4] suggesting a potential link between theFAKPI3KAkt signaling pathway and theMMP-2 andMMP-9 In addition we have further demonstrated by RT-PCRand Immunofluorescence staining that the expression andactivation of MMP-2 and MMP-9 in the mouse cornea atthe early stage of HSV-1 infection play an active role in thedevelopment of HSK To further elucidate the relationshipbetween the FAKPI3KAkt signaling pathway and the HSKwe plan to use the inhibitor

In summary our results provide in vivo evidence thatFAKPI3KAkt signaling pathway and MMP-2 and MMP-9may be involved in the development of HSK The discoveryof the association between FAKPI3KAkt signaling pathwayMMP-2 and MMP-9 and HSK may facilitate the study ofthe mechanism of HSK and studies of the FAKPI3KAKTsignaling pathway may provide new targets for the treatmentofHSKHowever in order to show the regulatory relationshipbetween FAKPI3KAkt signaling pathway and MMP-2 andMMP-9 further research is necessary

BioMed Research International 7

Table4Com

paris

onof

relativee

xpressionlevelsof

p-FA

KFA

Kp-PI3K

PI3K

p-AktA

ktM

MP-2andMMP-9in

cornea

ateach

timep

oint

(Xplusmns)

Timep

oint

p-FA

KFA

Kp-PI3K

PI3K

p-Akt

Akt

MMP-2

MMP-9

0d004plusmn001

054plusmn002

004plusmn001

063plusmn002

004plusmn001

063plusmn000

004plusmn000

006plusmn004

2d026plusmn010

ab056plusmn002

024plusmn001

ab064plusmn001

029plusmn004

ab062plusmn001

045plusmn013

ab033plusmn016

a

7d019plusmn010

b055plusmn002

009plusmn003

b064plusmn001

014plusmn002

ab061plusmn000

024plusmn011

ab020plusmn015

ab

14d

054plusmn005

a057plusmn002

053plusmn003

a067plusmn000

047plusmn001

a064plusmn001

074plusmn009

a050plusmn013

a

28d

035plusmn010

ab057plusmn001

039plusmn005

ab066plusmn001

035plusmn004

ab063plusmn000

056plusmn008

ab042plusmn010

a

F1551

151

12338

873

11907

1014

2531

584

Plt005

gt005

lt005

gt005

lt005

gt005

lt005

lt005

Notea Plt005comparedwith

0dbPlt005comparedwith

14d(one-w

ayANOVA

LSD

-ttest)

8 BioMed Research International

Data Availability

The data used to support the findings of this study arecurrently kept under raps while the research findings aresignificant Requests for data 12 months after publication ofthis article will be considered by the corresponding authoron reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Project approval number 81370986)

References

[1] A J Lepisto G M Frank and R L Hendricks ldquoHow herpessimplex virus type 1 rescinds corneal privilegerdquo ChemicalImmunology and Allergy vol 92 pp 203ndash212 2007

[2] L Xie H Zhai X Dong et al ldquoPrimary diseases of corneal per-foration in Shandong Province China a 10-year retrospectivestudyrdquo American Journal of Ophthalmology vol 145 no 4 pp662ndash666 2008

[3] A V Farooq and D Shukla ldquoHerpes simplex epithelial andstromal keratitis an epidemiologic updaterdquo Survey of Ophthal-mology vol 57 no 5 pp 448ndash462 2012

[4] Y-N Yang D Bauer S Wasmuth K-P Steuhl and A Heili-genhaus ldquoMatrix metalloproteinases (MMP-2 and 9) and tissueinhibitors of matrix metalloproteinases (TIMP-1 and 2) dur-ing the course of experimental necrotizing herpetic keratitisrdquoExperimental Eye Research vol 77 no 2 pp 227ndash237 2003

[5] Z L Teo L McQueen-Miscamble K Turner et al ldquoIntegrinlinked kinase (ILK) is required for lens epithelial cell survivalproliferation and differentiationrdquo Experimental Eye Researchvol 121 no 3 pp 130ndash142 2014

[6] X-M Yang Y-S Wang J Zhang et al ldquoRole of PI3KAkt andMEKERK inmediating hypoxia-induced expression of HIF-1120572and VEGF in laser-induced rat choroidal neovascularizationrdquoInvestigative Ophthalmology amp Visual Science vol 50 no 4 pp1873ndash1879 2009

[7] S P Narayanan J Suwanpradid W Zhang et al ldquoArginasedepletion modulates Akt and iNOS to reduce retinal degenera-tion in a mouse model of retinopathy of prematurityrdquo Gut vol58 no 1 p 147 2010 author reply 147-8

[8] A Heiligenhaus D Bauer D Meller K-P Steuhl and S C GTseng ldquoImprovement of HSV-1 necrotizing keratitis with amni-otic membrane transplantationrdquo Investigative Ophthalmology ampVisual Science vol 42 no 9 pp 1969ndash1974 2001

[9] N A Chatzizacharias G P Kouraklis and S E TheocharisldquoClinical significance of FAK expression in human neoplasiardquoHistology and Histopathology vol 23 no 5 pp 629ndash650 2008

[10] B Hu M J Jarzynka P Guo Y Imanishi D D Schlaepfer andS-Y Cheng ldquoAngiopoietin 2 induces glioma cell invasion bystimulating matrix metalloprotease 2 expression through the120572v1205731 integrin and focal adhesion kinase signaling pathwayrdquoCancer Research vol 66 no 2 pp 775ndash783 2006

[11] M Canel P Secades M Garzon-Arango et al ldquoInvolvementof focal adhesion kinase in cellular invasion of head and neck

squamous cell carcinomas via regulation ofMMP-2 expressionrdquoBritish Journal of Cancer vol 98 no 7 pp 1274ndash1284 2008

[12] L-C Chang C-H Huang C-H Cheng B-H Chen and H-C Chen ldquoDifferential effect of the focal adhesion kinase Y397Fmutant on v-Src-stimulated cell invasion and tumor growthrdquoJournal of Biomedical Science vol 12 no 4 pp 571ndash585 2005

[13] Q Z Wu L Z Zhang W S Nie et al ldquoActivity of matrixmetalloproteinases 2 and 9 in cultured rabbit corneal epitheliumcells stimulated by tumor necrosis factor alphardquo Genetics andMolecular Research GMR vol 14 no 2 p 6360 2015

[14] Y-N Yang F Wang W Zhou Z-Q Wu and Y-Q XingldquoTNF-120572 stimulates MMP-2 and MMP-9 activities in humancorneal epithelial cells via the activation of FAKERK signalingrdquoOphthalmic Research vol 48 no 4 pp 165ndash170 2012

[15] A P Bhatt and B Damania ldquoAKTivation of PI3KAKTmTORsignaling pathway by KSHVrdquo Frontiers in Immunology vol 3 p401 2012

[16] N J Buchkovich Y Yu C A Zampieri and J C Alwine ldquoTheTORrid affairs of viruses Effects of mammalian DNA viruseson the PI3K-Akt-mTOR signalling pathwayrdquo Nature ReviewsMicrobiology vol 6 no 4 pp 265ndash275 2008

[17] N Diehl and H Schaal ldquoMake yourself at home viral hijackingof the PI3KAkt signaling pathwayrdquo Viruses vol 5 no 12 pp3192ndash3212 2013

[18] N Cheshenko J B Trepanier P A Gonzalez E A EugeninW R Jacobs and B C Herold ldquoHerpes simplex virus type 2glycoprotein h interacts with integrin 120572v1205733 to facilitate viralentry and calcium signaling in human genital tract epithelialcellsrdquo Journal of Virology vol 88 no 17 pp 10026ndash10038 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

Table 3 The relative expression of MMP-2 and MMP-9 mRNA in the cornea at each time point (Xplusmns)

Time point MMP-2 MMP-90d 100plusmn000 100plusmn0002d 202plusmn015ab 181plusmn007ab

7d 159plusmn004ab 151plusmn005ab

14d 270plusmn026a 230plusmn015a

28d 199plusmn015ab 194plusmn002ab

F 5069 12216P lt005 lt005NoteaP lt 005 compared with 0d bP lt 005 compared with 14d (One-way ANOVA Dunnettrsquos t test)MMP matrix metalloproteinases

0d (Plt 005) The expression of p-Akt MMP-2 and MMP-9at 7 days after infection was significantly higher than that at 0days (Plt 005) At 14 days the expression of p-FAK p-PI3Kp-Akt andMMP-2 protein was higher than that at 2d 7d and28d and the difference was statistically significant (Plt 005)The expression ofMMP-9 protein at 7d was lower than that at14d and the difference was statistically significant (Plt 005)There was no significant difference in the expression levelsof FAK PI3K and Akt between tissues at all time points (Pgt005) (Table 4)

4 Discussion

HSK is one of the leading eye diseases in the world leadingto corneal blindness [3] Recurrent HSK will undoubtedlyaggravate the deterioration of the disease and eventually leadto blinding outcomes Although the research on molecularmechanisms related to the formation of HSK is increasingand deepening the pathogenesis of HSK is complex andits pathogenesis is still not fully understood There is noclinically satisfactory drug treatment that can prevent it fromrecurring or prevent it from developing into a bad outcomeTherefore it is imperative to explore drug treatment withmultiple pathways multiple targets and multiple therapeuticeffects

Recent research indicates that FAK not only participatesin the regulation of cell proliferation apoptosis migrationinvasion metastasis and adhesion but also participates in theregulation of the expression and activity ofMMPs and TIMPsthrough multiple signaling pathways [9ndash12] At the sametime our previous study found that there were two peaksin the phosphorylation of FAK in the corneal epithelium ofHSV-1-infected mouse corneal models The phosphorylationlevels of FAK increased on the 2d 14d and 28d afterinfection and decreased on the 7d compared with 2d [13]The expression pattern of MMP-2 andMMP-9 in the cornealmodel of HSV-1-infected mice in this study is very similarto our previous studies [4] In addition we also found thatTNF-120572 can increase the expression and secretion of MMP-2 and MMP-9 through the FAKERK signaling pathway invitro experiments [14] This further suggests the role of theFAK signaling pathway in HSK

PI3KAkt is an important signaling pathway downstreamof FAK that regulates many important biological signaling

pathways It has been reported that on the one handthe herpes virus can enhance its function of replicationtranscription and translation by regulating the PI3KAktsignaling pathwayOn the other hand the PI3KAkt signalingpathway can be used to facilitate virus entry replicationdelay and activation [15ndash17] Activation of PI3KAkt sig-naling can occur at multiple stages of the herpes virus lifecycle [16 17] Cheshenko reported that HSV can activateand release intracellular calcium and phosphorylate FAKvia the integrin signaling pathway which activates the AKTsignaling pathway and provides suitable conditions for virusentry into cells [18] Therefore we hypothesized that theFAKPI3KAkt signaling pathway and MMP-2 and MMP-9 may be involved in the development of HSK in thecornea

In this study we first studied the patterns of FAK p-FAKPI3K p-PI3K Akt and p-Akt in the FAKPI3KAkt signalingpathway and MMP-2 and MMP-9 in the HSK mouse modelBy Western blot we observed that the levels of p-FAK p-PI3K and p-Akt were upregulated on the 2d 7d 14d and28d after infection The upregulated level on the 7th daydecreased compared with that on the 2d day and reachedthe maximum on the 14d day At the same time the changesin the levels of MMP-2 and MMP-9 protein were similar tothose of p-FAK p-PI3K and p-Akt This is similar to ourprevious study [4] suggesting a potential link between theFAKPI3KAkt signaling pathway and theMMP-2 andMMP-9 In addition we have further demonstrated by RT-PCRand Immunofluorescence staining that the expression andactivation of MMP-2 and MMP-9 in the mouse cornea atthe early stage of HSV-1 infection play an active role in thedevelopment of HSK To further elucidate the relationshipbetween the FAKPI3KAkt signaling pathway and the HSKwe plan to use the inhibitor

In summary our results provide in vivo evidence thatFAKPI3KAkt signaling pathway and MMP-2 and MMP-9may be involved in the development of HSK The discoveryof the association between FAKPI3KAkt signaling pathwayMMP-2 and MMP-9 and HSK may facilitate the study ofthe mechanism of HSK and studies of the FAKPI3KAKTsignaling pathway may provide new targets for the treatmentofHSKHowever in order to show the regulatory relationshipbetween FAKPI3KAkt signaling pathway and MMP-2 andMMP-9 further research is necessary

BioMed Research International 7

Table4Com

paris

onof

relativee

xpressionlevelsof

p-FA

KFA

Kp-PI3K

PI3K

p-AktA

ktM

MP-2andMMP-9in

cornea

ateach

timep

oint

(Xplusmns)

Timep

oint

p-FA

KFA

Kp-PI3K

PI3K

p-Akt

Akt

MMP-2

MMP-9

0d004plusmn001

054plusmn002

004plusmn001

063plusmn002

004plusmn001

063plusmn000

004plusmn000

006plusmn004

2d026plusmn010

ab056plusmn002

024plusmn001

ab064plusmn001

029plusmn004

ab062plusmn001

045plusmn013

ab033plusmn016

a

7d019plusmn010

b055plusmn002

009plusmn003

b064plusmn001

014plusmn002

ab061plusmn000

024plusmn011

ab020plusmn015

ab

14d

054plusmn005

a057plusmn002

053plusmn003

a067plusmn000

047plusmn001

a064plusmn001

074plusmn009

a050plusmn013

a

28d

035plusmn010

ab057plusmn001

039plusmn005

ab066plusmn001

035plusmn004

ab063plusmn000

056plusmn008

ab042plusmn010

a

F1551

151

12338

873

11907

1014

2531

584

Plt005

gt005

lt005

gt005

lt005

gt005

lt005

lt005

Notea Plt005comparedwith

0dbPlt005comparedwith

14d(one-w

ayANOVA

LSD

-ttest)

8 BioMed Research International

Data Availability

The data used to support the findings of this study arecurrently kept under raps while the research findings aresignificant Requests for data 12 months after publication ofthis article will be considered by the corresponding authoron reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Project approval number 81370986)

References

[1] A J Lepisto G M Frank and R L Hendricks ldquoHow herpessimplex virus type 1 rescinds corneal privilegerdquo ChemicalImmunology and Allergy vol 92 pp 203ndash212 2007

[2] L Xie H Zhai X Dong et al ldquoPrimary diseases of corneal per-foration in Shandong Province China a 10-year retrospectivestudyrdquo American Journal of Ophthalmology vol 145 no 4 pp662ndash666 2008

[3] A V Farooq and D Shukla ldquoHerpes simplex epithelial andstromal keratitis an epidemiologic updaterdquo Survey of Ophthal-mology vol 57 no 5 pp 448ndash462 2012

[4] Y-N Yang D Bauer S Wasmuth K-P Steuhl and A Heili-genhaus ldquoMatrix metalloproteinases (MMP-2 and 9) and tissueinhibitors of matrix metalloproteinases (TIMP-1 and 2) dur-ing the course of experimental necrotizing herpetic keratitisrdquoExperimental Eye Research vol 77 no 2 pp 227ndash237 2003

[5] Z L Teo L McQueen-Miscamble K Turner et al ldquoIntegrinlinked kinase (ILK) is required for lens epithelial cell survivalproliferation and differentiationrdquo Experimental Eye Researchvol 121 no 3 pp 130ndash142 2014

[6] X-M Yang Y-S Wang J Zhang et al ldquoRole of PI3KAkt andMEKERK inmediating hypoxia-induced expression of HIF-1120572and VEGF in laser-induced rat choroidal neovascularizationrdquoInvestigative Ophthalmology amp Visual Science vol 50 no 4 pp1873ndash1879 2009

[7] S P Narayanan J Suwanpradid W Zhang et al ldquoArginasedepletion modulates Akt and iNOS to reduce retinal degenera-tion in a mouse model of retinopathy of prematurityrdquo Gut vol58 no 1 p 147 2010 author reply 147-8

[8] A Heiligenhaus D Bauer D Meller K-P Steuhl and S C GTseng ldquoImprovement of HSV-1 necrotizing keratitis with amni-otic membrane transplantationrdquo Investigative Ophthalmology ampVisual Science vol 42 no 9 pp 1969ndash1974 2001

[9] N A Chatzizacharias G P Kouraklis and S E TheocharisldquoClinical significance of FAK expression in human neoplasiardquoHistology and Histopathology vol 23 no 5 pp 629ndash650 2008

[10] B Hu M J Jarzynka P Guo Y Imanishi D D Schlaepfer andS-Y Cheng ldquoAngiopoietin 2 induces glioma cell invasion bystimulating matrix metalloprotease 2 expression through the120572v1205731 integrin and focal adhesion kinase signaling pathwayrdquoCancer Research vol 66 no 2 pp 775ndash783 2006

[11] M Canel P Secades M Garzon-Arango et al ldquoInvolvementof focal adhesion kinase in cellular invasion of head and neck

squamous cell carcinomas via regulation ofMMP-2 expressionrdquoBritish Journal of Cancer vol 98 no 7 pp 1274ndash1284 2008

[12] L-C Chang C-H Huang C-H Cheng B-H Chen and H-C Chen ldquoDifferential effect of the focal adhesion kinase Y397Fmutant on v-Src-stimulated cell invasion and tumor growthrdquoJournal of Biomedical Science vol 12 no 4 pp 571ndash585 2005

[13] Q Z Wu L Z Zhang W S Nie et al ldquoActivity of matrixmetalloproteinases 2 and 9 in cultured rabbit corneal epitheliumcells stimulated by tumor necrosis factor alphardquo Genetics andMolecular Research GMR vol 14 no 2 p 6360 2015

[14] Y-N Yang F Wang W Zhou Z-Q Wu and Y-Q XingldquoTNF-120572 stimulates MMP-2 and MMP-9 activities in humancorneal epithelial cells via the activation of FAKERK signalingrdquoOphthalmic Research vol 48 no 4 pp 165ndash170 2012

[15] A P Bhatt and B Damania ldquoAKTivation of PI3KAKTmTORsignaling pathway by KSHVrdquo Frontiers in Immunology vol 3 p401 2012

[16] N J Buchkovich Y Yu C A Zampieri and J C Alwine ldquoTheTORrid affairs of viruses Effects of mammalian DNA viruseson the PI3K-Akt-mTOR signalling pathwayrdquo Nature ReviewsMicrobiology vol 6 no 4 pp 265ndash275 2008

[17] N Diehl and H Schaal ldquoMake yourself at home viral hijackingof the PI3KAkt signaling pathwayrdquo Viruses vol 5 no 12 pp3192ndash3212 2013

[18] N Cheshenko J B Trepanier P A Gonzalez E A EugeninW R Jacobs and B C Herold ldquoHerpes simplex virus type 2glycoprotein h interacts with integrin 120572v1205733 to facilitate viralentry and calcium signaling in human genital tract epithelialcellsrdquo Journal of Virology vol 88 no 17 pp 10026ndash10038 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

Table4Com

paris

onof

relativee

xpressionlevelsof

p-FA

KFA

Kp-PI3K

PI3K

p-AktA

ktM

MP-2andMMP-9in

cornea

ateach

timep

oint

(Xplusmns)

Timep

oint

p-FA

KFA

Kp-PI3K

PI3K

p-Akt

Akt

MMP-2

MMP-9

0d004plusmn001

054plusmn002

004plusmn001

063plusmn002

004plusmn001

063plusmn000

004plusmn000

006plusmn004

2d026plusmn010

ab056plusmn002

024plusmn001

ab064plusmn001

029plusmn004

ab062plusmn001

045plusmn013

ab033plusmn016

a

7d019plusmn010

b055plusmn002

009plusmn003

b064plusmn001

014plusmn002

ab061plusmn000

024plusmn011

ab020plusmn015

ab

14d

054plusmn005

a057plusmn002

053plusmn003

a067plusmn000

047plusmn001

a064plusmn001

074plusmn009

a050plusmn013

a

28d

035plusmn010

ab057plusmn001

039plusmn005

ab066plusmn001

035plusmn004

ab063plusmn000

056plusmn008

ab042plusmn010

a

F1551

151

12338

873

11907

1014

2531

584

Plt005

gt005

lt005

gt005

lt005

gt005

lt005

lt005

Notea Plt005comparedwith

0dbPlt005comparedwith

14d(one-w

ayANOVA

LSD

-ttest)

8 BioMed Research International

Data Availability

The data used to support the findings of this study arecurrently kept under raps while the research findings aresignificant Requests for data 12 months after publication ofthis article will be considered by the corresponding authoron reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Project approval number 81370986)

References

[1] A J Lepisto G M Frank and R L Hendricks ldquoHow herpessimplex virus type 1 rescinds corneal privilegerdquo ChemicalImmunology and Allergy vol 92 pp 203ndash212 2007

[2] L Xie H Zhai X Dong et al ldquoPrimary diseases of corneal per-foration in Shandong Province China a 10-year retrospectivestudyrdquo American Journal of Ophthalmology vol 145 no 4 pp662ndash666 2008

[3] A V Farooq and D Shukla ldquoHerpes simplex epithelial andstromal keratitis an epidemiologic updaterdquo Survey of Ophthal-mology vol 57 no 5 pp 448ndash462 2012

[4] Y-N Yang D Bauer S Wasmuth K-P Steuhl and A Heili-genhaus ldquoMatrix metalloproteinases (MMP-2 and 9) and tissueinhibitors of matrix metalloproteinases (TIMP-1 and 2) dur-ing the course of experimental necrotizing herpetic keratitisrdquoExperimental Eye Research vol 77 no 2 pp 227ndash237 2003

[5] Z L Teo L McQueen-Miscamble K Turner et al ldquoIntegrinlinked kinase (ILK) is required for lens epithelial cell survivalproliferation and differentiationrdquo Experimental Eye Researchvol 121 no 3 pp 130ndash142 2014

[6] X-M Yang Y-S Wang J Zhang et al ldquoRole of PI3KAkt andMEKERK inmediating hypoxia-induced expression of HIF-1120572and VEGF in laser-induced rat choroidal neovascularizationrdquoInvestigative Ophthalmology amp Visual Science vol 50 no 4 pp1873ndash1879 2009

[7] S P Narayanan J Suwanpradid W Zhang et al ldquoArginasedepletion modulates Akt and iNOS to reduce retinal degenera-tion in a mouse model of retinopathy of prematurityrdquo Gut vol58 no 1 p 147 2010 author reply 147-8

[8] A Heiligenhaus D Bauer D Meller K-P Steuhl and S C GTseng ldquoImprovement of HSV-1 necrotizing keratitis with amni-otic membrane transplantationrdquo Investigative Ophthalmology ampVisual Science vol 42 no 9 pp 1969ndash1974 2001

[9] N A Chatzizacharias G P Kouraklis and S E TheocharisldquoClinical significance of FAK expression in human neoplasiardquoHistology and Histopathology vol 23 no 5 pp 629ndash650 2008

[10] B Hu M J Jarzynka P Guo Y Imanishi D D Schlaepfer andS-Y Cheng ldquoAngiopoietin 2 induces glioma cell invasion bystimulating matrix metalloprotease 2 expression through the120572v1205731 integrin and focal adhesion kinase signaling pathwayrdquoCancer Research vol 66 no 2 pp 775ndash783 2006

[11] M Canel P Secades M Garzon-Arango et al ldquoInvolvementof focal adhesion kinase in cellular invasion of head and neck

squamous cell carcinomas via regulation ofMMP-2 expressionrdquoBritish Journal of Cancer vol 98 no 7 pp 1274ndash1284 2008

[12] L-C Chang C-H Huang C-H Cheng B-H Chen and H-C Chen ldquoDifferential effect of the focal adhesion kinase Y397Fmutant on v-Src-stimulated cell invasion and tumor growthrdquoJournal of Biomedical Science vol 12 no 4 pp 571ndash585 2005

[13] Q Z Wu L Z Zhang W S Nie et al ldquoActivity of matrixmetalloproteinases 2 and 9 in cultured rabbit corneal epitheliumcells stimulated by tumor necrosis factor alphardquo Genetics andMolecular Research GMR vol 14 no 2 p 6360 2015

[14] Y-N Yang F Wang W Zhou Z-Q Wu and Y-Q XingldquoTNF-120572 stimulates MMP-2 and MMP-9 activities in humancorneal epithelial cells via the activation of FAKERK signalingrdquoOphthalmic Research vol 48 no 4 pp 165ndash170 2012

[15] A P Bhatt and B Damania ldquoAKTivation of PI3KAKTmTORsignaling pathway by KSHVrdquo Frontiers in Immunology vol 3 p401 2012

[16] N J Buchkovich Y Yu C A Zampieri and J C Alwine ldquoTheTORrid affairs of viruses Effects of mammalian DNA viruseson the PI3K-Akt-mTOR signalling pathwayrdquo Nature ReviewsMicrobiology vol 6 no 4 pp 265ndash275 2008

[17] N Diehl and H Schaal ldquoMake yourself at home viral hijackingof the PI3KAkt signaling pathwayrdquo Viruses vol 5 no 12 pp3192ndash3212 2013

[18] N Cheshenko J B Trepanier P A Gonzalez E A EugeninW R Jacobs and B C Herold ldquoHerpes simplex virus type 2glycoprotein h interacts with integrin 120572v1205733 to facilitate viralentry and calcium signaling in human genital tract epithelialcellsrdquo Journal of Virology vol 88 no 17 pp 10026ndash10038 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

Data Availability

The data used to support the findings of this study arecurrently kept under raps while the research findings aresignificant Requests for data 12 months after publication ofthis article will be considered by the corresponding authoron reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Project approval number 81370986)

References

[1] A J Lepisto G M Frank and R L Hendricks ldquoHow herpessimplex virus type 1 rescinds corneal privilegerdquo ChemicalImmunology and Allergy vol 92 pp 203ndash212 2007

[2] L Xie H Zhai X Dong et al ldquoPrimary diseases of corneal per-foration in Shandong Province China a 10-year retrospectivestudyrdquo American Journal of Ophthalmology vol 145 no 4 pp662ndash666 2008

[3] A V Farooq and D Shukla ldquoHerpes simplex epithelial andstromal keratitis an epidemiologic updaterdquo Survey of Ophthal-mology vol 57 no 5 pp 448ndash462 2012

[4] Y-N Yang D Bauer S Wasmuth K-P Steuhl and A Heili-genhaus ldquoMatrix metalloproteinases (MMP-2 and 9) and tissueinhibitors of matrix metalloproteinases (TIMP-1 and 2) dur-ing the course of experimental necrotizing herpetic keratitisrdquoExperimental Eye Research vol 77 no 2 pp 227ndash237 2003

[5] Z L Teo L McQueen-Miscamble K Turner et al ldquoIntegrinlinked kinase (ILK) is required for lens epithelial cell survivalproliferation and differentiationrdquo Experimental Eye Researchvol 121 no 3 pp 130ndash142 2014

[6] X-M Yang Y-S Wang J Zhang et al ldquoRole of PI3KAkt andMEKERK inmediating hypoxia-induced expression of HIF-1120572and VEGF in laser-induced rat choroidal neovascularizationrdquoInvestigative Ophthalmology amp Visual Science vol 50 no 4 pp1873ndash1879 2009

[7] S P Narayanan J Suwanpradid W Zhang et al ldquoArginasedepletion modulates Akt and iNOS to reduce retinal degenera-tion in a mouse model of retinopathy of prematurityrdquo Gut vol58 no 1 p 147 2010 author reply 147-8

[8] A Heiligenhaus D Bauer D Meller K-P Steuhl and S C GTseng ldquoImprovement of HSV-1 necrotizing keratitis with amni-otic membrane transplantationrdquo Investigative Ophthalmology ampVisual Science vol 42 no 9 pp 1969ndash1974 2001

[9] N A Chatzizacharias G P Kouraklis and S E TheocharisldquoClinical significance of FAK expression in human neoplasiardquoHistology and Histopathology vol 23 no 5 pp 629ndash650 2008

[10] B Hu M J Jarzynka P Guo Y Imanishi D D Schlaepfer andS-Y Cheng ldquoAngiopoietin 2 induces glioma cell invasion bystimulating matrix metalloprotease 2 expression through the120572v1205731 integrin and focal adhesion kinase signaling pathwayrdquoCancer Research vol 66 no 2 pp 775ndash783 2006

[11] M Canel P Secades M Garzon-Arango et al ldquoInvolvementof focal adhesion kinase in cellular invasion of head and neck

squamous cell carcinomas via regulation ofMMP-2 expressionrdquoBritish Journal of Cancer vol 98 no 7 pp 1274ndash1284 2008

[12] L-C Chang C-H Huang C-H Cheng B-H Chen and H-C Chen ldquoDifferential effect of the focal adhesion kinase Y397Fmutant on v-Src-stimulated cell invasion and tumor growthrdquoJournal of Biomedical Science vol 12 no 4 pp 571ndash585 2005

[13] Q Z Wu L Z Zhang W S Nie et al ldquoActivity of matrixmetalloproteinases 2 and 9 in cultured rabbit corneal epitheliumcells stimulated by tumor necrosis factor alphardquo Genetics andMolecular Research GMR vol 14 no 2 p 6360 2015

[14] Y-N Yang F Wang W Zhou Z-Q Wu and Y-Q XingldquoTNF-120572 stimulates MMP-2 and MMP-9 activities in humancorneal epithelial cells via the activation of FAKERK signalingrdquoOphthalmic Research vol 48 no 4 pp 165ndash170 2012

[15] A P Bhatt and B Damania ldquoAKTivation of PI3KAKTmTORsignaling pathway by KSHVrdquo Frontiers in Immunology vol 3 p401 2012

[16] N J Buchkovich Y Yu C A Zampieri and J C Alwine ldquoTheTORrid affairs of viruses Effects of mammalian DNA viruseson the PI3K-Akt-mTOR signalling pathwayrdquo Nature ReviewsMicrobiology vol 6 no 4 pp 265ndash275 2008

[17] N Diehl and H Schaal ldquoMake yourself at home viral hijackingof the PI3KAkt signaling pathwayrdquo Viruses vol 5 no 12 pp3192ndash3212 2013

[18] N Cheshenko J B Trepanier P A Gonzalez E A EugeninW R Jacobs and B C Herold ldquoHerpes simplex virus type 2glycoprotein h interacts with integrin 120572v1205733 to facilitate viralentry and calcium signaling in human genital tract epithelialcellsrdquo Journal of Virology vol 88 no 17 pp 10026ndash10038 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom