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• W3136171646 abstract "HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 41, No. 4Letter by Campanero and Redondo Regarding Article, “Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBLetter by Campanero and Redondo Regarding Article, “Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta” Miguel R. Campanero and Juan Miguel Redondo Miguel R. CampaneroMiguel R. Campanero https://orcid.org/0000-0003-1410-8621 Cell-Cell Communication and Inflammation Department, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas–Universidad Autónoma de Madrid, Spain (M.R.C.). Search for more papers by this author and Juan Miguel RedondoJuan Miguel Redondo https://orcid.org/0000-0001-5779-9122 Gene Regulation in Cardiovascular Remodeling and Inflammation Group, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain (J.M.R.). Search for more papers by this author Originally published24 Mar 2021https://doi.org/10.1161/ATVBAHA.121.315934Arteriosclerosis, Thrombosis, and Vascular Biology. 2021;41:e238–e239To the Editor:We have read with interest the report by Dr David Dichek et al claiming that jugular vein injection of high-titer lentiviral vectors does not transduce the aorta.1 After careful analysis of the report, we firmly believe that the experimental basis behind their negative results is likely conditioned by insufficient technical experience and training. Moreover, the 50% to 70% drop in the titer of lentivirus provided by us and the Fred Hutch Cancer Research Center strongly suggests that another contributing factor to the experimental failure may have been inappropriate lentivirus manipulation. Furthermore, the experimental design is flawed by the incorporation of an experimental group with only 2 mice (those inoculated with a low amount of active virus from our lab). Indeed, this weakness is recognized by the authors, who state: “Our study has some limitations. We used a small number of mice. Our lentivirus titration did not exactly match that of Dr Redondo’s laboratory. Our results do not exclude a low level of in vivo aortic transduction”. This is shocking, as in our view, it does not make sense that with such a weak basis, this report aims to contradict a methodology routinely used for over a decade in our laboratory and by several other groups. Thus, it is important to provide a historical perspective on the methodology and its wide use and to review every technical aspect of Dichek’s report.The protocol for aorta transduction used in this report was described by us and is based on the intrajugular inoculation of high-titer lentiviral vectors (>108 active infective particles in adult mice).2–4 We have used this protocol with vectors encoding GFP (green fluorescent protein)-tagged peptides that inhibit the activity of calcineurin, a phosphatase that mediates vascular damage. Immunohistochemistry analysis of aortic sections readily detected GFP in all aortic layers (Figure 5A in Esteban et al3). Moreover, expression of these peptides inhibited angiotensin II–induced abdominal aortic aneurysm in Apoe−/− mice as efficiently as pharmacological inhibition of calcineurin with cyclosporine A (Figures 4 and 5 in Esteban et al3). Furthermore, the inhibition of angiotensin II–induced activation of NFAT (nuclear factor of activated T-cells) transcription factors in all layers of the abdominal aorta, evidenced by southwestern histochemistry analysis (Figure 5C in Esteban et al3), was an independent experimental approach that demonstrated the presence of GFP-tagged peptides in the tissue.We also used the intrajugular route to deliver lentivirus encoding GFP plus either Adamts1-targeting siRNA or a control siRNA. Again, GFP immunohistochemistry analysis showed specific GFP staining in all layers of the aorta (Figure 2B and Figures IH and IVA in the Data Supplement in Oller et al4). The loss of Adamts1 staining in aortas from mice transduced with Adamts1-specific siRNA and the sharp drop in aortic Adamts1 expression detected by immunoblot analysis of these mice are 2 independent demonstrations shown in that report of the efficient transduction of the aorta (Figure 2B and 2C; Figures 1h and 4a in the Data Supplement in Oller et al4). Importantly, results from numerous independent experiments throughout that report show that Adamts1 silencing in wild-type mice produced a phenotype that recapitulates the aortopathy observed in Adamts1+/− mice. In total, the experiments shown in these studies have demonstrated efficient transduction of the aortas of >250 mice. These numbers are robust and contrast dramatically with the low number of mice used in Dichek’s report. Thus, our data unambiguously demonstrate that intrajugular inoculation of high-titer lentiviral vectors not only efficiently transduces the aorta but can also be used for phenotypic modulation of the aorta.The immunohistochemistry analysis in Dr Dichek’s report is far from adequate if the intention is to provide a counterpoint to previous studies. Several tissue sections in this report present numerous holes that are compatible with inadequate processing of the tissue samples. Moreover, the high number of viral genomes (12/cell) detected in the aorta fragment used as a positive control (n=1 aorta) is inconsistent with the very poor GFP staining of the aorta fragment used for histology (n=1 aorta) and suggests the use of a low-sensitivity staining protocol. The combination of inadequate tissue processing and low-sensitivity staining might itself explain the lack of thoracic aorta GFP staining in this report.Quantification data of an endogenous gene are missing in the tissues analyzed. This issue is particularly important in the case of aortic tissue because the protocol used to extract genomic DNA from the aorta differed from that used for the other tissues and might have yielded heavily fragmented genomic DNA. The strong adventitial GFP staining of the right carotid artery sections from lentivirus-GFP-infused mice shown in Figure 2 points to a potentially more important mistake in the inoculation: that a substantial proportion of the viral preparation was spilled around the inoculation site instead of being fully inoculated into the jugular vein. Indeed, this is the most frequent mistake made by personnel with insufficient training in this procedure.The experimental procedure is not difficult but requires adequate skills and training of personnel to fully inoculate the virus preparation into the bloodstream of a mouse jugular vein. Self-training is possible by inoculating 100 µL of PBS spiked with a dye such as Evans blue to facilitate visualization of any spilled-over material. Eleven team members from our labs have successfully used this technique over the past 10 years. We have also trained researchers from other groups, and some of them have already published results showing efficient transduction of the aorta, as is the case of the rigorous studies by Dr Arroyo’s (incorrectly attributed to us in Dr Dichek’s report) and Dr Ramiro’s groups.5,6 As Dr Dichek’s report might mislead readers to conclude that our laboratory is the only one to report successful transduction of the aorta with lentivirus, we have to stress that while we contributed to both studies, the lentiviral inoculation and the phenotypic characterization of the inoculated mice were exclusively performed by members of the leading authors’ teams in their laboratories. Indeed, Dr Dichek’s statement that our reports “conflict with several rigorous investigations of in vivo lentivirus tropism published in 2000 to 20028–13” is not only wrong—because those studies did not examine aortic transduction—but also biased because it oddly ignores 3 additional independent reports showing aorta transduction after tail vein inoculation of lentivirus.7–9 In total, 7 independent groups, at least, have reported transduction and phenotypic modulation of the aorta after lentivirus intravenous inoculation.3–10As we agree with Dr Dichek’s report that “efficient gene transfer to the vascular wall via intravenous injection would be useful for experimental vascular biology and gene therapy,” we have scheduled exchanges of personnel with two additional labs for training and in-house demonstration of this technique. Moreover, we invite other researchers wishing to use this technique to join our lab for training, just asking in return that they train other groups. We believe that the scientific community should not be discouraged from using this technique by the publication of a failed single experiment.Disclosures None.FootnotesFor Disclosures, see page e239.References1. Bi L, Wacker BK, Stamatikos A, Sethuraman M, Komandur K, Dichek DA. Jugular vein injection of high-titer lentiviral vectors does not transduce the aorta—brief report.Arterioscler Thromb Vasc Biol. 2021; 41:1149–1155. doi: 10.1161/ATVBAHA.120.315125LinkGoogle Scholar2. Alfranca A, Campanero MR, Redondo JM. New methods for disease modeling using lentiviral vectors.Trends Mol Med. 2018; 24:825–837. doi: 10.1016/j.molmed.2018.08.001CrossrefMedlineGoogle Scholar3. Esteban V, Méndez-Barbero N, Jiménez-Borreguero LJ, Roqué M, Novensá L, García-Redondo AB, Salaices M, Vila L, Arbonés ML, Campanero MR, et al. Regulator of calcineurin 1 mediates pathological vascular wall remodeling.J Exp Med. 2011; 208:2125–2139. doi: 10.1084/jem.20110503CrossrefMedlineGoogle Scholar4. Oller J, Méndez-Barbero N, Ruiz EJ, Villahoz S, Renard M, Canelas LI, Briones AM, Alberca R, Lozano-Vidal N, Hurlé MA, et al. Nitric oxide mediates aortic disease in mice deficient in the metalloprotease Adamts1 and in a mouse model of Marfan syndrome.Nat Med. 2017; 23:200–212. doi: 10.1038/nm.4266CrossrefMedlineGoogle Scholar5. Martín-Alonso M, García-Redondo AB, Guo D, Camafeita E, Martínez F, Alfranca A, Méndez-Barbero N, Pollán Á, Sánchez-Camacho C, Denhardt DT, et al. Deficiency of MMP17/MT4-MMP proteolytic activity predisposes to aortic aneurysm in mice.Circ Res. 2015; 117:e13–e26. doi: 10.1161/CIRCRESAHA.117.305108LinkGoogle Scholar6. de Yébenes VG, Briones AM, Martos-Folgado I, Mur SM, Oller J, Bilal F, González-Amor M, Méndez-Barbero N, Silla-Castro JC, Were F, et al. Aging-associated miR-217 aggravates atherosclerosis and promotes cardiovascular dysfunction.Arterioscler Thromb Vasc Biol. 2020; 40:2408–2424. doi: 10.1161/ATVBAHA.120.314333LinkGoogle Scholar7. Huang X, Yue Z, Wu J, Chen J, Wang S, Wu J, Ren L, Zhang A, Deng P, Wang K, et al. MicroRNA-21 knockout exacerbates angiotensin II-induced thoracic aortic aneurysm and dissection in mice with abnormal transforming growth factor-β-SMAD3 signaling.Arterioscler Thromb Vasc Biol. 2018; 38:1086–1101. doi: 10.1161/ATVBAHA.117.310694LinkGoogle Scholar8. Lovren F, Pan Y, Quan A, Singh KK, Shukla PC, Gupta N, Steer BM, Ingram AJ, Gupta M, Al-Omran M, et al. MicroRNA-145 targeted therapy reduces atherosclerosis.Circulation. 2012; 12611 suppl 1S81–S90. doi: 10.1161/CIRCULATIONAHA.111.084186LinkGoogle Scholar9. Xu X, Zhang F, Lu Y, Yu S, Sun W, Sun S, Cheng J, Ma J, Zhang M, Zhang C, et al. Silencing of NONO inhibits abdominal aortic aneurysm in apolipoprotein E-knockout mice via collagen deposition and inflammatory inhibition.J Cell Mol Med. 2019; 23:7449–7461. doi: 10.1111/jcmm.14613CrossrefMedlineGoogle Scholar10. DuPont JJ, McCurley A, Davel AP, McCarthy J, Bender SB, Hong K, Yang Y, Yoo JK, Aronovitz M, Baur WE, et al. Vascular mineralocorticoid receptor regulates microRNA-155 to promote vasoconstriction and rising blood pressure with aging.JCI Insight. 2016; 1:e88942. doi: 10.1172/jci.insight.88942CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails April 2021Vol 41, Issue 4 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/ATVBAHA.121.315934PMID: 33760632 Originally publishedMarch 24, 2021 PDF download Advertisement SubjectsAneurysmAnimal Models of Human DiseaseVascular BiologyVascular Disease" @default.
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