Retina Vascular Interstitial Cells in Retinal Arteriolar Annuli Are Altered During Hypertension David Ramos,1,2 Joana Catita,2–4 Mariana López-Luppo,2,3 Andreia Valença,1,2 Aina Bonet,2,3 Ana Carretero,1–3 Marc Navarro,1–3 Victor Nacher,1–3 Simon Mendez-Ferrer,5 Anna Meseguer,6–8 Alba Casellas,2,9,10 Luı́sa Mendes-Jorge,1,2 and Jesús Ruberte1–3 1 CIISA–Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, Universidade de Lisboa, Lisbon, Portugal 2 Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, Bellaterra, Spain 3 Department of Animal Health and Anatomy, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain 4 Department of Anatomy, Faculty of Veterinary Medicine, Universidade Lusófona de Humanidades e Tecnologias, Lisbon, Portugal 5 Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute and Department of Haematology, University of Cambridge, and NHS-Blood and Transplant, Cambridge, United Kingdom 6 Renal Physiopathology Group, CIBBM-Nanomedicine, Vall d’Hebron Research Institute (VHIR), Barcelona, Spain 7 Department of Biochemistry and Molecular Biology, Unitat de Bioquı́mica de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain 8 Red de Investigación Renal (REDINREN), Instituto Carlos III-FEDER, Madrid, Spain 9 Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain 10 CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain Correspondence: Jesús Ruberte, PURPOSE. It has been suggested that arteriolar annuli localized in retinal arterioles regulate Universitat Autònoma de Barcelona, retinal blood flow acting as sphincters. Here, the morphology and protein expression profile Edifici H, carrer de la Vallmoronta, of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild- Bellaterra, Barcelona 08193, Spain; type, b-actin–Egfp, and Nestin–gfp transgenic mice. Additionally, to study the effect of jesus.ruberte@uab.cat. hypertension, the KAP transgenic mouse has been used. Submitted: June 11, 2018 Accepted: December 10, 2018 METHODS. Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on Citation: Ramos D, Catita J, López- paraffin sections and whole mount retinas by immunofluorescence and histochemistry. Luppo M, et al. Vascular interstitial cells in retinal arteriolar annuli are RESULTS. The ultrastructural analysis of arteriolar annuli showed a different cell population altered during hypertension. Invest found between endothelial and muscle cells that matched most of the morphologic criteria Ophthalmol Vis Sci. 2019;60:473– established to define interstitial Cajal cells. The profile of protein expression of these vascular 487. https://doi.org/10.1167/ interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the iovs.18-25000 endothelial and smooth muscle cells, because they expressed b-actin, nestin, and CD44, but they did not express CD31 and a-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-b). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. CONCLUSIONS. A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease. Keywords: retina, arteriolar annuli, hypertensive retinopathy ypertension is an important public health challenge type of retinopathy and affects 3% to 14% of the adult H worldwide. This condition affected 972 million adults in population over 40 years of age.2,3 Arteriolar annuli have been described as conical accumula- 2000, and its prevalence is predicted to increase by 60% to a total of 1.56 billion people by 2025.1 Under elevated blood tions of periodic acid Schiff (PAS)-positive material with increased cellularity surrounding the opening of retinal pressure, retinal vasculature undergoes a series of pathologic arteriolar branching sites.4,5 It has been suggested that these changes, including generalized and focal arteriolar narrowing, structures regulate retinal blood flow by exerting a sphincter- intimal thickening, hyperplasia of the tunica media, and hyaline like activity.4,6,7 This hypothesis is supported by previous degeneration.2 Altogether, these symptoms are referred to as studies demonstrating constrictions at arteriolar branch hypertensive retinopathy, which is the second most common points.8,9 However, there is no general agreement concerning Copyright 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783 473 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 474 the cellular composition, structure, and function of arteriolar Immunohistochemistry annuli in the retina. Several cell types cooperate to provide an appropriate Paraffin-embedded eyes were sectioned (3 lm) along the eye vascular tone including endothelial, smooth muscle cells, and axis through the optic disc and cornea, deparaffinized, and pericytes. Recently, multipolar cells with thin processes, which rehydrated. Whole mount retinas were fixed in 10% neutral- have not been classified within the above mentioned cell types, buffered formalin for 2 hours at 48C or in cold acetone for 5 have been identified in the wall of different blood vessels.10–15 minutes, depending on antibodies used. After they were These cells were called vascular interstitial cells (VICs) because washed in PBS, paraffin sections and whole mount retinas of their structural and molecular similarities with interstitial were incubated overnight at 48C with the following markers: Cajal cells. anti-collagen IV (Millipore, Darmstadt, Germany) diluted at In this work, we aimed to finely define the morphology and 1:20, anti–platelet-derived growth factor-receptor b (PDGF-Rb) the protein expression profile of a new subtype of VICs (Abcam, Cambridge, UK) diluted at 1:100, anti–neuron glial 2 localized in the annuli of murine retinal arterioles. Because chondroitin sulphate (NG2) (Millipore) diluted at 1:200, anti–a- these annuli have been considered as discrete sites for blood smooth muscle actin (a-SMA) (Abcam) diluted at 1:100, anti–c- flow regulation, the second main objective of this study was to Kit (Abcam) diluted at 1:50, anti-anoctamin1 (Ano1) (Abcam) analyze whether VICs are altered during hypertensive retinop- diluted at 1:90, anti-CD44 (Abcam) diluted at 1:100, anti-CD31 athy, becoming thus a possible target for the treatment of this (BD Pharmingen, San Jose, CA, USA) diluted at 1:25, anti– disease. glutamine synthetase (GS) (Sigma-Aldrich Corp., St. Louis, MO, USA) diluted at 1:500, anti–enhanced green fluorescent protein (EGFP) (Molecular Probes, Eugene, OR, USA) at 1:300 dilution, MATERIALS AND METHODS and rhodamine-phalloidin (Sigma-Aldrich Corp.) at 5 lg/mL concentration. Retinas were washed several times in PBS and Eyes then incubated overnight at 48C with specific secondary antibodies: anti-rat IgG-biotinylated (Abcam), anti-goat IgG- Eyes from wild-type CD1 (ICR) mice together with b-actin- biotinylated (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, Egfp16 and Nestin-gfp transgenic mice17 served to analyze the USA), anti-rabbit Alexa Fluor 568 (Invitrogen, Carlsbad, CA, presence of VICs in arteriolar annuli under physiologic USA) and anti-rabbit Alexa Fluor 488 (Abcam). All secondary conditions. Additionally, to study the effect of hypertension in antibodies were diluted at 1:100. Once retinas were washed in arteriolar annuli, a model of elevated blood pressure, the kidney PBS, Streptavidin Alexa Fluor 488 (Invitrogen) or Streptavidin androgen-regulated protein (KAP) transgenic mouse, was used.18 Wild-type and b-actin–Egfp transgenic mice were Alexa Fluor 568 (Invitrogen) were used as fluorochromes at maintained at the Animal Facility of the Universitat Autònoma 1:100 dilution. Microscopic analysis was performed with a de Barcelona (UAB, Barcelona, Spain), whereas Nestin-gfp laser scanning confocal microscope (TCS SP2; Leica Micro- transgenic mice were kindly gifted by Grigori N. Enikolopov systems GmbH, Heidelberg, Germany). To assess retinal (Stony Brook University, New York, NY, USA). Hypertensive KAP morphology, deparaffinized eye sections were stained with transgenic mice were provided by Anna Meseguer (Vall hematoxylin and eosin and observed with a Nikon Eclipse d’Hebron Institut de Recerca–VHIR and Centre d’Investigacions E800 microscope (Nikon Corp.). en Bioquı́mica i Biologia Molecular–CIBBIM, Barcelona, Spain). Mice were maintained on 12-hour day/night cycles, with free Measurement of Arterial Pressure access to food and water. Principles of laboratory animal care Arterial pressure was measured using tail-cuff equipment were followed. Animal care and experimental procedures conformed to the ARVO Statement for the Use of Animals in adapted for mice (Letica, Barcelona, Spain), at several times Ophthalmic and Vision Research and were approved by Ethics between 0900 and 1200 hours, and pressure values were Committee in Animal and Human Experimentation of the considered acceptable when at least five consecutive measure- Universitat Autònoma de Barcelona (CEEAH/UAB). ments provided similar values. To ensure a proper assessment of blood pressure, mice were previously trained to be kept in a plastic restraining chamber for several minutes during several Retinal Digestion weeks. Retinal digestion was performed according to the technique described by Kuwabara and Cogan4 and Hammes et al.19 with Thickness of Arteriolar Tunica Media minor modifications. After fixation with neutral-buffered formalin, whole mount retinas were washed in filtered water The thickness of the arteriolar tunica media was measured in and subjected to a trypsin digestion (3% trypsin in 0.1 M tris confocal images of a-SMA immunolabeled arterioles. To avoid buffer) for approximately 90 minutes at 378C. Once digested, bias errors, each measure was determined four times per retinas were rinsed several times in filtered water to isolate arteriole in four different arterioles per retina. Retinas from retinal blood vessels, which were finally stained with three KAP transgenic and three littermate control mice were hematoxylin and PAS. Digested retinal vasculatures were studied. microscopically analyzed (Nikon Eclipse E800; Nikon, Tokyo, Japan). NADPH Diaphorase Activity Analysis and Quantification Transmission Electron Microscopy Analysis Nicotinamide adenine dinucleotide phosphate (NADPH) di- Retinal fragments from three mice were fixed in 2.5% aphorase activity was analyzed in four KAP transgenic and four glutaraldehyde and 2% paraformaldehyde, postfixed in 1% control littermate mice. Whole retinas were fixed in 4% osmium tetroxide, stained in aqueous uranyl acetate, dehy- paraformaldehyde for 3 hours at room temperature. After drated, and embedded in epoxy resin. Ultrathin sections (70 several washes in PBS and Tris HCl buffer 0.1 M, pH 8.7, retinas nm) of seven different arteriolar side-arm branching sites were were incubated for 90 minutes at 378C in a reactive solution stained using lead citrate and examined by a transmission containing 15 mM malic acid, 1 mM MnCl2, 1 mM NADP, 0.2 electron microscope (Jeol 1400; Jeol Ltd., Tokyo, Japan). mM nitro blue tetrazolium, and 0.2% Triton X-100 diluted in Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 475 FIGURE 1. Identification of arteriolar annuli in the mouse retina. (A, B) PAS/hematoxilin staining of pepsin/trypsin-digested retinas evidenced the presence of arteriolar annuli at the origin of side-arm branches of the first order arterioles. (C) Arteriolar annuli were characterized by increased PAS stain and hypercellularity. OD, optic disc; 1st art, first-order arteriole; 2nd art, second-order arteriole. Arrowheads point to arteriolar annuli. Scale bars: (B) 57.2 lm; (C) 14.9 lm. Tris buffer 0.1 M, pH 8.7. Then, retinas were washed in Tris arterioles (Figs. 1B, 1C).4,5,20,21 An increase in cellularity was buffer HCl 0.1 M, pH 8.7 and mounted in buffered glycerol. observed in mouse arteriolar annuli (Figs. 1B, 1C), similarly to Images were acquired with an optic microscope (Nikon findings reported in other mammals.5 Eclipse E800). Although it was previously suggested that an incompletely To quantify NADPH diaphorase activity, images were characterized cell type might reside in retinal arteriolar converted to black and white, inverted, and binarized. Then, branches,22 the exact nature of cells in arteriolar annuli has gray intensity was measured in 725.24-lm2 areas of 35 remained unknown. Therefore, we used transmission electron arteriolar annuli with the Nis Elements Imaging Software microscopy (TEM) and immunohistochemistry to characterize (Nikon). cells in arteriolar annuli. The ultrastructural analysis of arteriolar annuli evidenced unequivocally three distinct cellular types (Fig. 2A): endothelial Protein Quantification cells forming a continuous layer that covered the luminal To test whether hypertension is capable to affect VICs, Ano1 surface; smooth muscle cells occupying the abluminal surface; expression was analyzed in arteriolar annuli from five KAP and a morphologically distinct VIC-like cell population transgenic and five control littermate mice. Mean fluorescence embedded in a loose extracellular matrix localized between intensity was measured with Leica LAS AF Lite imaging endothelial and smooth muscle cells (Fig. 2A). Moreover, software (Leica) in 210.77-lm2 areas of 10 arteriolar annuli immunohistochemistry against a-SMA (Fig. 2B) and CD31 (Fig. per retina. 2C), two well-known markers of smooth muscle and endothe- lial cells, respectively,23,24 showed that VIC-like cells were different from endothelial and smooth muscle cells because Statistical Analysis these cells were CD31 and a-SMA negative (Figs. 2B, 2C). Results are shown as mean 6 SEM. Statistical analyses were performed by unpaired t-test. Differences between groups Morphologic Characterization of VICs in Retinal were considered statistically significant at P < 0.05. All Arteriolar Annuli statistical analyses were performed with the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Under TEM analysis, annuli presumptive VICs appeared as irregularly shaped cells with slender processes (Figs. 2, 3A). These observations match previous results reporting the RESULTS presence of a subset of cells with cytoplasmic processes in arteriolar branches of semi-corroded casts of mouse retinal Retinal Arteriolar Annuli Topography and Cellular vasculature.21 Presumptive VICs in arteriolar annuli also Composition showed nuclei with more than one lobe, numerous large and elongated mitochondria, and many cisternae of smooth and As previously described, arteriolar annuli in mouse retinas rough endoplasmic reticulum (Fig. 3A). Numerous caveolae appeared as conical accumulations of PAS-stained material were also found all along the nucleated region of presumptive restricted to the origin of side-arm branches of first-order VICs and in their cytoplasmic processes (Figs. 3B, 3D, 3E). Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 476 FIGURE 2. Cellular composition of arteriolar annuli. (A–C) TEM evidenced three distinct cellular types in arteriolar annuli: endothelial cells (EC, blue), smooth muscle cells (SMC, red), and VICs (green). Specific immunostaining of smooth muscle cells with a-SMA (D) and endothelial cells with CD31 (E) demonstrated that VICs did not belong to these cell types. BM, basement membrane (yellow); 1st art, first-order arteriole; 2nd art, second- order arteriole. Arrowheads point to arteriolar annuli. Scale bars: (A) 1.74 lm; (B) 5.87 lm; (C) 9.32 lm. Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 477 transmembrane glycoprotein CD44 can also be used as a marker to identify murine interstitial Cajal cells.28 VICs were positive for this marker in the wall of mouse arteriolar annuli (Fig. 4C). Several studies have evidenced the expression of NADPH diaphorase in gastrointestinal interstitial Cajal cells.36,37 NADPH diaphorase is an enzyme involved in nitric oxide synthesis and reflects nitric oxide synthase activity.38 NADPH activity was detected in VICs in retinal arteriolar annuli (Fig. 4D). Moreover, as previously described,39,40 NADPH diapho- rase activity was also detected in endothelial cells of the mouse retina (Fig. 4D). Altogether, these results suggest similarities between VICs in arteriolar annuli and gastrointestinal interstitial Cajal cells. In particular, the expression of Ano1 and NADPH diaphorase suggest that VICs might play a role in the motility of arteriolar annuli through pacemaker activity and nitrergic regulation of vascular tone, respectively. Similarities and Differences With Smooth Muscle Cells. VICs observed in other vascular trees from other body localizations share many morphologic characteristics with smooth muscle cells. However, whether VICs are interstitial Cajal cells or belong to the smooth muscle cell lineage remains debatable.10,11,29,41 Several authors have reported distinctive expression of FIGURE 3. Ultrastructural characterization of VICs. (A–C) TEM actin and intermediate filaments in interstitial Cajal cells, evidenced that VICs were characterized by multilobulated nuclei, compared with smooth muscle cells.14,29,37,42,43 As shown elongated mitochondria (mi), cytoplasmic processes, and gap junctions (Fig. 2B), VICs in arteriolar annuli did not express a-SMA, the (arrowheads). (D, E) Numerous caveolae (ca) and endoplasmic main functional marker of smooth muscle cells.44 To further reticulum (ER) were also observed in VICs. SMC, smooth muscle cells; differentiate both cellular types, we analyzed the expression of EC, endothelial cell; Lu, vascular lumen. Scale bars: (A) 175.39 nm; (B; filament proteins F-actin, b-actin, and nestin in mouse arteriolar upper inset) 441.86 nm; (B; lower inset) 347.94 nm. annuli. Phalloidin, a toxin from Amanita phalloides, can be used to Moreover, many gap junctions were observed coupling stain filamentous F-actin.45 VICs in retinal arteriolar annuli presumptive VICs with other annuli neighboring cells (Figs. displayed a distribution of filamentous actin different from 3B, 3C). All these morphologic features are shared with the smooth muscle cells (Fig. 5A). F-actin was scarcely expressed established TEM criteria to define interstitial Cajal cells25 and and mainly localized in the periphery of VICs. In contrast, F- strongly suggest that the uncovered cell population specifically actin occupied the whole cytoplasm of vascular smooth located between endothelial and smooth muscle cells in retinal muscle cells (Fig. 5A). arteriolar annuli is comprised by VICs. In addition, it has been demonstrated that arterial VICs show higher expression of b-actin, compared with vascular Characterization of Protein Profile of VICs in smooth muscle cells.14 To analyze b-actin expression in Retinal Arteriolar Annuli arteriolar annuli, we used the b-actin–Egfp transgenic mouse,16 which expresses the EGFP under the control of We further characterized VICs immunophenotypically through the chicken b-actin promoter. Although previous data immunofluorescence of arteriolar annuli. suggested that all cells except hair cells and erythrocytes Similarities and Differences With Interstitial Cajal express EGFP in this transgenic mouse,16 our results showed Cells. c-Kit, a proto-oncogen that encodes a transmembrane that green fluorescence was not homogenously distributed tyrosine kinase receptor, is one of the most frequently used through the entire retina. An intense green emission was markers to detect interstitial Cajal cells.26–28 However, VICs in observed in the inner limiting membrane, retinal blood retinal arteriolar annuli were negative for c-Kit (Fig. 4A). In this vessels, and the inner segments of photoreceptors (Fig. 5B). regard, several studies dealing with the presence of interstitial Interestingly, a detailed analysis of the retinal vasculature Cajal cells in the vasculature have shown that these cells can be showed an intense green fluorescence emission in VICs of both c-Kit positive15,29 or negative,11,12,14 depending on arteriolar annuli (Fig. 5C). A higher expression of b-actin was arteries and species under study. detected in VICs compared with smooth muscle cells of The protein Ano1, a calcium-activated chloride channel, has retinal arteriolar annuli. Definitely, this molecular pattern been reported as a highly selective molecular marker for characterized by low expression of contractile proteins, like interstitial Cajal cells in the gastrointestinal tract.30,31 Immu- a-SMA (Fig. 2B), together with high expression of cytoskeletal nohistochemical detection of Ano1 in the retina demonstrated proteins, like b-actin (Fig. 6C), suggests a lack of contractility a high expression of this protein in VICs in arteriolar annuli of VICs. This is in accordance with previous studies (Fig. 4B). A weaker Ano1 expression could also be observed in demonstrating that isolated VICs do not contract in response smooth muscle cells located in second-order retinal arterioles, to vasoactive drugs.10 but not in those localized in first-order retinal arterioles (Fig. The intermediate filament nestin can also be used as a 4B). Although Ano1 has been observed in smooth muscle cells marker for VICs.46–48 The Nestin-gfp transgenic mice can be of several murine blood vessels,32–35 our results indicated a used as a highly sensitive reporter system to detect nestin notable and specific increase in the expression of Ano1 in VICs positive cells.17 Therefore, we used this model to analyze in retinal arteriolar annuli. In addition to c-Kit and Ano1, the nestin expression in retinal arteriolar annuli. Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 478 FIGURE 4. Similarities and differences with interstitial Cajal cells. (A) No evidence of c-Kit expression was observed in VICs during confocal analysis of whole mount retinas double immunostained against c-Kit (green) and collagen IV (red). By contrast, confocal microscopy evidenced strong expression of Ano1 (B) and CD44 (C) in whole mount retinas stained by immunohistochemistry against collagen IV (red) and these molecular markers (green). (D) NADPH diaphorase was increased in VICs and endothelial cells (EC) of arteriolar annuli. SMC, smooth muscle cell. Arrowheads point to arteriolar annuli. Scale bars: (A) 14.4 lm; (B) 16.09 lm; (C) 14.19 lm; (D) 6.87 lm. Retinal paraffin sections stained with hematoxylin/eosin Within retinal arteriolar annuli, VICs were easily distin- showed that transgene expression did not alter retinal guishable due to their strong green fluorescence, confirming a architecture in Nestin-gfp transgenic mice (Fig. 6A). Consistent high expression of nestin (Fig. 6C). Given that GFP fluores- with previous observations,49 our immunohistochemical anal- cence occupied the nucleus, cell body, and cytoplasmic ysis of the retina showed that GFP colocalized with glutamino processes of VICs, the morphology and location of these cells synthetase, a specific marker of Müller cells50 (Fig. 6B). These within arteriolar annuli could be easily analyzed in Nestin-gfp results demonstrate that nestin is predominantly expressed by retinas. VICs presented an elongated cell body, mostly glial cells in the mature mouse retina. occupied by a multilobulated nucleus, from which ramified Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 479 FIGURE 5. Actin expression differentiates VICs from smooth muscle cells. (A) Phalloidin histochemistry evidenced that F-actin was differentially distributed in VICs with respect to smooth muscle cells. (B) Retinas from b-actin–Egfp mice showed strong green fluorescence in retinal blood vessels (arrows), inner limiting membrane (ILM), and the inner segments of photoreceptors (IS). (C) A deep analysis of the retinal vasculature of b- actin–Egfp mice showed that annuli VICs were characterized by strong expression of b-actin (green), whereas F-actin (red) appeared restricted to the periphery. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: (A) 5.86 lm; (B) 65.79 lm; (C) 6.46 lm. cytoplasmic processes that surrounded the lumen of the arteriolar annuli (Fig. 6C, inset). Similarities and Differences With Pericytes. Together with smooth muscle cells, pericytes constitute the contractile TABLE. Immunophenotypes of VICs, Interstitial Cajal Cells, Smooth cell population of blood vessels. Pericytes are completely Muscle Cells, and Pericytes embedded within the basement membrane of capillaries and Protein Interstitial Smooth postcapillary venules, and it is generally accepted that they are Marker VICs Cajal Cells Muscle Cells Pericytes not present in arterioles.51 We used several molecular markers that can be used to identify pericytes, including NG2, platelet- a-SMA  þ/ 85 þþþ 44 þ88 derived growth factor receptor beta (PDGFR-b), and desmin, b-Actin þþ þ86 þ/14,87 þ89 among others.52–55 VICs in retinal arteriolar annuli expressed NG2 þþ  14 þþþ53 both NG2 (Fig. 7A) and PDGFR-b (Fig. 7B), but did not express F-actin þ þþþ24 þþ88 desmin (Fig. 7C). Thus, although VICs do not seem to be PDGF-Rb þ   þþ55 contractile cells (based on the lack of a-SMA), these cells Ano1 þþþ þþ30,31 þþ32–35  exhibit pericyte markers, which are weakly or not expressed in Nestin þþþ þþ46  þ90 arteriolar smooth muscle cells (Figs. 5A, 5B). CD44 þ þ28   Taken together, these results suggest that VICs located in NADPH d þþ þþ36,37   mouse retinal arteriolar annuli, rather than belonging to the Results shown refer to normotensive conditions. Superscript smooth muscle cell lineage, represent a new subtype of numbers are reference sources. , no expression; þ, low expression; interstitial Cajal cells. The Table recapitulates the immunophe- þþ, moderate expression; þþþ, strong expression. Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 480 FIGURE 6. Nestin expression differentiates VICS from smooth muscle cells. (A) Hematoxilin/eosin staining of retinal paraffin sections showed that transgene expression did not cause morphologic alterations in retinal architecture of Nestin-gfp mice. (B) Confocal microscopy showed high colocalization (yellow) between GS (red) and EGFP (green), indicating that Müller cells highly expressed nestin in the adult retina. (C) Analysis of whole mount retinas from Nestin-gfp transgenic mice immunolabeled against collagen IV showed that VICs strongly express nestin. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; 1st art, first-order arteriole; 2nd art, second-order arteriole; Mü, Müller cell. Scale bars: (A) 60.4 lm; (B) 55.6 lm; (C) 4.52 lm. notypic characterization of VICs compared with interstitial annuli (VICs) is affected during hypertension, we analyzed Cajal cells, smooth muscle cells, and pericytes under normo- retinas from 8- to 11-month-old KAP transgenic male mice. tensive conditions. KAP transgenic male mice overexpressing KAP driven by its own promoter in a cell- and androgen-restricted manner VICs in Retinal Arteriolar Annuli Are Altered develop hypertension at 4 months of age.18 As expected, tail- cuff measurements of systolic and diastolic arterial pressure in During Hypertensive Retinopathy KAP transgenic male mice showed that these animals were As a response to elevated blood pressure, the retinal overtly hypertensive at 7 months of age (KAP-Tg SYSTOLIC ¼ circulation undergoes a series of pathophysiologic changes 154.52 6 5.63 versus CONTROL SYSTOLIC ¼ 80.52 6 3.64, N ¼ known as hypertensive retinopathy. This spectrum of vascular 4, P < 0.0001; KAP-Tg DIASTOLIC ¼ 120.76 6 5.90 versus signs includes generalized and focal arteriolar narrowing, CONTROL DIASTOLIC ¼ 61.59 6 3.25, N ¼ 4, P < 0.0001; Fig. arteriovenous nicking, retinal hemorrhages, cotton wool spots, 8A). and swelling of the optic disk.56 More specifically, retinal Retinas from KAP transgenic male mice did not show gross hypertension induces alterations in arteriolar smooth cells, morphologic alterations (Fig. 8B). However, a more detailed producing hyperplasia of the tunica media.57 To analyze analysis of retinal arterioles by immunofluorescent labeling of whether this newly found component of retinal arteriolar a-SMA showed a statistically significant increase of the tunica Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 481 FIGURE 7. Similarities and differences with pericytes. Confocal microscopy of whole mount retinas immunolabeled against collagen IV (red) and pericyte specific markers (green) evidenced weak expression of NG2 (A) and PDGF-Rb (B). By contrast, desmin was not expressed in VICs (C). Arrowhead point to arteriolar annuli. Scale bars: (A) 10.46 lm; (B) 12.39 lm; (C) 7.63 lm. media thickness in KAP transgenic retinal arterioles compared can be markedly different.60 In fact, it has been reported that with control littermates (1483 6 0.088 lm in KAP-Tg mice, 12 photoreceptor degeneration may be caused by hypertensive arterioles measured in three different animals versus 1075 6 choroidopathy61 and that hypertensive changes in choroidal 0.061 lm in WT mice, 12 arterioles measured in three different vascularization, including arteriolar narrowing,62 occur later animals; P ¼ 0.0003; Figs. 8C, 8D). Focal areas of arteriolar than changes in retinal vascularization. In this regard, our narrowing were also observed in the retina of KAP transgenic results would suggest that KAP-Tg mice develop a strong mice (Fig. 8E). hypertensive retinopathy with a mild hypertensive choroidop- Degeneration of photoreceptors has been observed in rats athy. with sustained systemic hypertension.58,59 However, photore- Altogether, these results suggest that KAP transgenic male ceptors were not apparently affected histologically in KAP-Tg mice develop vascular lesions compatible with hypertensive mice (Fig. 8B). To analyze in more detail photoreceptor retinopathy and can be used as a model for the study of this morphology, TEM was used. The photoreceptor ultrastructure was maintained in retinas from KAP-Tg mice (Fig. 8F), and only disease. slight mitochondrial alterations were occasionally observed in It has been previously reported that Ano1 expression is the photoreceptor inner segments (Fig. 8G). reduced in the basilar artery during hypertension.63 Therefore, It is well-known that retinal circulation provides blood to we analyzed the expression of Ano1 in VICs in arteriolar annuli the ganglion cell and inner nuclear layers, whereas photore- of KAP transgenic mice. Whole mount retinas doubly ceptors are nourished mainly by the choroidal circulation. As immunostained for Ano1 and collagen IV showed that Ano1 retinal and choroidal vascular systems present great anatomical expression was markedly diminished in arteriolar annuli VICs and physiological differences, their responses to hypertension of KAP transgenic mice compared with age- and sex-matched Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 482 FIGURE 8. KAP-transgenic (KAP-Tg) mice develop vascular lesions compatible with hypertensive retinopathy. (A) Tail-cuff measurements of systolic (SAP) and diastolic (DAP) arterial pressure of 7-month-old KAP-Tg mice and control littermates evidenced that KAP-Tg mice were hypertensive. (B) Paraffin sections stained with hematoxylin and eosin evidenced no gross morphologic alterations in retinas from KAP-Tg mice. (C, D) Immunohistochemistry against a-SMA evidenced a significant increase in the thickness of the tunica media of the retinal arterioles of KAP-Tg mice compared with wild-type (WT) mice. (E) Focal narrowing areas (arrowheads) were observed in retinal arterioles of KAP-Tg mice during confocal analysis of whole mount retinas immunostained against collagen IV (red). (F) TEM showed no evident ultrastructural alterations of photoreceptors in retinas from KAP-Tg mice. (G) A deeper analysis evidenced slight mitochondrial morphologic alterations in the inner segment of KAT-Tg photorreceptors. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, photoreceptor inner segment; OS, photoreceptor outer segment. ***P < 0.001. Scale bars: (B) 34.5 lm; (C) 5.78 lm; (E) 37.76 lm; (F) 4 lm; (G) 0.67 lm. control mice (23.64 6 6.456 arbitrary units, n ¼ 5 versus 64.27 nitric oxide synthase, we studied NADPH diaphorase activity in 6 9.443 arbitrary units, n ¼ 5; P ¼ 0.0075; Fig. 9). retinal arteriolar annuli from KAP transgenic mice. Our results KAP gene overexpression induces hypertension partly by demonstrated that NADPH diaphorase activity was markedly activating the renin-angiotensin system.64 Furthermore, in- reduced in retinal arteriolar annuli, both in VICs and in creased production of angiotensin II, the active agent of the endothelial cells from KAP transgenic male mice compared renin-angiotensin system, decreases nitric oxide activity by with age- and sex-matched control mice (0.5153 3 107 þ nitric oxide synthase uncoupling.65 Therefore, to analyze the 0.1488 3 107 arbitrary units, n ¼ 4 versus 2.872 3 107 þ 0.5902 Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 483 FIGURE 9. Ano1 expression is downregulated in VICs during hypertensive retinopathy. (A) Confocal microscopy analysis showed decreased Ano1 expression in whole mount retinas from KAP-Tg mice (B). (C) Quantification of fluorescence intensity demonstrated that the decrease in Ano1 expression during hypertensive retinopathy was statistically significant. Arrowheads point to arteriolar annuli. *P < 0.05. Scale bars: (A) 17.16 lm; (B) 14.85 lm. 3 107, n ¼ 4; P ¼ 0.0002; Fig. 10). Thus, our results suggest that where the effect of circulating hormones is almost negligi- nitric oxide synthesis might be reduced in retinal arteriolar ble.39,68,69 annuli during hypertensive retinopathy. Given that nitric oxide In this paper, a novel type of vascular cells that could play a is a survival factor for VICs,65,66 these results suggest a possible role in the motility of retinal arteriolar annuli, and therefore, alteration of VICs during hypertensive retinopathy. participate in the retinal blood flow regulation, has been Altogether, these results suggest a loss of function of described morphologically and immunophenotypically. Ac- calcium-activated chloride channels and nitric oxide synthesis cording to TEM analysis, these cells were located within the in VICs during hypertensive retinopathy that could affect the vascular wall, between endothelial and smooth muscle cells. control of the retinal vascular tone at the level of arterial This specific localization and their morphologic features make annuli. them compatible with VICs. Furthermore, their ultrastructure and molecular phenotype suggested that VICs in arteriolar annuli, despite sharing some markers with smooth muscle cells DISCUSSION and pericytes, belong to a new subtype of interstitial Cajal cells. It is now well accepted that blood vessels are far more The presence of interstitial Cajal cells in the blood vessel sophisticated than simple tubes conducting blood. In fact, wall has been under discussion since 1953, when Meyling70 these highly organized and complex structures are able to described the autonomic interstitial cells. Also Dahl and autonomously regulate the blood flow to provide oxygen and Nelson71 reported, in 1964, the presence of cells with irregular nutrients according to tissue needs.67 This autonomous control nuclei and multipolar processes in several arteries of the of blood flow is even more important in the retina, which does central nervous system. Nowadays, interstitial Cajal-like cells not contain sympathetic innervation of blood vessels and have been described in a number of vessels, including portal Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 484 FIGURE 10. NADPH diaphorase activity is decreased in arteriolar annuli of KAP-Tg mice. (A) A great decrease in NADPH diaphorase activity was observed in retinas from KAP-Tg mice compared with wild-type (WT) mice. (B) Quantification of NADPH diaphorase activity by measuring staining intensity evidenced statistical significance. Arrowheads point to arteriolar annuli. ***P < 0.001. Scale bars: 12.7 lm. vein, mesenteric arteries, aorta, carotid artery, middle cerebral Interstitial Cajal cells regulate gastrointestinal motility artery, and pulmonary veins.10–15 through their pacemaker activity,15 and VICs in retinal The nature of interstitial Cajal cells has remained contro- arteriolar annuli show morphologic and molecular character- versial. Cajal believed that interstitial gastrointestinal cells were istics (like gap junctions and Ano1 expression) that have been primitive neurons, whereas other researchers suggested that proposed to play a role for pacemaker activity.77,78 Further- these cells were specialized smooth muscle cells or even more, we have shown that VICs have NADPH diaphorase fibroblast-like cells.72 Developmental studies suggested com- activity and therefore can potentially synthesize nitric oxide, mon precursors for interstitial Cajal and smooth muscle thus suggesting that VICs could play a role in nitrergic regulation of vascular tone. Overall, these findings indicated cells.73,74 Recently, CD44þ vascular multipotent stem cells that VICs could play a role in the motility of arteriolar annuli. able to differentiate into smooth muscle cells and pericytes Mechanical distortion of interstitial Cajal cells increases have been identified within the adult human arterial adventi- their pacemaker activity.79 Due to their location, interstitial tia.75 These multipotent mesenchymal cells show similar Cajal cells can monitor the intestine contractility and transmit molecular phenotype to that of VICs in retinal arteriolar this information to the enteric nervous system.79 The precise annuli, as they express CD44, NG2, and nestin. Furthermore, it location of VICs, between endothelial and smooth muscle cells has been reported that interstitial Cajal cells can revert to in the arteriolar annuli, could also make these cells able to smooth muscle cell type when the Kit signaling pathway is sense variations in the vascular tone. Furthermore, it has been blocked.76 Altogether, these results suggest that interstitial postulated that adhesion molecules, such as CD44, couple Cajal cells, VICs, smooth muscle cells, and pericytes may be extracellular and nuclear matrix scaffolds by means of part of the same vascular cell lineage. cytoskeleton filaments that transduce changes in the basement Downloaded from iovs.arvojournals.org on 07/06/2020 Arterial Retinal Annuli and Hypertension IOVS j February 2019 j Vol. 60 j No. 2 j 485 membrane to the nucleus.80 CD44 is expressed by VICs, 2. Wong TY, McIntosh R. Hypertensive retinopathy signs as risk suggesting that these cells are endowed with a molecular indicators of cardiovascular morbidity and mortality. Br Med mechanism to transform mechanical distortion into biological Bull. 2005;73–74:57–70. responses. 3. Meyer-Rüsenberg B, Pavlidis M, Stupp T, Thanos S. Patholog- The autonomous control of retinal blood flow operates only ical changes in human retinal ganglion cells associated with within a certain range of perfusion pressure; this autoregula- diabetic and hypertensive retinopathies. Graefe’s Arch Clin tion becomes ineffective above physiologic thresholds. Conse- Exp Ophtahlmol. 2006;245:1009–1018. quently, arterial hypertension causes derangement of retinal 4. Kuwabara T, Cogan DG. Studies of retinal vascular patterns blood flow and arteriosclerosis.81 Therefore, we studied the part I. Normal architecture. Arch Ophthalmol. 1960;64:904– effects of hypertension on arteriolar annuli (and, more 911. specifically, on VICs) through the use of a well-established 5. Henkind P, De Oliveira LF. Retinal arteriolar annuli. Invest mouse model of arterial hypertension: the KAP transgenic Ophthalmol Vis Sci. 1968;7:584–591. mice.18,64 6. Pannarale L, Onori P, Ripani M, Gaudio E. Precapillary The fine analysis of arteriolar annuli in KAP transgenic mice patterns and perivascular cells in the retinal microvasculature. demonstrated that VICs are altered during hypertensive A scanning electron microscope study. J Anat. 1996;188:693– retinopathy, because there was a marked decrease in Ano1 703. expression and NADPH diaphorase activity in these cells. 7. Ninomiya H, Inomata T. Microvasculature of the mouse eye: Previous reports concerning the status of Ano1 during scanning electron microscopy of vascular corrosion casts. J hypertension are discordant, with some studies arguing for Exp Anim Sci. 2006;43:149–159. reduced Ano1 expression63 or altered allostery,82 whereas 8. Thuránzsky K. Der Blutkreislauf der Netzhaut: Intravital- other studies reported increased Ano1 activity.35,83 However, mikroskopische und Histologische Studien an der Katzen- hypertension-related downregulation of Ano1 has been always retina. Budapest: Ungarische Akademie Wissenchaften; 1957. observed in the vasculature of the central nervous system, both 9. Friedman E, Smith TR, Kuwabara T. Retinal microcirculation in brain63 and in the retina.82 Thus, differences between in vivo. Inv Ophthalmol. 1964;3:217–226. previous reports may reflect modifications of Ano1 dynamics 10. Povstyan OV, Gordienko DV, Harhun MI, Bolton TB. Identifi- to adapt the vascular response to the specific needs of each cation of interstitial cells of Cajal in the rabbit portal vein. Cell tissue. Furthermore, studies in Ano1 knockout mice have Calcium. 2003;33:223–239. demonstrated that the absence of this protein greatly reduces 11. Pucovský V, Moss RF, Bolton TB. Non-contractile cells with smooth muscle contraction.77 Therefore, alterations in Ano1 thin processes resembling interstitial cells of Cajal found in expression observed in retinas from KAP transgenic mice the wall of guinea-pig mesenteric arteries. J Physiol. 2003; might indicate a loss of function of calcium-dependent chloride 552:119–133. channels that could lead to abnormal vascular motility. 12. Bobrishev YV. Subset of cells immunopositive for neurokinin- As previously discussed, VICs present NADPH diaphorase 1 receptor identified as arterial interstitial cells of Cajal in activity, suggesting that these cells might play a role in nitrergic human large arteries. Cell Tissue Res. 2005;321:45–55. regulation of the vascular tone. Nitric oxide is a potent 13. Morel E, Meynoret D, Thivolet-Bejuy F, Chevalier P. Identifi- vasodilator that has been implicated in the regulation of blood cation and distribution of interstitial Cajal cells in human pressure and regional blood flow, and its expression is altered pulmonary veins. Heart Rhythm. 2008;5:1063–1067. during hypertension.84 Therefore, the NADPH abnormalities observed in KAP transgenic mice might diminish nitric oxide 14. Harhun MI, Szewczyk K, Laux H, et al. 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