diagnostics Article Relationship between Retinal Microvasculature, Cardiovascular Risk and Silent Brain Infarction in Hypertensive Patients Rosa Forés 1,2, * , Josep M. Manresa 1 , Victor M. López-Lifante 1,3 , Antonio Heras 1,2 , Pilar Delgado 4,5 , Xose Vázquez 6 , Susana Ruiz 7 , Maria Teresa Alzamora 1,2 and Pere Toran 1 1 Unitat de Suport a la Recerca Metropolitana Nord, Institut Universitari d’Investigació en Atenció Primària Jordi Gol (IDIAP Jordi Gol), 08303 Mataró, Spain; jmmanresa@idiapjgol.info (J.M.M.); vmlopezli.mn.ics@gencat.cat (V.M.L.-L.); aheras.bnm.ics@gencat.cat (A.H.); maiteal2007@gmail.com (M.T.A.); ptoran.bnm.ics@gencat.cat (P.T.) 2 Riu Nord-Riu Sud Primary Healthcare Centre, Santa Coloma de Gramenet, Gerència d’Àmbit d’Atenció Primària Metropolitana Nord, Institut Català de la Salut, 08921 Barcelona, Spain 3 Palau Healthcare Centre, Palau-Solità Plegamans, Gerència d’Àmbit d’Atenció Primària Metropolitana Nord, Institut Català de la Salut, 08124 Barcelona, Spain 4 Neurovascular Research Lab, Vall D’Hebron Research Institute, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; pilar.delgado@vhir.org 5 Dementia Unit, Neurology Service, Vall D’Hebron University Hospital, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain 6 Hospital Municipal de Badalona, Badalona Serveis Assistencials, Secció d’Oftalmologia, Badalona,  08911 Barcelona, Spain; xvazquez@bsa.cat 7  Germans Trias i Pujol University Hospital, Badalona, 08916 Barcelona, Spain; sruizbilbao@gmail.com * Correspondence: rfores.bnm.ics@gencat.cat; Tel.: +34-934662096 Citation: Forés, R.; Manresa, J.M.; López-Lifante, V.M.; Heras, A.; Abstract: Objective: The aims of this study are to analyze the role of artery-vein ratio AVR assessment Delgado, P.; Vázquez, X.; Ruiz, S.; using VesselMap 2 software (Imedos Systems) and cardiovascular risk evaluation by means of Alzamora, M.T.; Toran, P. REGICOR in the prediction of silent brain infarction (SBI) in middle-age hypertensive patients from Relationship between Retinal the ISSYS study. Material and Methods: A cross-sectional study with 695 patients with hypertension Microvasculature, Cardiovascular Risk and Silent Brain Infarction in aged 50 to 70 years who participated in the project Investigating Silent Strokes in HYpertensives: a Hypertensive Patients. Diagnostics Magnetic Resonance Imaging Study (ISSYS), was conducted in two Primary Care Centres of Barcelona. 2021, 11, 937. https://doi.org/ Participants agreed to a retinography and an MRI to detect silent brain infarction (SBI). The IMEDOS 10.3390/diagnostics11060937 software was used for the semiautomatic caliber measurement of retinal arteries and veins, and the AVR was considered abnormal when <0.66. The REGICOR score was calculated for all patients. Academic Editor: Gino Results: Multivariate logistic regression analysis was used to evaluate the impact of AVR and Seravalle Seravalle REGICOR scores on SBI. The OR (odds ratio) for a high REGICOR score and an abnormal AVR were 3.16 and 4.45, respectively. When analysing the interaction of both factors, the OR of an abnormal Received: 16 April 2021 AVR and moderate REGICOR score was 3.27, whereas with a high REGICOR score it reached 13.07. Accepted: 21 May 2021 Conclusions: The measurement of AVR in patients with hypertension and with a high REGICOR Published: 24 May 2021 score can contribute to the detection of silent brain infarction. Publisher’s Note: MDPI stays neutral Keywords: silent brain infarction; cardiovascular risk; hypertension; retinal microvasculature with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Silent brain infarction (SBI) is a brain injury diagnosed with magnetic resonance Copyright: © 2021 by the authors. imaging (MRI). Initially described by Fisher [1], epidemiological evidence shows the Licensee MDPI, Basel, Switzerland. association between SBI and cognitive dysfunction [2], dementia [3], symptomatic stroke, This article is an open access article and increased overall mortality [4]. A study by Gupta and colleagues [5] suggests that one distributed under the terms and in five adults over 60 with no history of stroke may have SBI, and SBI increases the risk of conditions of the Creative Commons stroke more than twofold. Attribution (CC BY) license (https:// The factors most strongly associated with SBI are age, hypertension (HT), carotid creativecommons.org/licenses/by/ stenosis, chronic kidney disease, and metabolic syndrome [6]. In turn, HT is a cardio- 4.0/). vascular risk factor associated with increased risk of heart disease, encephalopathy, and Diagnostics 2021, 11, 937. https://doi.org/10.3390/diagnostics11060937 https://www.mdpi.com/journal/diagnostics Diagnostics 2021, 11, 937 2 of 7 kidney disease [7]. The WHO estimates a global prevalence of 1.13 billion people with hypertension, one of the main causes of premature death. In 2015, one in four men and one in five women had hypertension [7]. Menéndez et al. observed in 2016 that 42.6% (49.9% men and 37.1% women) of the Spanish population aged 18 years and over was hypertensive [8]. Recent publications address the effect of hypertension on the retinal microvasculature [9,10], with studies linking milder degrees of retinopathy and abnormal caliber of retinal vessels with increased risk of stroke [11–13], heart disease [14], stroke and heart disease [15], and cognitive decline [16,17]. Decreasing the incidence of cardiovascular disease remains a public health priority. Different scales adapted to specific populations have been created to assess cardiovascular risk [18,19]. The REGICOR scale, the most used in our setting, is a validation of the FRAMINGHAM [20] scale adapted to the Mediterranean population. The REGICOR scale [18] includes the following variables: age, sex, smoking, diabetes, total cholesterol, HDL cholesterol, and systolic and diastolic blood pressure. While fundoscopy has been routinely used to screen for diabetic retinopathy in pa- tients with diabetes, patients with hypertension are not regularly screened for hypertensive retinopathy. However, innovative computer software has already been developed to semi- automatically interpret eye fundus lesions and to measure the retinal microvasculature. This program will facilitate the use of fundoscopy to evaluate patients with hyperten- sion [21–24] and to screen for asymptomatic cardiovascular disease [25]. One measurement used is the retinal artery-vein ratio (AVR), which calculates the ratio between the mean arteriolar and venular width measured in the area of interest around the optic disc. The aims of this study are to analyze the role of AVR assessment using VesselMap 2 software (Imedos Systems GmbH, Jena, Germany) and cardiovascular risk evaluation by means of REGICOR in the prediction of SBI in middle-age hypertensive patients from the ISSYS study. 2. Methods The study subjects were participants of Investigating Silent Strokes in Hypertensives: a Magnetic Resonance Imaging Study (ISSYS) [26], where the methodology of the study is described. The ISSYS study was conducted on a randomized sample of patients with hypertension between the ages of 50 and 70 usually managed in their primary care center, to determine the prevalence of SBI. Patients were randomly selected from a sample of 27,000 subjects living in the northern areas of Barcelona after stratifying by age and sex. Participants received a phone call inviting them to a visit where study researchers evaluated eligibility [26]. Inclusion criteria: patients diagnosed with hypertension for at least one year; age between 50 and 70 years; informed consent to perform a retinography. Exclusion criteria: prior history of stroke or dementia; contraindication to performing MRI; suspected white coat hypertension; terminal illness; anticipated difficulties for follow up; physical and functional limitation to obtain and interpret the retinography: media opacity, retinal photocoagulation, myopia magna, functional single eye and eye movement disorders. 2.1. Procedure During the ISSYS project visit, subjects were asked to undergo a retinography ex- amination in one of the two designated Primary Care Centers between March 2011 and July 2012. Each participant underwent a retinography of both eyes centered on the optic disc/macula, at 45º, without mydriasis and under similar light conditions. Papilla-centered images were also taken when possible. A TOPCON TRC-NW65 retinograph was used, which consisted of the following: a Nikon D7000 camera for TRC-NW6, IbaseDigiCaptura, a 19” TFT monitor, computer with Windows 7 operating system, type I, ATE-600, and the VesselMap 2 software (Version 3.10) (Imedos Systems GmbH, Jena, Germany) to visualize During the ISSYS project visit, subjects were asked to undergo a retinography exam- ination in one of the two designated Primary Care Centers between March 2011 and July 2012. Each participant underwent a retinography of both eyes centered on the optic disc/macula, at 45º, without mydriasis and under similar light conditions. Papilla-cen- Diagnostics 2021, 11, 937 tered images were also taken when possible. A TOPCON TRC-NW65 retinograph3 of was 7 used, which consisted of the following: a Nikon D7000 camera for TRC-NW6, IbaseDi- giCaptura, a 19” TFT monitor, computer with Windows 7 operating system, type I, ATE- 600, and the VesselMap 2 software (Version 3.10) (Imedos Systems GmbH, Jena, Germany) and measure and to visualize the retinal measure vasculature the retinalimages (developed vasculature imagesby IMEDOS Systems). (developed by IMEDOS TheSystems). images were stored in jpg format on a purpose-built server. The images were stored in jpg format on a purpose-built server. Two Twoexperienced experiencedophthalmologists ophthalmologistsevaluated evaluatedthe theretinographies retinographiesand andrecorded recordedthe the following lesions: abnormal AVR, abnormal arteriovenous crossings, microaneurysms, following lesions: abnormal AVR, abnormal arteriovenous crossings, microaneurysms, flame flamehemorrhages, hemorrhages,softsoftexudates, exudates,hard hardexudates, exudates,and andpapilledema. papilledema.The Thesubjective subjectiveoph- oph- thalmoscopic AVR measurement was replaced by the semiautomatic caliber measurement thalmoscopic AVR measurement was replaced by the semiautomatic caliber measure- of arteries and veins by the VesselMap software, made by a technician prior to the training ment of arteries and veins by the VesselMap software, made by a technician prior to the (Figure 1). With this system, the vessels are manually located and the AVR calculation is training (Figure 1). With this system, the vessels are manually located and the AVR calcu- automatically provided. Values <0.66 are considered abnormal [27,28]. lation is automatically provided. Values <0.66 are considered abnormal [27,28]. Figure 1. Semi-automatic measurement of artery and vein diameters by the VesselMap software. Figure 1. Semi-automatic measurement of artery and vein diameters by the VesselMap software. https://imedos.com/en/products/sva/ (accessed on 16 April 2021). https://imedos.com/en/products/sva/ (accessed on 16 April 2021). 2.2.Statistical 2.2. StatisticalAnalysis Analysis Qualitativevariables Qualitative variablesare aredescribed describedwith withabsolute absolutefrequencies frequenciesandandpercentages, percentages,while while meanand mean and standard standard deviation deviation were wereused usedtotodescribe describecontinuous continuous variables. Pearson’s variables. Chi- Pearson’s square test was used for comparison of qualitative variables, and Student’s Chi-square test was used for comparison of qualitative variables, and Student’s t-test t-test for com- parison for of continuous comparison variables. of continuous We conducted variables. a multivariate We conducted logistic logistic a multivariate regression analysis regression where SBI analysis wasSBI where the was dependent variablevariable the dependent while abnormal AVR, REGICOR while abnormal Risk andRisk AVR, REGICOR the and com- bination the of bothofwere combination boththe independent were factors.factors. the independent The level Theoflevel significance was 5%. of significance wasFor all 5%. For all analyses, analyses, we usedwethe used the statistical SPSS SPSS statistical packagepackage for Windows, for Windows, version version 26.0. 26.0. 3.3.Results Results Figure Figure22shows shows that that from from the initial 1035 the initial 1035 patients, patients, 39 39were wereexcluded excludeddueduetotoatrial atrialfi- fibrillation, 58 because the MRI could not be performed, and 243 because we could brillation, 58 because the MRI could not be performed, and 243 because we could not lo-not locate them, their refusal cate them, to undergo their refusal the retinography, to undergo and other the retinography, exclusion and other criteria. exclusion Finally, criteria. the Finally, retinography was performed on 695 participants and SBI was diagnosed in 76 (prevalence the retinography was performed on 695 participants and SBI was diagnosed in 76 (preva- oflence 10.15%). of 10.15%). Diagnostics 2021, 11, 937 4 of 7 Diagnostics 2021, 11, x FOR PEER REVIEW 4 of 8 Figure 2. Flow Chart. * AF: Atrial Fibrillation; † MRI: Magnetic Resonance Imaging; ‡ MI: Myocardial Infarction; § SBI:Chart. Figure 2. Flow Silent*Brain Infarction. AF: Atrial Fibrillation; † MRI: Magnetic Resonance Imaging; ‡ MI: Myocar- dial Infarction; § SBI: Silent Brain Infarction. Table 1 describes participants’ characteristics based on the absence or presence of SBI. Of these characteristics, Table we should underscore 1 describes participants’ the following characteristics four: based on the REGICOR absence > 10 in of or presence 20.7% SBI. and 34.3% (p = 0.014); AVR < 0.66 in 12.7% and 38.8% (p < 0.001) in patients without Of these characteristics, we should underscore the following four: REGICOR > 10 in 20.7% and with SBI, respectively. and 34.3% (p = 0.014); AVR < 0.66 in 12.7% and 38.8% (p < 0.001) in patients without and with SBI, respectively. Table 1. Description of characteristics of the 695 participants based on the presence or absence of SBI. Table 1. Description of characteristics of the 695 participants based on the presence or absence of NO SBI * SBI TOTAL SBI. N = 628 N = 67 N = 695 p NO SBI * SBI TOTAL Age 62.4 (5.6) 64.6 (4.7) 62.7 (5.6) 0.002 Men N = 628 N285= 67 (45.4%) N = 69549 (73.1%)p 334 (48.1%) 0.000 Age Former smoker 62.4 (5.6) 64.694(4.7) (15.0%) 62.7 (5.6)11 (16.4%) 0.002 105 (15.1%) 0.753 Men Dyslipidemia †285 (45.4%) 438 (70.1%) 49 (73.1%) 52 (78.8%) 334 (48.1%) 0.000 490 (70.9%) 0.139 REGICOR 0.014 Former smoker 94 (15.0%) 11 (16.4%) 105 (15.1%) 0.753 Low < 5 170 (27.1%) 10 (14.9%) 180 (25.9%) Dyslipidemia † 438 (70.1%) Moderate 5–10 52 (78.8%) 328 (52.2%) 490 (70.9%) 0.139 34 (50.7%) 362 (52.1%) REGICOR High > 10 130 (20.7%) 0.014 23 (34.3%) 153 (22.0%) Low < 5 Detected Alterations in 170 (27.1%) 10 (14.9%) 180 (25.9%) Moderate 5–10 Retinography 328 (52.2%) 34 (50.7%) 362 (52.1%) AVR ‡ < 0.66 80 (12.7%) 26 (38.8%) 106 (15.3%) 0.000 High > 10 130 (20.7%) Arteriovenous nicking 23 (34.3%) 407 (64.8%) 153 (22.0%) 45 (67.2%) 452 (65.0%) 0.701 Detected Alterations in Retinography Mycroaneurysms 14 (2.2%) 3 (4.5%) 17 (2.4%) 0.258 AVR ‡ < 0.66 80 (12.7%) Flame hemorrhages 26 (38.8%) 0 (0.0%) 106 (15.3%) 0.000 1 (1.5%) 1 (0.1%) — Arteriovenous nicking Soft exudades 407 (64.8%) 6 (1.0%) 452 (65.0%) 45 (67.2%) 0 (0.0%) 0.701 6 (0.9%) 0.422 Hard exudates 6 (1.0%) 2 (3.0%) 8 (1.2%) 0.39 Mycroaneurysms 14 (2.2%) 3 (4.5%) 17 (2.4%) 0.258 * SBI: Silent Brain I* SBI: Silent Brain Infarction; † Dyslipidemia: 3 + 1 missings; ‡ AVR: Artery-Vein Ratio. Flame hemorrhages 0 (0.0%) 1 (1.5%) 1 (0.1%) --- Soft exudades 6 (1.0%) 0 (0.0%) 6 (0.9%) 0.422 A multivariate logistic regression analysis was performed to calculate the risk of SBI Hard exudates 6 (1.0%) 2 (3.0%) 8 (1.2%) 0.39 with abnormal AVR and the REGICOR risk score (Table 2A). The results show an Odds * SBI: Silent Brain I* SBI: Silent Brain Infarction; † Dyslipidemia: 3 + 1 missings; ‡ AVR: Artery- Ratio of 3.16 with a high REGICOR score and of 4.45 with an abnormal AVR. In the model Vein Ratio. with the interaction of both factors, an abnormal AVR plus a moderate REGICOR risk resulted in an Odds Ratio of 3.27, while with an abnormal AVR plus a high REGICOR risk A multivariate logistic regression analysis was performed to calculate the risk of SBI the Odds Ratio increased to 13.07 (Table 2B). with abnormal AVR and the REGICOR risk score (Table 2A). The results show an Odds Ratio of 3.16 with a high REGICOR score and of 4.45 with an abnormal AVR. In the model with the interaction of both factors, an abnormal AVR plus a moderate REGICOR risk Diagnostics 2021, 11, 937 5 of 7 Table 2. Model obtained from the multivariate logistic regression analysis for the risk of presenting silent cerebral infarct. A-Adjusted Model β Coefficient OR (CI 95%) a p Constant −3.252 REGICOR Low < 5 Reference Moderate 5–10 0.652 1.92 (0.91–4.04) 0.086 High > 10 1.150 3.16 (1.3–7.00) 0.005 RAV < 0.66 b,c 1.493 4.45 (2.56–7.73) <0.001 B-Interaction Model β Coefficient OR (CI 95%) a p Constant −2.570 AVR > 0.66 + Low REGICOR Reference AVR < 0.66 + Moderate REGICOR 1.184 3.27 (1.53–6.97) 0.002 AVR <0.66 + High REGICOR 2.570 13.07 (5.71–29.90) <0.001 CI 95%: Confidence Interval 95%; a Dependent Variable: Silent brain infarction (diagnosed by MRI); b Abnormal AVR when <0.66; c AVR obtained with the Vesselmaps2 software version 3.10 (Imedos). 4. Discussion We believe that the results of this study can be extrapolated to the population between 50 and 70 years of age with hypertension in our setting, because the ISSYS study is a randomized, population-based study and the baseline characteristics of participants who underwent and did not undergo retinography did not significantly differ. [29] SBI prevalence in our study was over 10%, a value in the lowest range amongst population-based epidemiological studies [6], and lower than the prevalence described in cohorts of patients with hypertension. These differences could be attributed to the younger average age and better control of hypertension in the current cohort [29]. Our findings corroborate results observed in other population-based studies, [11] which suggest that hypertensive retinopathy is an independent cardiovascular risk factor. Yatsuda et al. [12] observed that the narrowing of the central artery and widened central retinal vein caliber, focal arteriolar narrowing, and arteriovenous crossings were predictors of lacunar stroke in a cohort of adults followed for an average of 11 years. In a meta-analysis by Dumitrascu et al., [30] venular dilation was associated with the appearance of lacunar stroke. Our study shows that the semi-automatic measurement of AVR < 0.66 correlates better with SBI than any other signs of hypertensive retinopathy, possibly thanks to the removal of the observer’s subjectivity [31]. Ikram et al. [32] describe the association between widened retinal venules and the progression of microvascular brain lesions in a general population over 55 years of age. However, this association fades when retinal circulation is measured with the AVR. Sub- sequent studies that use automated vessel caliber measurement indicate that even in adolescents, hypertension has a greater impact on arterioles than on venules [33]. Most studies agree that due to as yet poorly defined underlying mechanisms, abnormalities in arterioles and venules are associated with different cardiometabolic risk factors: narrower arterioles with higher blood pressure; wider venules with atherosclerosis, inflammation, higher cholesterol levels, and higher body mass index [34]. Other factors to consider are gender, since arteriolar and venular diameters are smaller in women than men, and aging, which decreases the density of retinal vessels, reduces the thickness of the inner retinal layer and the speed of retinal blood flow, particularly in the venules [28]. In this study we could not evaluate the effect of the different retinopathy lesions such as crossings and microaneurysms, which were detected in low numbers. Furthermore, the semiautomatic reading data produced by the VesselMap software does not discriminate between the diameters of the arteries and small veins to elucidate the most significant components in the variation of the AVR. However, with the addition of the REGICOR score to the AVR, we incorporated factors known to affect the diameter of arterioles and venules such as age, sex, blood pressure, and cholesterol. Diagnostics 2021, 11, 937 6 of 7 The findings of this study suggest that AVR measurements in patients with hyper- tension are useful to screen people at high risk of SBI, particularly in patients with high REGICOR risk. To this end, the automation of retinal vasculature measurements during retinography remains crucial. In conclusion, we recommend retinography screening to measure the AVR in all patients with hypertension and a REGICOR score >10 and to evaluate the possibility of carrying out a MRI for the detection of SBI. The diagnosis of SBI would streamline treatment and follow-up, particularly the prevention of symptomatic stroke in this high-risk group of patients. Author Contributions: Conceptualization: R.F., J.M.M., P.T.; Data curation: J.M.M.; Formal Statistical analysis: J.M.M.; Investigation: R.F., A.H., P.D., X.V., S.R., M.T.A., V.M.L.-L.; Methodology: J.M.M., P.T.; Supervision: R.F., J.M.M., P.T.; Validation: R.F., J.M.M., P.T., A.H., V.M.L.-L., M.T.A., X.V., S.R.; Original draft: R.F., J.M.M., V.M.L.-L.; Writing-review and editing: R.F., J.M.M., V.M.L.-L., A.H., P.D., X.V., S.R., M.T.A., P.T. All authors have read and agreed to the final version of the manuscript. Funding: This project was co-funded by the Basque Government and the European Regional Devel- opment Fund. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki. Ethical review and approval were included in the YSSYS study protocol. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Fisher, C.M. Lacunes: Small, deep cerebral infarcts. Neurology 1998, 50, 841. [CrossRef] [PubMed] 2. Longstreth, W.T., Jr.; Dulberg, C.; Manolio, T.A.; Lewis, M.R.; Beauchamp, N.J., Jr.; O’Leary, D.; Carr, J.; Furberg, C.D. Incidence, manifestations, and predictors of brain infarcts defined by serial cranial magnetic resonance imaging in the elderly. Cardiovascular Health Study Stroke 2002, 33, 2376–2382. [CrossRef] [PubMed] 3. 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