Three-Dimensional Visualization of Hypoxia-Induced Pulmonary Vascular Remodeling in Mice

HomeCirculationVol. 144, No. 17Three-Dimensional Visualization of Hypoxia-Induced Pulmonary Vascular Remodeling in Mice Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBThree-Dimensional Visualization of Hypoxia-Induced Pulmonary Vascular Remodeling in Mice Takayuki Fujiwara, MD, PhD, Norifumi Takeda, MD, PhD, Hironori Hara, MD, PhD, Satoshi Ishii, MD, Genri Numata, MD, PhD, Hiroyuki Tokiwa, MD, PhD, Sonoko Maemura, MD, PhD, Takaaki Suzuki, MD, Hiroshi Takiguchi, MD, Yoshiaki Kubota, MD, PhD, Kinya Seo, PhD, Asuka Sakata, MD, PhD, Seitaro Nomura, MD, PhD, Masaru Hatano, MD, PhD, Kazutaka Ueda, MD, PhD, Mutsuo Harada, MD, PhD, Haruhiro Toko, MD, PhD, Eiki Takimoto, MD, PhD, Hiroshi Akazawa, MD, PhD, Satoshi Nishimura, MD, PhD and Issei Komuro, MD, PhD Takayuki FujiwaraTakayuki Fujiwara Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Department of Computational Diagnostic Radiology and Preventive Medicine (T.F.), The University of Tokyo, Bunkyo-ku, Japan. Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan (T.F., S.N.). Search for more papers by this author , Norifumi TakedaNorifumi Takeda Norifumi Takeda, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Email E-mail Address: [email protected] https://orcid.org/0000-0003-4818-3347 Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Hironori HaraHironori Hara Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Satoshi IshiiSatoshi Ishii Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Genri NumataGenri Numata https://orcid.org/0000-0001-8155-5304 Department of Advanced Translational Research and Medicine in Management of Pulmonary Hypertension (G.N., H. Toko), The University of Tokyo, Bunkyo-ku, Japan. Search for more papers by this author , Hiroyuki TokiwaHiroyuki Tokiwa Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Sonoko MaemuraSonoko Maemura Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Takaaki SuzukiTakaaki Suzuki Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Hiroshi TakiguchiHiroshi Takiguchi Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Yoshiaki KubotaYoshiaki Kubota https://orcid.org/0000-0001-6672-4122 Department of Anatomy, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan (Y.K.). Search for more papers by this author , Kinya SeoKinya Seo https://orcid.org/0000-0003-0004-7370 Department of Cardiovascular Medicine, Stanford University, CA (K.S.). Search for more papers by this author , Asuka SakataAsuka Sakata Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan (A.S.). Search for more papers by this author , Seitaro NomuraSeitaro Nomura Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Department of Therapeutic Strategy for Heart Failure (S.N., M. Hatano), The University of Tokyo, Bunkyo-ku, Japan. Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan (T.F., S.N.). Search for more papers by this author , Masaru HatanoMasaru Hatano Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Department of Therapeutic Strategy for Heart Failure (S.N., M. Hatano), The University of Tokyo, Bunkyo-ku, Japan. Search for more papers by this author , Kazutaka UedaKazutaka Ueda Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Mutsuo HaradaMutsuo Harada Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Department of Advanced Clinical Science and Therapeutics, Graduate School of Medicine (M. Harada), The University of Tokyo, Bunkyo-ku, Japan. Search for more papers by this author , Haruhiro TokoHaruhiro Toko Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Department of Advanced Translational Research and Medicine in Management of Pulmonary Hypertension (G.N., H. Toko), The University of Tokyo, Bunkyo-ku, Japan. Search for more papers by this author , Eiki TakimotoEiki Takimoto Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Hiroshi AkazawaHiroshi Akazawa https://orcid.org/0000-0002-3574-9607 Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author , Satoshi NishimuraSatoshi Nishimura Search for more papers by this author and Issei KomuroIssei Komuro Correspondence to: Issei Komuro, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Email E-mail Address: [email protected] https://orcid.org/0000-0002-0714-7182 Department of Cardiovascular Medicine, The University of Tokyo Hospital, Bunkyo-ku, Japan (T.F., N.T., H.H., S.I., H. Tokiwa, S.M., T.S., H. Takiguchi, S.N., M. Hatano, K.U., M. Harada, H. Toko, E.T., H.A., I.K.). Search for more papers by this author Originally published25 Oct 2021https://doi.org/10.1161/CIRCULATIONAHA.121.056219Circulation. 2021;144:1452–1455Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary arterial remodeling, including the progressive obliteration of arterioles, which can lead to increased pulmonary vascular resistance and fatal respiratory and circulatory failure. The histological analysis of PH vessels by 2-dimensional imaging methods has revealed constrictive inward remodeling with the proliferation of smooth muscle cells (SMCs) and endothelial cells (ECs). Although 3-dimensional (3D) tissue visualization systems that use confocal and multiphoton microscopy allow imaging of complex microstructures in several organs, imaging of changes in vascular architecture has remained challenging because factors, such as tissue opacity, light scattering, and absorbance,1 limit the depth of light penetration to ≈100 to 200 µm. Recent remarkable progress in tissue-clearing techniques has been able to overcome these technical barriers to visualize whole organs without thin sectioning. Specifically, the clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC) technique is a newly developed tissue-clearing method that enables 3D imaging of whole organs.1To analyze vascular remodeling in PH, we developed a new 3D visualization system of murine pulmonary vasculature using the CUBIC tissue-clearing method and multiphoton microscopy. All experiments were approved by the Ethics Committee for Animal Experiments of the University of Tokyo and strictly adhered to stipulated guidelines of the University of Tokyo for animal experiments. The data that support the findings described here are available from the corresponding authors on reasonable request.For sample preparation, mice were transcardially and sequentially perfused with 4% (wt/vol) paraformaldehyde in phosphate-buffered saline and 50% (v/v) CUBIC-1 reagent. Postcaval lobes of the right lung were excised and continuously immersed in the CUBIC-1 reagent at 37 °C, either for 5 days for whole-mount staining or for 1 day for fluorescent protein labeling (Figure [A]). Next, for whole-mount immunostaining, lungs were immersed in 20% (wt/vol) sucrose in phosphate-buffered saline, frozen in optimal cutting temperature compound at −80 °C overnight to increase antibody penetration,2 thawed at room temperature, and then subjected to whole-mount immunostaining with primary antibodies diluted in 2% (v/v) Triton X-100–phosphate-buffered saline for 3 days. The fluorescent protein–labeled and –stained samples were finally immersed in CUBIC-2 reagent for 1 day before imaging by multiphoton excitation fluorescence microscopy with tile scanning.Download figureDownload PowerPointFigure. Morphological and quantitative assessment of pulmonary vasculature using 3D images.A, Experimental protocol for tissue clearing and whole-mount immunostaining. CUBIC-1 reagent was prepared as a mixture of 25 wt% urea, 25 wt% N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine, and 15 wt% Triton X-100 in deionized water. CUBIC-2 reagent was prepared as a mixture of 50 wt% sucrose, 25 wt% urea, 10 wt% 2,2′,2′′-nitrilotriethanol, and 0.1% (v/v) Triton X-100 in deionized water. B, Bright-field images of a murine lung lobe before and after optical clearing using the CUBIC technique. Scale bar, 1 mm. C, 3D-reconstructed images of the vasculature in the postcaval lobe of pulmonary endothelial-specific tdTomato-expressing mice (L1-Cre; Rosa26-lsl-tdTomato). Images of tissue-cleared, immunofluorescent-labeled, or fluorescent protein–labeled samples were acquired using multiphoton excitation fluorescence microscopy (SP8, Leica Microsystems GmbH) equipped with a 5× (HCX PL Fluotar 5×/0.15; numeric aperture=0.15; working distance=13.7 mm), 10× (HCX PL Fluotar 10×/0.30; numeric aperture=0.3; working distance=11 mm), or 20× (HCX APO 20×/0.75 IMM CORR CS2; numeric aperture=0.75; working distance=0.68 mm). The pixel size of each image obtained using a 5×, 10×, or 20× objective lens was x=4.54, 2.27, and 1.13 μm and y=4.54, 2.27, and 1.13 μm, respectively; z-step was set to 13.2, 6.6, and 2.2 μm, respectively. Samples were immersed in CUBIC-2 reagent during image acquisition. Samples were excited at wavelengths of 800 and 900 nm using a Ti:sapphire laser (Chameleon Vision II; Coherent). Image processing and 3D reconstruction (maximum intensity projection and volume rendering) were performed using the Leica application suite X (Leica Microsystems GmbH). Smooth muscle cells (SMCs) were stained with fluorescein isothiocyanate–conjugated anti–α smooth muscle actin (αSMA) antibody (Merck KGaA), and tdTomato fluorescence visualized endothelial cells (ECs). The image in the Center was acquired using a 5× objective lens, whereas others were captured using a 20× objective lens. Scale bar, 1 mm in the Center, 200 μm in the second and fourth panels from the left, and 50 μm in the first and fifth panels from the left. D and E, 2D (D) and 3D (E) image analysis in normoxia (Nx) and hypoxia-induced pulmonary hypertension (Hx-PH). SMCs were stained with Cy3-conjugated anti-αSMA antibody (Merck KGaA). PH was induced in male C57BL/6J mice by conditioning them at 8.5% oxygen at sea-level atmospheric pressure for 3 weeks. Scale bar, 100 μm in D; 500 μm in E (200 μm in Inset). F, Calculating the SMC elongation index, defined as the ratio of total length of vascular SMC branch to lung volume, for the quantitative assessment of SMC branching and elongation. The total length of vascular SMC branch arising from the trunk was calculated by adding up divided vessel segments in each z-stack image that were manually traced and measured to prevent overlap of consecutive parts obtained in adjacent images. The volume of interest was calculated as integral of the area, which was manually traced and measured. G, SMC elongation index (n=4 per group). Student t test was used to compare Nx and Hx-PH mice. H and I, 2D (H) and 3D (I) EC lineage-tracing analysis in Nx and Hx-PH. Scale bar, 100 μm in H; 500 μm in I (100 μm in Inset). J, Calculating angiogenesis index, defined as the ratio of total neovessel length to lung surface area, for the quantitative assessment of angiogenesis near the lung surface. Total neovessel length was calculated by adding up the divided vessel segments in each z-stack image taken at the lung surface facing the lens; these were manually traced and measured to ensure that there was no overlapping of consecutive parts obtained from adjacent images. The surface area of interest was obtained by calculating the integral of manually calculated lung circumference. K, Angiogenesis index (n=3 per group). Student t test was used to compare Nx and Hx-PH mice. CUBIC indicates clear unobstructed brain/body imaging cocktails and computational analysis; DAPI, 4′,6-diamidino-2-phenylindole; 2D, 2-dimensional; 3D, 3-dimensional; OCT, optimal cutting temperature; and PBS, phosphate-buffered saline.The lung tissue was successfully transparentized (Figure [B]). Fluorescence attributable to ECs expressing tdTomato or fluorescein isothiocyanate–conjugated anti–α smooth muscle actin antibody staining enabled 3D visualization of whole-lung vasculature, at full depth and at a single-cell resolution, in pulmonary EC-specific tdTomato-expressing mice (L1-Cre;Rosa26-lsl-tdTomato; Figure [C]); therefore, we analyzed 3D SMC remodeling in a murine model of hypoxia-induced PH. Conventional histopathologic analysis revealed greater numbers of α smooth muscle actin–stained arterioles (Figure [D]), which had been previously characterized as distal arteriole muscularization of nonmuscularized vessels and medial hypertrophy in PH. In addition, 3D imaging of α smooth muscle actin–stained SMCs could depict a previously uncharacterized SMC branching and elongation into the peripheral lung in hypoxia-induced PH (Figure [E]), which was quantitatively assessed as the SMC elongation index (Figure [F and G]).To further characterize SMC remodeling and verify the involvement of angiogenesis with the proliferation of existing SMCs surrounding EC sprouts, we performed 3D EC lineage-tracing experiments in hypoxia-induced PH mice by crossing Rosa26-lsl-tdTomato with mice carrying a VE-cadherin-CreERT2–inducible endothelial Cre driver. A single dose of tamoxifen (5 mg/kg body weight) was administered at 5 weeks of age, and mice were subjected to 3D imaging after 3 weeks of normoxic or hypoxic conditioning at 11 weeks of age. The 3D EC lineage-tracing analysis clearly showed EC sprouting and elongation during PH development, mainly near the lung surface, which was not apparent with 2-dimensional EC lineage-tracing imaging (Figure [H and I]). This angiogenetic response was quantitatively evaluated as the angiogenesis index (Figure [J and K]).Here, we described novel vascular remodeling in a murine model of hypoxia-induced PH using our new 3D visualization system and quantification methods. Immediate and sequential perfusion of fixative solution and 50% (v/v) CUBIC-1 reagent were crucial for better tissue transparency and clear 3D visualization.1 Tissue freezing and thawing in optimal cutting temperature compound2 and the use of higher detergent concentration (2% of Triton X-100) in antibody solution3 were needed to increase antibody penetration and signal intensities. Because we could not optimize conditions for visualizing ECs using anti-CD31 and anti-CD102 antibodies or isolectin-B4, ECs were 3D imaged using EC-specific tdTomato fluorescence. We propose that this 3D system can provide significant advantages in merging morphology and phenotypes in PH, and possibly other conditions. In neuroscience, comprehensive 3D imaging has been used to detect tumor invasion and metastasis, neural excitation, and cell senescence.4,5 Covisualization of other cell types such as immune cells with defined markers for their origin, differentiation, and function would reveal both spatial heterogeneity and their contribution to vascular remodeling in PH, leading to a better understanding of the mechanisms of PH.AcknowledgmentsWe are grateful to the laboratory members in the Department of Cardiovascular Medicine, the University of Tokyo, for their valuable technical assistance, especially A. Ogawa, Y. Ishiyama, and K. Akiba. We thank Prof Miyazono, Dr Oh, and Dr Beppu for valuable discussion.Sources of FundingThis work was supported by the Practical Research Project for Rare/Intractable Diseases from Japan Agency for Medical Research and Development (grants 20ek0109487h0001 to Drs Fujiwara, Takeda, and Komuro and 21ek0109560h0001 to Drs Fujiwara and Takeda), grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (to Dr Fujiwara), MSD Life Science Foundation (to Drs Fujiwara and Takeda), SENSIN Medical Research Foundation (to Dr Takeda), Novaltis Pharma Grants for Basic Research (to Dr Takeda), Bristol-Meyers Squibb Foundation (to Dr Takeda), and Nippon Shinyaku Co, Ltd (to Dr Fujiwara).Disclosures None.Footnoteshttps://www.ahajournals.org/journal/circFor Sources of Funding and Disclosures, see page 1455.Correspondence to: Issei Komuro, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Email [email protected]co.jpNorifumi Takeda, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Email [email protected]ecc.u-tokyo.ac.jpReferences1. Tainaka K, Kubota SI, Suyama TQ, Susaki EA, Perrin D, Ukai-Tadenuma M, Ukai H, Ueda HR. Whole-body imaging with single-cell resolution by tissue decolorization.Cell2014; 159:911–924. doi: 10.1016/j.cell.2014.10.034CrossrefMedlineGoogle Scholar2. Liang H, Akladios B, Canales CP, Francis R, Hardeman EH, Beverdam A. Cubic protocol visualizes protein expression at single cell resolution in whole mount skin preparations.J Vis Exp2016; 114:54401. doi: 10.3791/54401Google Scholar3. Susaki EA, Shimizu C, Kuno A, Tainaka K, Li X, Nishi K, Morishima K, Ono H, Ode KL, Saeki Y, et al.. Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.Nat Commun2020; 11:1982. doi: 10.1038/s41467-020-15906-5CrossrefMedlineGoogle Scholar4. Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, Keller PJ. Tissue clearing and its applications in neuroscience.Nat Rev Neurosci2020; 21:61–79. doi: 10.1038/s41583-019-0250-1CrossrefMedlineGoogle Scholar5. Omori S, Wang TW, Johmura Y, Kanai T, Nakano Y, Kido T, Susaki EA, Nakajima T, Shichino S, Ueha S, et al.. Generation of a p16 reporter mouse and its use to characterize and target p16high cells in vivo.Cell Metab2020; 32:814–828.e6. doi: 10.1016/j.cmet.2020.09.006CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails October 26, 2021Vol 144, Issue 17Article InformationMetrics Download: 874 © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.121.056219PMID: 34694894 Originally publishedOctober 25, 2021 Keywordsmicroscopy, fluorescence, multiphotonendothelial cellsmyocytes, smooth muscleneovascularization, pathologicPDF download Advertisement SubjectsAngiogenesisBasic Science ResearchVascular Biology