Section A
Comprehensive Reading
理解性阅读

Label-free optical biomarkers detect early calcific ^① aortic ^② valve ^③ disease in a wild-type mouse model 非标记光学生物标记物检测野生型小鼠模型早期钙化性主动脉瓣病变

① calcific ［kælˈsɪfɪk］adj.钙化的

② aortic ［eɪˈɔːtɪk］adj.主动脉的

③ valve ［vælv］n.瓣膜

Ishita Tandon ¹ ，Shelby Johns ¹ ，Alan Woessner ¹ ，Jessica Perez ¹ ，Delaney Cross ¹ ，Asya Ozkizilcik ¹ ，Timothy J Muldoon ¹ ，Srikanth Vallurupalli ² ，Muralidhar Padala ³ ，Kyle P Quinn ¹ ，Kartik Balachandran ^1*

*Correspondence：kbalacha@uark.edu

¹ Department of Biomedical Engineering，University of Arkansas，122 John，A.White Jr.Engineering Hall，Fayetteville，AR 72701，USA

Full list of author information is available at the end of the article.

BMC Cardiovasc Disord 2020 Dec 11；20（1）：521

PMID：33308143

PMCID：PMC7731510

DOI：10.1186/s12872-020-01776-8

© The Author（s）2020.Open Access.This article is licensed under a Creative Commons Attribution 4.0 International License，which permits use，sharing，adaptation，distribution and reproduction in any medium or format，as long as you give appropriate credit to the original author（s）and the source，provide a link to the Creative Commons licence，and indicate if changes were made.The images or other third party material in this article are included in the article’s Creative Commons licence，unless indicated otherwise in a credit line to the material.If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use，you will need to obtain permission directly from the copyright holder.To view a copy of this licence，visit http：//creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver（http：//creativeco mmons.org/publicdomain/zero/1.0/）applies to the data made available in this article，unless otherwise stated in a credit line to the data.

Abstract

Background： Calcific aortic valve disease（CAVD）pathophysiology is a complex，multistage process，usually diagnosed at advanced stages after significant anatomical ^① and hemodynamic ^② changes in the valve.Early detection of disease progression is thus pivotal in the development of prevention and mitigation ^③ strategies.In this study，we developed a diet-based，non-genetically modified mouse model for early CAVD progression，and explored the utility of two-photon excited fluorescence ^④ （TPEF）microscopy for early detection of CAVD progression.TPEF imaging provides label-free，non-invasive，quantitative metrics ^⑤ with the potential to correlate with multiple stages of CAVD pathophysiology including calcium deposition ^⑥ ，collagen ^⑦ remodeling and osteogenic ^⑧ differentiation ^⑨ .

① anatomical ［ˌænəˈtɒmɪkl］adj.解剖（学）的

② hemodynamic ［ˌhemədaɪˈnæmɪk］adj.血液动力学的

③ mitigation ［ˌmɪtɪˈɡeɪʃn］n.减轻，缓解

④ 双光子激发荧光

⑤ metrics ［ˈmetrɪks］n.指标

⑥ deposition ［ˌdepəˈzɪʃn］n.沉积物

⑦ collagen ［ˈkɒlədʒən］n.胶原

⑧ osteogenic ［ˌɒsti:ədʒenɪk］adj.成骨的

⑨ differentiation ［ˌdɪfəˌrenʃɪˈeɪʃn］n.分化

Methods： Twenty-week-old C57BL/6J mice were fed either a control or pro-calcific diet for 16 weeks and monitored via echocardiography ^① ，histology， immunohistochemistry ^② ，and quantitative polarized ^③ light imaging.Additionally，TPEF imaging was used to quantify tissue autofluorescence（A）at 755 nm，810 nm and 860 nm excitation，to calculate TPEF 755-860 ratio（A _860/525 /（A _755/460 + A _860/525 ））and TPEF collagen-calcium ratio（A _810/525 /（A _810/460 + A _810/525 ））in the murine valves.In a separate experiment，animals were fed the above diets till 28 weeks to assess for later-stage calcification.

① echocardiography ［，ekəʊkɑːdɪˈɒɡrəfɪ］n.超声心动图

② immunohistochemistry ［ˈɪmjʊnəʊˌhɪstəʊˈkemɪstrɪ］n.免疫组织化学

③ polarized ［ˈpəʊləraɪzd］adj.偏振的

Results ：Pro-calcific mice showed evidence of lipid ^④ deposition at 4 weeks and calcification at 16 weeks at the valve commissures.The valves of pro-calcific mice also showed positive expression for markers of osteogenic differentiation， myofibroblast ^⑤ activation，proliferation，inflammatory cytokines ^⑥ and collagen remodeling.Pro-calcific mice exhibited lower TPEF autofluorescence ratios，at locations coincident with calcification，that correlated with increased collagen disorganization and positive expression of osteogenic markers.Additionally，locations with lower TPEF autofluorescence ratios at 4 and 16 weeks exhibited increased calcification at later 28-week time points.

④ lipid ［ˈlɪpɪd］n.脂质

⑤ myofibroblast ［maɪɔːˈfaɪbrɒblɑːst］n.肌成纤维细胞

⑥ cytokine ［ˈsaɪtəʊˌkaɪn］n.细胞因子

⑦ valvulopathy ［vælvjʊˈlɒpəθɪ］n.瓣膜病

Conclusions ：This study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology.

Keywords ：Calcific aortic valve disease，Valve calcification，Two-photon excited fluorescence microscopy，Wild-type mouse model

Background

Calcific aortic valve disease（CAVD）is the most common form of valvulopathy ^⑦ in the western world，with a prevalence ^⑧ of 13.3% in people above 75 years of age ［1，2］.CAVD is associated with a 50% elevated risk of morbidity ^⑨ and mortality ^⑩ ［3］.Once severe aortic stenosis ^⑪ develops，valve replacement，either via surgical or transcatheter approaches，is the current treatment standard，as early detection，prevention，and mitigation strategies have yet to be clinically adopted ［1，2，4］.There is thus a need to develop better diagnostic and therapeutic approaches for CAVD.

⑧ prevalence ［ˈprevələns］n.患病率

⑨ morbidity ［mɔːˈbɪdɪtɪ］n.发病率，病态

⑩ mortality ［mɔːˈtælətɪ］n.死亡率，死亡

⑪ stenosis ［stɪˈnəʊsɪs］n.狭窄

The early pathogenic processes in CAVD are defined by the phenotypic ^① transformation of valve endothelial ^② （VEC）and interstitial ^③ （VIC）cells via cellular activation，osteogenic differentiation ［5-7］，as well as maladaptive extracellular matrix ^④ （ECM）remodeling ［8，9］.CAVD pathophysiology is marked by infiltration ^⑤ of cytokines，lipid deposition，and calcific nodule ^⑥ formation ［4，10，11］.These early pathogenic processes are simulated ^⑦ using twoand three- dimensional ^⑧ in vitro models and in vivo models that mimic the CAVD process and valve milieu ^⑨ to varying degrees of accuracy ［7，12-15］. Wild-type diet-based mouse CAVD models have previously demonstrated deposition of monocyte-macrophages ^⑩ ，lipids，and lipoproteins ［16，17］，but to our knowledge no such diet-based models exhibit early calcification as demonstrated by positive Alizarin ^⑪ Red S（ARS）staining，as well as positive osteopontin and Runt-related transcription factor 2（RUNX2）expression ［14，17］ ^⑫ .Assmann and colleagues have shown that a diet regimen ^⑬ supplemented with vitamin D， cholesterol ^⑭ and dicalcium ^⑮ phosphate ^⑯ caused significant calcification of the aortic root in Wistar rats ［18］.Vitamin D in combination with cholesterol has been separately shown to increase vascular calcification in rats［19］.Vitamin D is responsible for maintaining serum calcium levels in humans and both deficiency and excess of Vitamin D has been shown to promote cardiovascular calcification ［20-22］.Higher calcium phosphate and elevated serum calcium are also established risk factors for calcification progression ［21，22］.We exploited a similar dietary regimen to develop a wild-type mouse model for early CAVD.

① phenotypic ［ˌfi:nəˈtɪpɪk］adj.表型的

② endothelial ［ˌendəʊˈθiːlɪəl］adj.内皮的

③ interstitial ［ˌɪntəˈstɪʃl］adj.间质的

④ matrix ［ˈmeɪtrɪks］n.基质

⑤ infiltration ［ˌɪnfɪlˈtreɪʃn］n.渗透

⑥ nodule ［ˈnɒdjuːl］n.小节结

⑦ simulate ［ˈsɪmjuleɪt］v.模拟

⑧ dimensional ［dɪˈmenʃənəl］adj.维度的

⑨ milieu ［mɪlˈjɜː］n.环境

⑩ monocyte-macrophage ［ˈmɒnəsaɪt ˈmækrəfeɪdʒ］单核巨噬细胞

⑪ Alizarin ［əˈlɪz（ə）rɪn］n.茜草色素

⑫ 野生型饮食基础的小鼠CAVD模型先前已经显示出单核巨噬细胞、脂质和脂蛋白的沉积，但据我们所知，这种饮食基础的模型没有表现出早期钙化，如茜素红S（ARS）染色阳性、骨桥蛋白和Run相关转录因子2（RUNX2）的阳性表达。本句为并列复合句，并列连词but连接了两个并列句；前句的主干是models have demonstrated deposition，后句的主干是no such diet-based models exhibit calcification，as引导方式状语从句，as与demonstrated之间省略了they are，因此后一个句子是复合句，意思是像……所展示/证实的那样。

⑬ regimen ［ˈredʒɪmən］n.方案

⑭ cholesterol ［kəˈlestərɒl］n.胆固醇

⑮ dicalcium ［daɪˈkælsɪəm］n.二钙化物

⑯ phosphate ［ˈfɒsfeɪt］n.磷酸盐

Multiple biochemical and imaging markers are associated with independent events such as endothelial dysfunction，inflammatory cytokine infiltration，matrix remodeling，and mineral deposition to assess either the presence or severity of calcification ［2，4，10，11，23］.However，there is a dearth ^① of label-free imaging biomarkers to monitor the progression of calcification in the aortic valve，both in clinics and research laboratories ［23］.Clinically，a stenotic aortic valve usually is detected by auscultation ^② of a systolic ^③ murmur ^④ and confirmed by echocardiography，and relies on late hallmarks of CAVD like hemodynamic malfunction and impaired geometry of the valve ［24］.Imaging techniques such as cardiac magnetic resonance ^⑤ imaging and cardiac computed tomography ^⑥ have also been shown to be sensitive to the late hallmarks of the disease；however，there is a specific lack of tools to detect early CAVD progression ［24-26］.Two-photon excited fluorescence（TPEF）microscopy has recently shown potential in providing label-free quantitative metrics that might associate with osteogenic differentiation ［27］. TPEF allows quantification of the endogenous fluorescence ratios of the cellular co-factors fl avin ^⑦ adenine ^⑧ （FAD）and nicotinamide ^⑨ adenine（NADH） dinucleotides ^⑩ in their oxidized and reduced forms，respectively and is known as the optical redox ratio（i.e.FAD/（FAD + NADH））［27］ ^⑪ .It was previously established in vitro that osteogenic differentiation of mesenchymal ^⑫ stem cells is associated with a reduction in TPEF-derived optical redox ratios ［27］. We have also demonstrated that this TPEF-based optical redox ratios in VICs was reduced as cellular stretching increased from normal to pathologic magnitudes ［13］ . ^⑬ Additionally，optical redox ratio altered and correlated with temporal changes in VIC phenotype during osteogenic de-differentiation，in vitro ［28］.In addition to NADH and FAD，molecules such as collagen，calcium and lipids within tissues will contribute to the autofluorescence emission ^⑭ in the visible range ［27，29-32］.Recently，TPEF has been used to identify endogenous fluorescence from calcium deposition that correlated with mineralization in ApoE ^-/- mice and calcified human valves ［29］.

① dearth ［dɜːθ］n.缺乏

② auscultation ［ˌɔːskəlˈteɪʃn］n.听诊

③ systolic ［ˌsɪˈstɒlɪk］adj.（心脏）收缩（期）的

④ murmur ［ˈmɜːmə（r）］n.杂音

⑤ resonance ［ˈrezənəns］n.共振

⑥ tomography ［təˈmɒɡrəfɪ］n.X 线断层摄影术

⑦ flavin ［ˈfleɪvɪn］n.黄素

⑧ adenine ［ˈædəˌnɪn］n.腺嘌呤

⑨ nicotinamide ［nɪkəˈtɪnəmaɪd］n.烟酰胺，尼克酰胺

⑩ dinucleotide ［daɪˈnjuːklɪətaɪd］n.二核苷酸

⑪ TPEF允许细胞辅因子黄素腺嘌呤（FAD）和烟酰胺腺嘌呤（NADH）二核苷酸分别以氧化和还原形式进行内源荧光比率的定量，称为光学氧化还原比［即FAD /（FAD + NADH）］。本句是个简单句型，主干为TPEF allows quantification…and is known as…，有两个谓语部分，of…dinucleotides为介词of引导的介词短语修饰前面的quantification，而其中的of引导的介词短语作后置定语修饰前面的ratios，in their oxidized and reduced forms作句子的方式状语，说明以……在读到is的时候，有的作者会找不到它的主语，造成理解上的困难。

⑫ mesenchymal ［mesˈeŋkɪməl］adj.间质的

⑬ 我们还证明了，随着从正常幅度到病理程度细胞拉伸的增加，这个VICs中基于TPEF的光学氧化还原比率降低。句中as引导的是伴随状语从句。

⑭ emission ［ɪˈmɪʃn］n.发射，散

In this study，based on the above prior work，we hypothesized that TPEF autofluorescence markers would coincide with traditional phenotypic markers that indicate early progression of CAVD.We tested this hypothesis using ex vivo imaging of valves from a diet-based wild-type C57BL/6J mouse model that demonstrated early CAVD progression with lipid and calcium deposition，and matrix remodeling. In our model，the aortic valve commissures showed lower TPEF autofluorescence ratios，which was concurrent ^① with increased cell activation，inflammatory cytokine expression，cell proliferation and markers for osteogenic differentiation，and which was predictive of increased calcification at later time points . ^②

① concurrent ［kənˈkʌrənt］adj.并发的，一致的

② 在我们的模型中，主动脉瓣连合处显示了较低的TPEF自体荧光比率，这与细胞活化、炎症细胞因子表达、细胞增殖和成骨分化标记物的增加同时发生，并预示着后期钙化增加。which引导的非限定性定语从句，两个which均指代the aortic valve commissures showed lower TPEF autofluorescence ratios。

Methods

Experimental animals for early-CAVD model

All animal experiments were performed in compliance with the University of Arkansas Institutional Animal Care and Use Committee（IACUC）approval and conform to all appropriate guidelines.Male wild-type C57BL/6J mice were obtained from The Jackson Laboratory（Bar Harbor，ME）and housed at most four per cage under optimum temperature，humidity and light cycle.Adult mice aged 20 weeks ［33］were randomized and fed an open standard diet，with either a control or pro-calcific diet supplementation（Research Diets，New Brunswick，NJ）.The differences in the dietary supplements ［18］are outlined in Table 1.The amount of food intake was monitored per cage and the mice were weighed every three to four days till 16 weeks.In a separate experiment，animals were fed the above diets till 28 weeks to assess for later-stage calcification.

Echocardiography for animal model

Echocardiography was performed for all the mice in the study using standard protocols ［5］.At the 4，8，12，16 and 28-week time points，mice were anesthetized ^① using inhalative 4% isoflurane ^② with 2 L/Min oxygen.Mice were maintained at 1% isoflurane with 2 L/Min oxygen through a nose cone on a heated pad for the entire echocardiography procedure.After hair removal from the ventral ^③ side，an Agilent SONOS 5500（Agilent Technologies，Santa Clara，CA） ultrasound ^④ machine was used to obtain and analyze 2D and M-mode images to measure the diastolic（LVIDd）and systolic（LVIDs）left ventricle internal dimensions ^⑤ .The heart rate（HR）was monitored and recorded simultaneously via Covidien Kendall 230 conductive adhesive ^⑥ hydrogel ^⑦ electrodes ^⑧ （Dublin，Ireland）. Ejection Fraction （ EF ） ^⑨ ，End Diastolic Volume（EDV），End Systolic Volume（ESV）and Cardiac Output（CO）were assessed using Simpson’s method，via the below Eqs.［5］.At least seven animals per condition per time point were assessed.

① anesthetize ［æˈniːsθətaɪz］vt.使麻醉

② isoflurane ［ˌaɪsəʊˈfluəreɪn］n.异氟醚

③ ventral ［ˈventrəl］adj.腹侧的

④ ultrasound ［ˈʌltrəsaʊnd］n.超声

⑤ dimension ［dɪˈmenʃn］n.尺寸

⑥ adhesive ［ədˈhiːsɪv］adj.黏合的

⑦ hydrogel ［ˈhaɪdrə（ʊ）dʒel］n.水凝胶

⑧ electrode ［ɪˈlektrəʊd］n.电极

⑨ ejection fraction ［i（ː）ˈdʒɛkʃən ˈfrækʃn］射血分数

Pathophysiological assessment and tissue processing

At the aforementioned time points，the animals were euthanized ^⑩ by cervical ^⑪ dislocation，and their hearts were extracted ^⑫ ，washed thoroughly in cold， sterile ^⑬ PBS and frozen in optimum cutting temperature compound（Sakura Finetek USA Inc，Torrance，CA）.Serial 5-8 µm transverse ^⑭ sections of the aortic valve were obtained using a Leica CM1860 cryotome（Buffalo grove，IL）.Blood was collected in heparin ^① coated ^② tubes and plasma was stored at -80℃freezer for further processing.

⑩ euthanize ［ˈjuːθənaɪz］vt.使安乐死

⑪ cervical ［ˈsɜːvɪkl］adj.颈部的

⑫extract ［ˈekstrækt］v.提取，选录

⑬ sterile ［ˈsteraɪl］adj.无菌的

⑭ transverse ［ˈtrænzvɜːs］adj.横向的

① heparin ［ˈhepərɪn］n.肝素

② coated ［ˈkəʊtɪd］adj.上涂料的

Histology and Immunohistochemistry

Hematoxylin ^③ and eosin（H&E；IHC World，Woodstock，MD），Oil red O（Electron Microscopy Sciences，Hatfield，PA），Alizarin Red S（ARS；Sigma-Aldrich，St.Louis，MO）and Picrosirius ^④ Red（PSR；Electron Microscopy Sciences，Hatfield，PA）staining were performed using standard protocols ［5，18］.Oil red O was utilized to assess lipid deposition，ARS was used for detecting calcium deposition and PSR stain was used to analyze tissue collagen fibers.Immunohistochemistry was performed using primary antibodies（all from Abcam，Cambridge，MA）against α -SMA（1∶100）， vimentin ^⑤ （1∶300），RUNX2（1∶200），osteopontin（1∶200），BMP4（1∶100），Ki67（1∶500），TGF β 1（1∶50）.Appropriate Alexa fluor-488 and -594（1∶200；Life Technologies，Carlsbad，CA）conjugated secondary antibodies were used for immunolabeling.Negative controls were labeled only with secondary antibodies（Additional file 1：Fig.1）to assess nonspecific binding.A Nikon（Tokyo，Japan）epifluorescence microscope in conjunction with NIS Elements software was used to obtain both bright-field and fluorescent images.A Nikon C-SP simple polarizer eclipse（model no.MBB75370）was attached to the microscope and polarized light microscopy was utilized to image the PSR stained sections.

③ hematoxylin ［ˌhiːməˈtɒksɪlɪn］n.苏木精

④ picrosirius ［paɪkrəʊˈsɪərɪəs］n.天狼星

⑤ vimentin ［ˈvɪməntɪn］n.波形蛋白

To quantify the ARS positive calcification，images were loaded onto Fiji-ImageJ and converted to 8-bit format. A constant threshold ^⑥ was applied and the plugin ^⑦ “Analyze Particles ” was used to output the percentage area positive for the ARS staining（Additional file 1：Fig.2）in leaflets，commissures，and root separately by outlining as described for TPEF metrics in Additional file 1：Fig.3D. ^⑧ Analysis of PSR stained sections was performed using a custom MATLAB code（MathWorks，Natick，MA）for the aortic root，commissures and leaflet regions separately ［5］.Collagen fiber thickness was assessed based on color and binned into four categories—thick（red），thick intermediate（orange），thin intermediate（yellow）and thin（green）［5］.At least three to four biological replicates per group were analyzed.For immunohistochemistry，three individual researchers，blinded to the study，qualitatively assessed

⑥ threshold ［ˈθreʃhəʊld］n.阈值

⑦ plugin ［plʌgɪn］n.插件

⑧ 通过附件1：图3D中对TPEF指标的概述，应用恒定阈值、使用插件“粒度分析”分别输出主动脉瓣小叶、连合处和根部ARS染色阳性百分比面积（附件1：图2）。句中to output… 是前两个短句的目的状语，by outlining as described… 做方式状语

three biological replicates per group and provided a score of 0 for no or low expression，1 for moderate expression，or 2 for high expression.Scores of the three individuals were averaged and assigned a “-” if scored as 0，“ + ” if the mean score was from 0-0.66，“ + + ” if scored from 0.66 to 1.33 or “ + + + ” if scored from 1.33 to 2.The averaged mean score from the three individuals was used to perform correlation analysis with TPEF metrics.

Quantitative polarized light imaging

Quantitative polarized light imaging（QPLI）was used to image PSR stained sections，to measure collagen fiber orientation，and thickness through phase ^① retardation ^② .Briefly，a custom-built trans-illumination based QPLI microscope（Olympus BX51，Olympus Corp.，Tokyo，Japan）setup with a rotating ^③ polarizer and circular analyzer was utilized ［34-37］.Image collection was completed using a 20 × objective（UPlanFL N 20×，0.5 NA，Olympus Corp.）in order to accommodate the size of the samples，and full field images were produced via image stitching.The images were analyzed using a custom MATLAB code for the aortic root，commissures and leaflet regions separately（Additional file 1：Fig.3A）.Average light retardation，which is proportional ^④ to the thickness of collagen at each pixel ^⑤ location，was assessed as the primary QPLI metric.A rotating polarizer and circular analyzer enabled the assessment of variance ^⑥ in collagen fiber direction at each pixel ［36，37］，and directional variance of fiber orientations was computed to evaluate the relative strength of collagen fiber alignment ^⑦ in the mean direction ［37，38］.At least three to four biological replicates per group were analyzed.

① phase ［feɪz］n.时期，阶段

② retardation ［ˌriːtɑːˈdeɪʃn］n.迟延

③ rotating ［rəʊˈteɪtɪŋ］adj.旋转的

④ proportional ［prəˈpɔːʃənl］adj.成比例的

⑤ pixel ［ˈpɪksl］n.像素

⑥ variance ［ˈveərɪəns］n.方差

⑦ alignment ［ˌɔːrɪənˈteɪʃn］n.方向，取向

Two photon excited fluorescence（TPEF）imaging

TPEF imaging was carried out using a custom-built resonant-scanning setup ^⑧ （Thorlabs，Newton，NJ）with a Mai-Tai ultrafast Ti：Sapphire tunable laser source（Spectra-Physics，Santa Clara，CA），［13］with a（20x，0.75 NA）water immersion ^⑨ objective ^⑩ （Nikon，Japan）.Tissue sections utilized for TPEF imaging were frozen，unprocessed and unfixed.It has been previously shown that TPEF autofluorescence ratio of ex vivo frozen tissue sections correlates highly with in vivo measurements ［39］. The average intensity of the region of interest at a given excitation and emission wavelength is represented as A _{excitation/emission} . ^① TPEF autofluorescence was collected at 755nm excitation with 460nm/40nm emission for A _755/460 ，860nm excitation with 525nm/45nm emission for A _860/525 （Additional file 1：Fig.3B），810 nm excitation with 460 nm/40 nm emission for A _810/460 and 525nm/45nm emission for A _810/525 （Additional file 1：Fig.3C）［27，29］.Laser power of 40mW and photomultiplier ^② tube（PMT）gain was kept consistent throughout imaging.Two TPEF autofluorescence metrics were calculated using a custom MATLAB code.The aortic root，commissures and leaflet regions were manually outlined and analyzed separately（Additional file 1：Fig.3D）.The following equations were utilized to obtain the autofluorescence intensity ratios at each pixel.

⑧ setup ［ˈsetʌp］n.装置

⑨ immersion ［ɪˈmɜːʃn］n.沉浸

⑩ objective ［əbˈdʒektɪv］n.物镜

① 在给定的激发和发射波长下，感兴趣区域的平均强度表示为A _{激发/发射} 。the average intensity of the region of interest为整个句子主语，is represented as的意思是表示为……。

② photomultiplier ［ˌfəʊtəʊˈmʌltɪplaɪə］n.光电倍增管

It should be noted that both these TPEF autofluorescence ratios are influenced by the collagen，lipids，mineralized calcific deposits，NADH and FAD ［29，30］.TPEF 755-860 ratio has often been used as an optical redox ratio when NADH and FAD are the only significant fl uorophores ^③ ［27，32，40］.Four biological replicates were utilized for the TPEF imaging per experimental treatment group for the mice.

③ fluorophore ［ˈfluərəˌfɔ:］n.荧光团

Statistical analysis

All data was represented as mean ± standard error of the mean（SEM）.Two-way analysis of variance（ANOVA）was utilized for comparison between the treatment groups at different time points for the murine samples with Tukey’s post-hoc tests if data passed normality tests.A P -value of less than 0.05 was considered statistically significant.The exact P -value is reported wherever relevant，if it was less than 0.1.The Kruskal-Wallis ANOVA on ranks ^④ non-parametric test was utilized if data did not pass the normality tests.Pearson’s correlation coefficient ^⑤ was used for the correlation analysis of normally distributed data and Spearman’s Rank correlation was used for the correlation analysis of non-parametric data including immunohistochemistry scoring.All scoring of immunohistochemistry images was performed in a blinded manner.JMP（SAS Institute，Cary，NC）and SigmaPlot（Systat Software Inc.，San Jose，CA）were utilized for the data analysis and graphing.

④ ANOVA on ranks秩次方差分析

⑤ correlation coefficient ［ˌkɒrəˈleɪʃn ˌkəʊɪˈfɪʃnt］相关系数

Results

Mice on pro-calcific diet had lipid and calcium deposition at the aortic valve commissures

Histology was employed to assess the morphology ^① of the aortic valve and typical hallmarks of CAVD progression such as lipid deposition and calcification ［11，17，18，41］.Hematoxylin and Eosin staining revealed plaque ^② -like structures in the aortic root proximal ^③ to the commissures at 4 weeks in the mice fed a pro-calcific diet（Fig.1a-d）.Oil Red O staining was performed to confirm if the procalcific mice did indeed have deposition of lipid vacuoles ^④ .The control mice did not stain positive for Oil Red O（Fig.1e，f），however，the pro-calcific mice showed positive Oil Red O staining at 4 weeks at the valve commissures（Fig.1g，h）.Qualitatively，no increase in Oil Red O-positive lipid deposition was observed in the pro-calcific groups at 16 weeks compared to the 4-week time point.Alizarin Red S（ARS）staining was performed to assess calcium deposition.Positive ARS staining was not observed in the control mice for all time points（Fig.1i，j）.Pro-calcific mice showed positive ARS staining in the commissures at the 16-week time point（Fig.1k，l）.

① morphology ［mɔːˈfɒlədʒɪ］n.形态学

② plaque ［plæk］n.斑（块）

③ proximal ［ˈprɒksɪməl］adj.近端的

④ vacuole ［ˈvækjuəʊl］n.液泡

Mice on pro-calcific diet had increased expression of markers for cell activation and osteogenic dedifferentiation

Immunohistochemistry was performed to further investigate the early and late markers of aortic valve disease progression ［18，42］.Control mice showed lower α -SMA expression and higher vimentin expression suggesting a quiescent phenotype of the valve cells under this treatment（Fig.2a，b，a'，b'）［43］.Higher α -SMA and lower vimentin（Fig.2h，i，h'，i'）expression was observed in the pro-calcific mice，suggesting an activated cell phenotype ［43］.There was also higher osteopontin ^① expression（Fig.2d，k，d'，k'）at the commissures of pro-calcific mice as compared to control mice，suggesting an osteogenic phenotype in these samples ［2，18］.The expression of RUNX2 in pro-calcific mice was also qualitatively more pronounced at the 16-week time point（Fig.2c'，j'）as compared to the 4-week time point（Fig.2c，j）suggesting disease progression over the course of this timespan ^② ［2］.Osteopontin and BMP4 expression（Fig.2e，l，e'，l'）showed an increase in pro-calcific mice only at 4 weeks suggesting early changes.Control mice had lower Ki67 expression as compared to pro-calcific mice at the commissures，suggesting that the valvular cells in the pro-calcific groups were more proliferative ［5，39］（Fig.2f，m，f'm'）.TGF β 1，a marker for inflammatory cytokines ［42］，expressed positively at the commissures in pro-calcific mice（Fig.2g，n，g'，n'）at 16 weeks.

① osteopontin ［ɒsti:əʊˈpɒntɪn］n.骨桥蛋白

② timespan ［ˈtaɪmspən］n.时间跨度

③ lesion ［ˈliːʒn］n.损害，病变

Mice on pro-calcific diet showed increased collagen remodeling compared to control diet

To further characterize structural markers for disease progression，we assessed collagen remodeling via PSR staining and quantitative polarized light imaging（QPLI）techniques.PSR staining was performed to enhance the birefringence ^① of collagen ［44］present in the mouse valves（Fig.3a）. The PSR stained sections were first imaged through a linear polarizer to analyze the thickness of collagen fibers，a metric that is usually associated with collagen fiber diameter，where the increase in thickness corresponds to a shift ^② from green（thinner）to red（thicker）fibers . ^③ It is important to note that this shift from green to red is also dependent on the packing density and alignment ^④ of collagen fibers ［45］.A lower percentage of thinne（rgreen）fi bers was observed in the leaflets（ P = 0.057）（ Fig.3b）in the pro-calcific valves at 4 weeks as compared to the control valves.A higher percentage of thicke（rred）fi bers was observed in the commissures（ P = 0.057）（ Fig.3c）in the pro-calcific valves at 4 weeks as compared to the control valves.However，at 16 weeks these differences disappeared.No differences were observed in fiber thickness between the roots of control versus pro-calcific mice（ P ＞ 0.1）（ Fig.3d）.

① birefringence ［ˌbaɪrɪˈfrɪndʒəns］n.双折射

② shift ［ʃɪft］n.转变

③ PSR染色切片首先通过线性偏振器成像，以分析胶原纤维的厚度，胶原纤维的厚度通常是与胶原纤维直径有关的指标，厚度的增加对应着绿色（较薄）纤维向红色（较厚）纤维的转变。本句的主干是sections were imaged，不定式短语to analyze…fibers是目的状语部分，where的意思是in which （in the collagen fibers），corresponds to的意思是与……相一致，对应。

④ alignment ［əˈlaɪnmənt］n.队列，排列

Via QPLI，average retardation（Fig.3e）showed a significant reduction in the leaflets of procalcific mice as compared to the control mice at 16 weeks（ P = 0.0455）（Fig.3f）.Compared to the control mice at 4 weeks，the average retardation was significantly higher in control mice at 16 weeks（ P = 0.0269）.The commissures showed no statistically significant differences in the average retardation（ P ＞ 0.1）（Fig.3g）.The aortic root tended to have an increased average retardation in the pro-calcific group as compared to the control mice at 4 weeks（ P = 0.092）（Fig.3h）.

Average local directional variance in the collagen fiber orientation was used as a metric to assess the alignment of collagen fibers（Fig.3i）［38］.Average local directional variance increased in the leaflets of the pro-calcific mice as compared to the control mice at 16 weeks（ P = 0.061）（Fig.3j）.There were no significant differences（ P ＞ 0.1）observed in the commissures（Fig.3k）and roots（Fig.3l）.

Average collagen positive pixel density（Fig.3m）was assessed as an additional parameter for collagen remodeling ［38］.The leaflets showed significantly reduced collagen positive pixel density in the pro-calcific group at 16 weeks，as compared to the control mice at 16 weeks（ P = 0.0147）and pro-calcific mice at 4 weeks（ P = 0.0747）（Fig.3n）.There were no significant differences（ P ＞ 0.1）observed in the commissures（Fig.3o）and roots（Fig.3p）.

Changes in individual A _755/460 ，A _860/525 ，A _810/460 and A _810/525 autofluorescence intensities

The A _755/460 and A _860/525 autofluorescence intensities did not show significant differences in the leaflets（Fig.4a，d），commissures（Fig.4b，e），and roots（Fig.4c，f）（all P ＞ 0.1）for control and procalcific mice at 4 and 16 weeks.The A _755/460 intensity tended to increase at the commissures of the 16-week pro-calcific mice compared to the 16-week control mice（ P = 0.0597）and 4-week pro-calcific mice（ P = 0.0806）.The A _755/460 intensity also tended to increase at the roots of the 16-week procalcific mice compared to the 16-week control mice（ P = 0.0869）.The A _810/460 （ P ＞ 0.1）and A _810/525 （ P ＞ 0.1）autofluorescence did not change in the leaflets（Fig.4g，j）.The A _810/460 intensity increased significantly at the commissures of the 16-week pro-calcific mice compared to the 16-week control mice（ P = 0.0115）and 4-week pro-calcific mice（ P = 0.0304）and also increased with respect to 4-week control mice（ P = 0.0514）（Fig.4h）.The A _810/525 intensity increased significantly at the commissures of the 16-week pro-calcific mice compared to the 16-week control mice（ P = 0.0174）（Fig.4k）.The A _810/460 intensity increased significantly at the roots of the 16-week pro-calcific mice compared to the 16-week control mice（ P = 0.0045）and 4-week pro-calcific mice（ P = 0.0154）and also increased with respect to 4-week control mice（ P = 0.0775）（Fig.4i）.The A _810/525 intensity increased significantly at the roots of the 16-week pro-calcific mice compared to the 16-week control mice（ P = 0.0059）and 4-week pro-calcific mice（ P = 0.0463）（Fig.4l）.The A _810/460 intensity correlated significantly with the A _755/460 intensity for commissures（ R = 0.9482， P ＜ 0.0001）and roots（ R = 0.6938， P = 0.6938，and roots（ures（ica R = 0.2028， P = 0.4512）.The A _810/525 intensity correlated significantly with the A _860/525 intensity for commissures（ R = 0.8644， P ＜ 0.0001）and roots（ R = 0.6501， P = 0.0064）but not for leaflets（ R = 0.0302， P = 0.9117）.

TPEF autofluorescence ratios altered with calcific disease progression in murine aortic valves

Using the above autofluorescence intensities，we first obtained TPEF 755-860 ratio maps（Fig.5a）.Mouse valve sections from the control treatment group，without positive ARS staining and without positive expression for the osteogenic markers RUNX2 and osteopontin，and procalcific group mouse valves with positive ARS staining and positive expression for these same osteogenic markers，were considered for analysis.The TPEF 755-860 ratio at the leaflets of 16-week pro-calcific mice was significantly lower than 4-week control（ P = 0.035）and pro-calcific mice（ P = 0.033）and also lower than 16-week control mice（ P = 0.0768）（Fig.5b）.The TPEF 755-860 ratio at the commissures（Fig.5c）in the pro-calcific mice at 16 weeks was significantly lower，when compared to the 16-week control（ P = 0.0075），4-week control（ P = 0.0175）and pro-calcific mice（ P = 0.0069），also coinciding with positive ARS and RUNX expression.The TPEF 755-860 ratio decreased significantly at the roots（Fig.5d）（ P = 0.0492）at 16 weeks compared to the 4-week mice.

The TPEF Col-Cal ratio was assessed to evaluate differences in the relative contribution of collagen and calcium autofluorescence［29］.The TPEF Col-Cal ratio at the commissures decreased significantly in the 16-week pro-calcific mice when compared to 4-week control mice（ P = 0.0303）and pro-calcific mice（ P = 0.0498），but did not change with respect to 16-week control mice P = 0.1375）（Fig.5f）.The TPEF Col-Cal ratio did not show any differences between treatment groups in the leaflets（ P ＞ 0.1）（Fig.5e）and roots（ P ＞ 0.1）（Fig.5g）. Given ^① both TPEF autofluorescence ratios may be influenced by collagen，lipids，mineralized calcific deposits，NADH，and FAD ［29，30］，we sought to assess if TPEF Col-Cal and 755-860 ratio were correlated.However，there was no significant correlation observed between the TPEF Col-Cal ratio and the TPEF 755-860 ratio in leaflets（ R = 0.1928， P = 0.4743）（Additional file 1：Fig.4A），commissures（ R = 0.3473， P = 0.1876）（Additional file 1：Fig.4B）and roots（ R = 0.4522， P = 0.0786）（Additional file 1：Fig.4C）.

① given ［ˈɡɪvn］prep.考虑到

TPEF autofluorescence ratios negatively correlated with increased calcific disease progression in murine aortic valves

To quantify the relationship between TPEF autofluorescence ratio alterations and increased disease progression，TPEF autofluorescence ratios were correlated with immunohistochemistry scores and collagen-remodeling metrics obtained via QPLI.The TPEF 755-860 ratio correlated negatively with RUNX2 expression（Spearman’s ρ = -0.7165， P = 0.0455）（Fig.6a）and Ki67 expression（Spearman’s ρ = -0.8729， P = 0.0103）（Fig.6b），suggesting pro-calcific mice had increased RUNX2 and Ki67 expression with decreased TPEF 755-860 ratio at the commissures.The A _810/460 （Fig.6c）and A _810/525 （Fig.6d）intensities also positively correlated with RUNX2 expression（Spearman’s ρ = 0.9142， P = 0.0015 and Spearman’s ρ = 0.8648， P = 0.0056，respectively）at the commissures，suggesting RUNX2-expressing diseased areas may exhibit higher collagen and calcium deposition.

The TPEF 755-860 ratio also correlated positively with average retardation（ R = 0.5272， P = 0.0434）for pro-calcific mice and negatively with average directional variance（ R = -0.3909， P = 0.0438）（Fig.6e），while average retardation correlated negatively with average directional variance（ R = -0.6707， P = 0.0001）（Fig.6f）.At 4 weeks，TPEF Col-Cal ratio also correlated negatively with retardation（ R = -0.6139， P = 0.0067）suggesting decrease in Col-Cal ratio coincided ^② with increased retardation and thicker collagen fibers.At 4 weeks，slightly thicker fibers were found in pro-calcific mice.We additionally found that at 16 weeks，A _810/460 and A _810/525 intensities also positively correlated with average local variance（ R = 0.7458， P = 0.021 and R = 0.728， P = 0.0262，respectively）.Further，A _810/460 （Fig.6g）and A _810/525 （Fig.6h）intensities positively correlated with collagen positive pixel density（ R = 0.4937， P = 0.0089 and R = 0.5018， P = 0.0077，respectively）.

② coincide ［ˌkəʊɪnˈsaɪd］vi.一致，同时发生

Lower 755-860 Ratio may be predictive of increased calcification

To assess if presentation of lower 755-860 ratio preceded increased calcification，the custom diet was continued to be fed to mice until week 28，in a separate trial.Control and pro-calcific mice at 28 weeks were assessed for calcification via ARS staining（Fig.7a）and ARS positive percentage area was quantified for leaflets，commissures，and root.It was observed that the leaflets of pro-calcific mice at 28 weeks showed significantly increased ARS positive calcification as compared to 16-week control（ P = 0.0393），pro-calcific（ P = 0.0305）mice and 4-week control mice（ P = 0.0113）（Fig.7b）.It was also observed that the commissures（Fig.7c）and roots（Fig.7d）of pro-calcific mice at 28 weeks showed significantly increased ARS positive calcification as compared to 4-and 16-week mice and 28-week control mice（ P ＜ 0.0001）.Additionally，commissures of pro-calcific mice at 16 weeks showed significantly increased ARS positive calcification as compared to 4-week mice（ P ＜ 0.01）and 16（ P = 0.0004）and 28（ P = 0.0006）week control mice（Fig.7c）.Indeed，the ARS positive percentage area correlated negatively with TPEF 755-860 ratio for commissures（Spearman’s ρ = -0.8857， P = 0.0188）and roots（Spearman’s ρ = -0.9429， P = 0.0048）suggesting a decreasing TPEF 755-860 ratio could indicate increasing calcification.

Discussion

The most common small animal model for CAVD is the hypercholesterolemic ^① mouse with apoE and/or low density lipoprotein receptor knockouts ［14］.Other approaches involve genetically-modified mouse models with NOTCH1 ^+/- （NOTCH1 heterozygous ^② ），Postn ^-/- （ periostin ^③ deletion），or NOS3 ^-/- （endothelial nitric ^④ oxide synthase ^⑤ deletion）phenotypes［14，46］.These genetic mouse models are typically combined with a high fat and high cholesterol western diet ［14，46］.Most of these models have been shown to develop valve calcification in aged mice，or require twenty weeks or more of treatment ［41，47］.Another prior CAVD mouse model was created using a guidewire inflicted ^⑥ aortic valve tissue injury and was shown to develop calcification by 12 weeks following injury ［41］.In the current study，we developed and validated a diet-based wild-type small animal model for early progression of valve calcification，without genetic manipulation ^⑦ which showed early hallmarks of CAVD by 4 and 16 weeks.Male mice were chosen due to the higher prevalence of CAVD in males ［48］.We observed positive ARS staining of calcification at the commissural walls，similar to what Assmann et al.observed in a rat model ［18］.However，unlike the rat model in Assmann et al.we did not observe evidence of calcification in the aortic roots.

① hypercholesterolemic ［haɪpəkəʊlestərəʊˈlemɪk］adj.高胆固醇血的

② heterozygous ［ˌhetərəˈzaɪɡəs］adj.杂合的

③ periostin ［ˌpɪrɪˈɒstɪn］n.骨膜蛋白

④ nitric ［ˈnaɪtrɪk］adj.含氮的

⑤ synthase ［ˈsɪnθeɪz］n.合酶

⑥ inflict ［ɪnˈflɪkt］vt.造成

⑦ manipulation ［məˌnɪpjuˈleɪʃn］n.操纵

The mice in the pro-calcific diet groups also had evidence of lipid deposition near the commissural walls at 4 weeks as has been previously reported in mice on a high-fat diet with added cholesterol ［14］.Two previous studies reported valve mineralization in wild-type dietbased mouse models without genetic manipulation approximately 16 weeks after initiation of diet ［16，17］.However，the diet employed in these studies incorporated ^⑧ 58.7% fat，which is significantly more than the western diet，and also contained additional carbohydrates ^⑨ .In addition，demonstration of CAVD in these earlier studies relied solely on positive von Kossa particulate ^⑩ staining，which can be confounded ^⑪ in a mouse model by melanocytes ^⑫ or lipofuscin ^⑬ -containing granules ^⑭ with appearance similar to that of von Kossa positive calcification ［17］.In our model，we have shown positive ARS staining concurrent with the location of increased activation，proliferation and osteogenesis，suggesting improved utility of our model ［14］.To our knowledge，this is the only wild-type diet-based mouse model for early CAVD that has shown ARS-positive calcium staining.

⑧ incorporate ［ɪnˈkɔːpəreɪt］vt.包含

⑨ carbohydrate ［ˌkɑːbəʊˈhaɪdreɪt］n.碳水化合物

⑩ particulate ［pɑːˈtɪkjələt］n.微粒

⑪ confound ［kənˈfaʊnd］v.混淆

⑫ melanocyte ［ˈmelənə（ʊ）ˌsaɪt］n.黑素细胞

⑬ lipofuscin ［̗lɪpəˈfʌsɪn］n.脂褐素

⑭ granule ［ˈɡrænjuːl］n.颗粒

Subtle increases in TGF β 1，which have been known to influence VIC proliferation，differentiation and apoptosis，were observed in the pro-calcific mice at 4 and 16 weeks.This observation，together with increased Ki67，osteopontin，and RUNX2 expression suggest increased proliferation and osteogenic differentiation are occurring in the tissues，which are known hallmarks of CAVD ［42］and has been previously reported in various other mouse models［5，11，14，17，18，41，46，47］.Additionally，concurrent expression of TGF β 1， α -SMA and Ki67 also suggests that increased TGF β 1 could have led to the increased proliferation of the myofibroblastic ^① phenotype of VICs in our disease model ［49］.

① myofibroblastic ［maɪɔːfaɪbrɒbˈlɑːstɪk］adj.肌纤维母细胞的

Via QPLI，we observed that the 4-week pro-calcific mice had more mature collagen fibers and a higher positive pixel density with minimal changes in collagen fiber orientation compared to controls，suggesting increased collagen remodeling ［14，45，47］.In the 16-week pro-calcific mice，we observed a decrease in collagen thickness and density with an increased variance in collagen orientation in the leaflets.This was corroborated ^② by the negative correlation between average retardation and average local directional variance.This led us to speculate that by 16 weeks，there was either greater collagen degradation or de novo collagen synthesis ^③ ，leading to thinner fibers and increased variance in fiber orientation.Collagen remodeling is typically regulated by multiple factors including matrix metalloproteinases（MMPs）， cathepsins ^④ or other pro-fibrotic factors like TGF β and twist-1 ［50］.In fact，increased MMP activity co-localized with calcification at the commissures in rats fed with a similar diet in other studies ［18］.ECM changes have been associated with changes in phenotype ［51］.Moreover，in our study，the pro-calcific mice demonstrated a more myofibroblastic phenotype，as evidenced by increased expression of α -SMA，which could further contribute towards matrix remodeling.Pro-calcific mice also had a higher percentage of matured collagen fibers in diseased regions at 4 weeks，consistent with CAVD progression ［14，45，47］.

② corroborate ［kəˈrɒbəreɪt］vt.证实

③ synthesis ［ˈsɪnθəsɪs］n.合成

④ cathepsin ［kəˈθepsɪn］n.组织蛋白酶

Previously，TPEF microscopy has been used to demonstrate collagen remodeling through SHG imaging ［45］and calcification through autofluorescence imaging ［29］in the valve at the tissue level.TPEF microscopy can also be used to obtain an optical redox ratio（［FAD］/（［FAD］+ ［NADH］）which reflects the metabolic state of the cell ［13，27，39］.It has been shown in vitro that mesenchymal ^⑤ stem cells undergoing osteogenic differentiation exhibit decreased optical redox ratios ［27］.We have also shown previously that VICs under pathological stretch have reduced optical redox ratios compared to unstretched cells ［13］.We have also demonstrated that when VICs are subjected to osteogenic conditions in vitro，their optical redox ratio decreased and correlated with gene expression of osteogenic markers like RUNX2，osteopontin and osteocalcin ［28］.In the current study，we measured the TPEF 755-860 ratio（A _860/525 /（A _755/460 + A _860/525 ））from ex vivo tissue sections by obtaining the autofluorescence intensities at 755nm and 860nm excitation，which correspond to the NADH and FAD excitation wavelengths.However，we did not term it an optical redox ratio，as tissue autofluorescence measured via the TPEF 755-860 ratio can be attributable to multiple molecules including collagen，lipids，and mineralized calcific deposits ［29，30］.The TPEF 755-860 ratio could thus have been influenced by collagen and calcium in the tissue ［29］.We therefore assessed the TPEF Col-Cal ratio，where the individual A _810/460 and A _810/525 intensities correspond to collagen and calcium，respectively ［29］.The TPEF Col-Cal ratio increased significantly in aortic valve roots and commissures for the 16-week pro-calcific mice suggesting an increase in collagen and calcium autofluorescence.

⑤ mesenchyma ［mesˈeŋkɪmə］n.间叶细胞

In our study，we found that the TPEF autofluorescence ratio rather than the TPEF Col-Cal ratio was more sensitive to disease progression.While TPEF Col-Cal ratio did not show statistical significance between control versus pro-calcific mice，TPEF 755-860 ratio was sensitive to both time，presence of disease conditions，and their interaction.TPEF Col-Cal ratio only showed differences in the commissures，while TPEF 755-860 ratio showed significant changes in the leaflets，commissures and roots.We speculate ^① the difference in sensitivity ^② between TPEF 755-860 and TPEF Col-Cal ratios might be because of increased sensitivity of the TPEF 755-860 ratio to NADH.It should be noted，however，that both these TPEF autofluorescence ratios are influenced by collagen，lipids，mineralized calcific deposits，NADH，and FAD ［29，30］.

① speculate ［ˈspekjuleɪt］vi.推测

② sensitivity ［ˌsensəˈtɪvətɪ］n.敏感性

Additionally，we observed that TPEF 755-860 ratio correlated with RUNX2 and Ki67 negatively suggesting increased osteogenesis and proliferation was marked by reduced TPEF 755-860 ratio，which is consistent with previous studies ［13，27，32，39，52］.It is also interesting to note that A _810/460 and A _810/525 intensities positively correlated with RUNX2 expression leading us to believe that RUNX2-expressing diseased areas may exhibit higher collagen and calcium deposition.Indeed，A _810/460 and A _810/525 intensities also positively correlated with average directional variance and collagen positive pixel density.This suggested that the increase in A _810/460 and A _810/525 intensities indeed allude ^③ to increased collagen ［45］and calcium ［29］deposition during disease progression.TPEF 755-860 ratio which showed a decrease during disease progression correlated negatively with average directional variance further evidencing，thinner disorganized fibers during disease progression.This may suggest increased de novo collagen and calcium deposition with increased CAVD progression.Similar correlation between cell redox state and collagen synthesis is consistent with other studies ［27，53，54］.The ratio of blue∶green（i.e.460nm∶525nm emission）TPEF autofluorescence at 800nm excitation has been shown to be sensitive to mineralization and altered CAVD progression ［29］.Our current study is the first instance when a TPEF 755-860 ratio has been shown to demonstrate tissue level changes occurring during early CAVD progression in a mouse model that was more sensitive than the TPEF Col-Cal ratio.Like we had previously demonstrated in our in vitro studies ［13，28，52］，in this study also we observed TPEF autofluorescence ratios decreased at 16 weeks in the procalcific mice commissures and correlated with increased calcification，osteogenic differentiation and proliferation.This is also similar to the findings of Quinn et al.where the osteogenic differentiation of the mesenchymal stem cells correlated with the reduction in an optical redox ratio ［27］and Jones et al.where increased proliferation correlated with a reduction in redox ratio in skin wound healing ［39］.TPEF 755-860 ratio not only correlated with osteogenic progression and increased proliferation but also seemed to have a predictive quality for calcification.This further provides an incentive to explore TPEF 755-860 as an early biomarker for CAVD progression.

③ allude ［əˈluːd］vi.暗指

Limitations

One limitation of this study was that the autofluorescence signal from various endogenous fluorophores like NADH，FAD，lipids，collagen， elastin ^① ，and mineralization that are likely present in valves ［29，32］was not decoupled ^② .Additionally，this study was conducted ex vivo，considering the limitations of current TPEF imaging probes ^③ to assess cardiac valves.Although intravital ^④ pericardial imaging has been a possibility in recent past ［55-57］，clinical translation of TPEF imaging in valves is yet to be realized.Enhanced probe flexibility and miniaturization might open avenues for pre-clinical studies on larger animal models in the future ［58，59］.Finally，we acknowledge that the sample size for our quantitative analyses was low.However，it should be noted that our effect sizes were sufficient to reach significant conclusions.

① elastin ［ɪˈlæstɪn］n.弹性蛋白

② decouple ［diːˈkʌpl］vt.解耦

③ probe ［prəʊb］n.探针

④ intravital ［̗ɪntrəˈvaɪtəl］adj.活体的

Future directions

Decoupling the signals from various endogenous fluorophores（e.g.NADH，FAD，lipids，collagen，elastin，and mineralization）that are likely present in valves could provide a detailed quantitative assessment of different aspects of disease progression ［29，32］. More robust ^⑤ studies incorporating correlation between the TPEF metrics and established metrics of disease progression both in vivo and in vitro，would further demonstrate the utility of TPEF metric to track disease progression . ^① However，our study provides sufficient evidence to suggest that the TPEF 755-860 autofluorescence ratio can be further explored to serve as a label-free metric for assessing CAVD progression.

⑤ robust ［rəʊˈbʌst］adj.强健的

① 将TPEF指标与已建立的体内和体外疾病进展指标相结合的更稳健的研究将进一步证明TPEF指标在追踪疾病进展方面的实用性。本句的主干是studies would further demonstrate the utility of TPEF metric to track disease progression，意思是研究将进一步证明追踪疾病进展的TPEF指标的实用性，句中动词不定式短语to track disease progression作TPEF metric的后置定语。这句话的主语长而复杂，其中心词是studies，有前置定语more robust和后置定语incorporating correlation between the TPEF metrics and established metrics of disease progression both in vivo and in vitro，后置定语的意思是整合体内外TPEF指标和已经建立起来的疾病进展指标之间的关系。

Conclusions

In conclusion，we created a wild-type mouse model for early CAVD where the mice showed lipid deposition and collagen remodeling at 4 weeks and early evidence for calcification at 16 weeks.The valvular apparatus showed signs of proliferation，osteogenic differentiation，and increased inflammatory cytokine infiltration.The diseased valves also showed altered TPEF autofluorescence signatures，which were consistent with the findings in previous studies ［27-29］and correlated with osteogenic progression，ECM remodeling and calcification.Additionally，28-week ARS stains revealed presence of calcification in leaflets that was previously absent until 16 weeks.Enhanced calcification in the commissures and roots and negative correlation of TPEF 755-860 ratio with ARS positive calcification suggests lower autofluorescence ratios may be potentially explored as a predictive biomarker for calcification.This study suggests that with further validation ^② ，and imaging probe miniaturization，quantitative TPEF metrics hold promise to serve as a label-free tool to assess the progression of CAVD.

② validation ［ˌvælɪˈdeɪʃn］n.验证

Supplementary information

Supplementary information accompanies this paper at https：//doi.org/10.1186/s12872-020-01776-8 .

Additional file 1. Supplemental methods and results.

Abbreviations

CAVD：Calcific aortic valve disease；VEC：Valve endothelial cell；VIC：Valve interstitial cell；ECM：Extracellular matrix；TPEF：Two-photon excited fluorescence；SHG：Second harmonic generation；FAD：Flavin adenine dinucleotide；NADH：Nicotinamide adenine dinucleotide reduced；QPLI：Quantitative polarized light imaging；PSR：Picrosirius red；ARS：Alizaring Red S staining；EF：Ejection fraction；CO：Cardiac output；EDV：End-diastolic volume；ESV：Endsystolic volume；LVIDs：Left ventricle internal diameter systolic；LVIDd：Left ventricle internal diameter diastolic；H & E：Hematoxylin and eosin； α -SMA：Alpha smooth muscle actin；BMP4：Bone morphogenic protein 4；TGF β 1：Transforming growth factor beta 1；MMP：Matrix metalloproteinase；ANOVA：Analysis of variance；SEM：Standard error of the mean.

Acknowledgements

None.

Authors' contributions

I.T.and K.B.were involved in the conception and design of the study.I.T.，S.J.，A.W.，J.P.，A.O.and D.C.helped in the data acquisition and analysis.I.T.and K.B.wrote the manuscript.T.J.M.，S.V.，M.P.，K.P.Q.and K.B.reviewed and revised the manuscript.All authors have read and approved the final manuscript.

Funding

This work was supported by the National Science Foundation ［CMMI-1452943 to K.B.，CBET-1846853 to K.P.Q.］，and the American Heart Association ［18AIREA33900098 to K.B.，19PRE34370061 to I.T.］.This work was also supported by funding from National Institutes of Health ^① ［R00EB017723，R01AG056560 to K.P.Q.］.The custom-built multiphoton imaging setup was supported by the Arkansas Biosciences Institute to T.J.M.This work was also supported by the National Heart Lung and Blood Institute at the National Institutes of Health［1R01HL13514501A1，1R01HL13366701A1，1R01HL14032501A1，and 1R01HL14471401A1 to M.P.］.The above funding bodies had no role in design and execution of this study.

① National Institutes of Health 美国国立卫生研究院

Availability of data and materials

The data generated and/or analyzed during the current study are included in this article and supplementary information and also available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This study was approved by all our relevant institutional ethics committees.Research does not involve human participants，human material，or human data.All animal experiments were performed in compliance with the University of Arkansas Institutional Animal Care and Use Committee （ IACUC ） ^② approval and conform to all applicable provisions of the USDA Animal Welfare Act and Regulations，the PHS Policy on Humane and Use of Laboratory Animals，and other federal，state，and local regulations relating to the use of vertebrate animals in research.

② University of Arkansas Institutional Animal Care and Use Committee（IACUC）阿肯色大学动物保护和使用机构委员会

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹ Department of Biomedical Engineering，University of Arkansas，122 John A.White Jr.Engineering Hall，Fayetteville，AR 72701，USA. ² Division of Cardiology，University of Arkansas for Medical Sciences，Little Rock，AR 72205，USA. ³ Division of Cardiothoracic Surgery，Joseph P.Whitehead Department of Surgery，Emory University，Atlanta，GA 30322，USA.

Received：21 August 2020

Accepted：8 November 2020

Published online：11 December 2020

References

1.Maganti K，Rigolin VH，Sarano ME，Bonow RO.Valvular Heart Disease：Diagnosis and Management.Mayo Clin Proc.2010；85（5）：483-500.

2.Rajamannan NM，Evans FJ，Aikawa E，Grande-Allen KJ，Demer LL，Heistad DD，et al.Calcific aortic valve disease：not simply a degenerative process：a review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group.Executive summary：calcific aortic valve disease-2011 update.Circulation.2011；124（16）：1783-91.

3.Otto CM，Lind BK，Kitzman DW，Gersh BJ，Siscovick DS.Association of aortic-valve sclerosis with cardiovascular mortality and morbidity in the elderly.N Engl J Med.1999；341（3）：142-7.

4.Lerman DA，Prasad S，Alotti N.Calcific aortic valve disease：molecular mechanisms and therapeutic approaches.Eur Cardiol.2015；10（2）：108-12.

5.Perez J，Diaz N，Tandon I，Plate R，Martindale C，Balachandran K.Elevated Serotonin interacts with angiotensinii to result in altered valve interstitial cell contractility and remodeling.Cardiovasc Eng Technol.2017.

6.Yip CY，Chen JH，Zhao R，Simmons CA.Calcification by valve interstitial cells is regulated by the stiffiness of the extracellular matrix.Arterioscler Thromb Vasc Biol.2009.

7.Hjortnaes J，Shapero K，Goettsch C，Hutcheson JD，Keegan J，Kluin J，et al.Valvular interstitial cells suppress calcification of valvular endothelial cells.Atherosclerosis.2015；242（1）：251-60.

8.Sung DC，Bowen CJ，Vaidya KA，Zhou J，Chapurin N，Recknagel A，et al.Cadherin-11 overexpression induces extracellular matrix remodeling and calcification in mature aortic valves.Arterioscler Thromb Vasc Biol.2016；36（8）：1627-37.

9.Rodriguez KJ，Piechura LM，Porras AM，Masters KS.Manipulation of valve composition to elucidate the role of collagen in aortic valve calcification.BMC Cardiovasc Disord.2014；14：29.

10.Mathieu P，Boulanger M-C.Basic mechanisms of calcifc aortic valve disease.Can J Cardiol.2014；30（9）：982-93.

11.Small A，Kiss D，Giri J，Anwaruddin S，Siddiqi H，Guerraty M，et al.Biomarkers of calcific aortic valve disease.Arterioscler Thromb Vasc Biol.2017；37（4）：623-32.

12.Lam NT，Lam H，Sturdivant NM，Balachandran K.Fabrication of a matrigelcollagen semi-interpenetrating scaffold for use in dynamic valve interstitial cell culture.Biomed Mater.2017.

13.Lam NT，Muldoon TJ，Quinn KP，Rajaram N，Balachandran K.Valve interstitial cell contractile strength and metabolic state are dependent on its shape.Integr Biol（Camb）.2016；8（10）：1079-89.

14.Sider KL，Blaser MC，Simmons CA.Animal models of calcifc aortic valve disease.Int J Infamm.2011；2011：364310.

15.Sider KL，Zhu C，Kwong AV，Mirzaei Z，de Lange CF，Simmons CA.Evaluation of a porcine model of early aortic valve sclerosis.Cardiovasc Pathol.2014；23（5）：289-97.

16.Mehrabian M，Demer LL，Lusis AJ.Differential accumulation of intimal monocyte-macrophages relative to lipoproteins and lipofuscin corresponds to hemodynamic forces on cardiac valves in mice.Arterioscler Thromb Vasc Biol.1991；11（4）：947-57.

17.Drolet M-C，Roussel E，Deshaies Y，Couet J，Arsenault M.A high fat/high carbohydrate diet induces aortic valve disease in C57BL/6J mice.J Am Coll Cardiol.2006；47（4）：850-5.

18.Assmann A，Zwirnmann K，Heidelberg F，Schifer F，Horstkötter K，Munakata H，et al.The degeneration of biological cardiovascular prostheses under pro-calcific metabolic conditions in a small animal model.Biomaterials.2014；35（26）：7416-28.

19.Tang FT，Chen SR，Wu XQ，Wang TQ，Chen JW，Li J，et al.Hypercholesterolemia accelerates vascular calcification induced by excessive vitamin d via oxidative stress.Calcif Tissue Int.2006；79（5）：326-39.

20.Hsu JJ，Tintut Y，Demer LL.Vitamin D and osteogenic differentiation in the artery wall.CJASN.2008；3（5）：1542-7.

21.Nicoll R，Howard JM，Henein MY.A review of the effect of diet on cardiovascular calcification.Int J Mol Sci.2015；16（4）：8861-83.

22.Kamath AR，Pai RG.Risk factors for progression of calcifc aortic stenosis and potential therapeutic targets.Int J Angiol.2008；17（2）：63-70.

23.Beckmann E，Grau JB，Sainger R，Poggio P，Ferrari G.Insights into the use of biomarkers in calcific aortic valve disease.J Heart Valve Dis.2010；19（4）：441-52.

24.Lindman BR，Bonow RO，Otto CM.Current management of calcific aortic stenosis.Circ Res.2013；113（2）：223-37.

25.Kanwar A，Thaden JJ，Nkomo VT.Management of patients with aortic valve stenosis.Mayo Clin Proc.2018；93（4）：488-508.

26.Izquierdo-Gómez MM，Hernández-Betancor I，García-Niebla J，Marí-López B，Laynez-Cerdeña I，Lacalzada-Almeida J.Valve calcification in aortic stenosis：etiology and diagnostic imaging techniques.BioMed Res Int .2017；2017：5178631.

27.Quinn KP，Sridharan GV，Hayden RS，Kaplan DL，Lee K，Georgakoudi I.Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation.Sci Rep.2013；3：3432.

28.Tandon I，Kolenc OI，Cross D，Vargas I，Johns S，Quinn KP，et al.Label-free metabolic biomarkers for assessing valve interstitial cell calcifc progression.Sci Rep.2020；10（1）：10317.

29.Baugh LM，Liu Z，Quinn KP，Osseiran S，Evans CL，Huggins GS，et al.Non-destructive two-photon excited fluorescence imaging identifies early nodules in calcific aortic-valve disease.Nat Biomed Eng.2017；1（11）：914-24.

30.Croce AC，Bottiroli G.Autofluorescence spectroscopy and imaging：a tool for biomedical research and diagnosis.EJH.2014；58（4）：2461.

31.Zipfel WR，Williams RM，Webb WW.Nonlinear magic：multiphoton microscopy in the biosciences.Nat Biotechnol.2003；21（11）：1369-77.

32.Kolenc OI，Quinn KP.Evaluating cell metabolism through autofluorescence imaging of NAD（P）H and FAD.Antioxid Redox Signal.2019；30（6）：875-89.

33.Krishnamurthy VK，Guilak F，Narmoneva DA，Hinton RB.Regional structure-function relationships in mouse aortic valve tissue.J Biomech.2011；44（1）：77-83.

34.Glazer AM，Lewis JG，Kaminsky W.An automatic optical imaging system for birefringent media.Proc R Soc Lond Ser A Math Phys Eng Sci 1996 .1955；452：2751-65.

35.Tower TT，Neidert MR，Tranquillo RT.Fiber Alignment Imaging During Mechanical Testing of Soft Tissues.Ann Biomed Eng.2002；30（10）：1221-33.

36.Quinn KP，Winkelstein BA.Altered collagen fber kinematics defne the onset of localized ligament damage during loading.J Appl Physiol.2008；105（6）：1881-8.

37.Woessner AE，McGee JD，Jones JD，Quinn KP.Characterizing differences in the collagen fiber organization of skin wounds using quantitative polarized light imaging.Wound Repair Regener.2019；27（6）：711-4.

38.Quinn KP，Golberg A，Broelsch GF，Khan S，Villiger M，Bouma B，et al.An automated image processing method to quantify collagen fibre organization within cutaneous scar tissue.Exp Dermatol.2015；24（1）：78-80.

39.Jones JD，Ramser HE，Woessner AE，Quinn KP.In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing.Communications Biology.2018；1（1）：198.

40.Zipfel WR，Williams RM，Christie R，Nikitin AY，Hyman BT，Webb WW.Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.Proc Natl Acad Sci.2003；100（12）：7075.

41.Honda S，Miyamoto T，Watanabe T，Narumi T，Kadowaki S，Honda Y，et al.A novel mouse model of aortic valve stenosis induced by direct wire injury.Arterioscl Thrombo Vascul Biol.2014；34（2）：270-8.

42.Chester AH.Molecular and cellular mechanisms of valve calcification.Aswan Heart Centre Science &Practice Series.2011.

43.Latif N，Quillon A，Sarathchandra P，McCormack A，Lozanoski A，Yacoub MH，et al.Modulation of human valve interstitial cell phenotype and function using a fibroblast growth factor 2 formulation.PLoS ONE.2015；10（6）：e0127844.

44.Wegner KA，Keikhosravi A，Eliceiri KW，Vezina CM.Fluorescence of picrosirius red multiplexed with immunohistochemistry for the quantitative assessment of collagen in tissue sections.J Histochem Cytochem.2017；65（8）：479-90.

45.Hutson HN，Marohl T，Anderson M，Eliceiri K，Campagnola P，Masters KS.Calcific aortic valve disease is associated with layer-specific alterations in collagen architecture.PLoS ONE.2016；11（9）：0163858.

46.Miller JD，Weiss RM，Heistad DD.Calcific aortic valve stenosis：methods，models，and mechanisms.Circ Res.2011；108（11）：1392-412.

47.Weiss RM，Ohashi M，Miller JD，Young SG，Heistad DD.Calcific aortic valve stenosis in old hypercholesterolemic mice.Circulation.2006；114（19）：2065-9.

48.Thomassen HK，Ciof G，Gerdts E，Einarsen E，Midtbø HB，Mancusi C，et al.Echocardiographic aortic valve calcification and outcomes in women and men with aortic stenosis.Heart.2017；103（20）：1619-24.

49.Liu AC，Gotlieb AI.Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells.Am J Pathol.2008；173（5）：1275-85.

50.Leopold JA.Cellular mechanisms of aortic valve calcification.Circul Cardiovasc Interv.2012；5（4）：605-14.

51.Kim D-H，Provenzano PP，Smith CL，Levchenko A.Matrix nanotopography as a regulator of cell function.J Cell Biol.2012；197（3）：351-60.

52.Lam NT，Tandon I，Balachandran K.The role of fibroblast growth factor 1 and 2 on the pathological behavior of valve interstitial cells in a three dimensional mechanically-conditioned model.J Biol Eng.2019；13（1）：45.

53.Hernandez-Munoz R，Diaz-Munoz M，Chagoya de Sanchez V.Possible role of cell redox state on collagen metabolism in carbon tetrachlorideinduced cirrhosis as evidenced by adenosine administration to rats.Biochem Biophys Acta.1994；1200（2）：93-9.

54.Meleshina AV，Dudenkova VV，Bystrova AS，Kuznetsova DS，Shirmanova MV，Zagaynova EV.Twophoton FLIM of NAD（P）H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation.Stem Cell Res Therapy.2017；8（1）：15.

55.Jones JS，Small DM，Nishimura N.In vivo calcium imaging of cardiomyocytes in the beating mouse heart with multiphoton microscopy.Front Physiol.2018；9：969.

56.Matsuura R，Miyagawa S，Fukushima S，Goto T，Harada A，Shimozaki Y，et al.Intravital imaging with twophoton microscopy reveals cellular dynamics in the ischeamia-reperfused rat heart.Sci Rep.2018；8（1）：15991.

57.Allan-Rahill NH，Lamont MRE，Chilian WM，Nishimura N，Small DM.Intravital microscopy of the beating murine heart to understand cardiac leukocyte dynamics.Front Immunol.2020；11：92.

58.Ducourthial G，Leclerc P，Mansuryan T，Fabert M，Brevier J，Habert R，et al.Development of a real-time fexible multiphoton microendoscope for label-free imaging in a live animal.Sci Rep.2015；5：18303.

59.Zhao Y，Sheng M，Huang L，Tang S.Design of a fiber-optic multiphoton microscopy handheld probe.Biomed Opt Express.2016；7（9）：3425-37.

Activities 2-1

1.Define the following terms by analyzing the word parts.

1）hemodynamic_____________ 2）echocardiography_____________________

3）immunohistochemistry___________ 4）myofibroblast_________________________

5）valvulopathy______________ 6）monocyte____________________________

7）ultrasound_______________ 8）osteopontin__________________________

9）hypercholesterolemic____________ 10）periostin____________________________

2.Translate the following Chinese sentences into English.

1）手术中，进行了L ₃ 至L ₅ 椎板切开术，并完成了自终丝生长的可切除肿瘤的全切除。

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2）近年来，作者在撰写研究文章时，必须对研究背景、研究目标，包括所用动物的种类或品系、主要方法、主要发现和研究结论进行准确的总结。

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3.Read an article on animal research in PubMed and pay attention to the format. 5/p9L9maerRyIoc6WfLnyZW85W1iu6yNKypEaP1qA6G+KHvNjjNB/TKUNZBI7xwX