Abstract
Objectives: Vitamin D insufficiency has been associated with faster progression of atherosclerosis and increased cardiovascular disease risk, but limited data are available in HIV-infected people. So, we examined potential correlation between vitamin D status and atherosclerosis in people living with HIV.
Methods: A cross-sectional study was performed including adult HIV-infected patients on stable antiretroviral therapy, aged 40–60 years, and with a recent carotid ultrasonography. Subclinical atherosclerosis was defined as a carotid intima-media thickness (IMT) ≥0.9 mm at any site. Patients with diabetes mellitus or atherosclerotic cardiovascular disease were excluded.
Results: On the whole, 188 patients were enrolled: 86.2% were men and the mean age was 49.1 years. The mean CD4 T lymphocyte count was 567 cells/mm3, 176 (93.6%) had plasma HIV RNA <20 copies/mL, 51.1% were smoker, 29.2% had hypertension, 27.7% metabolic syndrome, and 44.7% LDL cholesterol >150 mg/dL. The mean serum concentration of vitamin D was 35.2 ng/mL, and 84 (44.6%) patients had a vitamin D insufficiency (<30 ng/mL). Subclinical atherosclerosis was reported in 105 (55.8%) and the mean vitamin D concentration was significantly lower among patients with subclinical atherosclerosis than among those without (18.2 vs 41.3 ng/mL, p < 0.001). Moreover, the multivariate linear regression analysis adjusted by confounding factors showed an independent association between subclinical atherosclerosis and vitamin D insufficiency, age >50 years, smoking, hypertension, metabolic syndrome, higher BMI, higher LDL cholesterol, longer duration of HIV infection, lower nadir CD4 cell count, and longer exposure to boosted protease inhibitors.
Conclusion: In our study, vitamin D insufficiency is significantly associated with subclinical atherosclerosis, so its role in HIV-associated cardiovascular disease should be further evaluated as a possible target for intervention.
Introduction
Vitamin D insufficiency is very common both in the general population and in HIV-infected persons, and the overall estimated prevalence of hypovitaminosis D in people living with HIV ranges from 70 to 90%.Citation1–3
The active metabolite of vitamin D is a steroid hormone involved in calcium-bone homeostasis and copious metabolic processes, and plays a critical role in the normal functioning of immune and cardiovascular systems.Citation4,Citation5 Considering the numerous metabolic, anti-inflammatory and anti-neoplastic effects of the vitamin D, a possible impact of its deficiency on the HIV-related comorbidities has been suggested, so the knowledge of risk factors associated with the hypovitaminosis D is useful to prevent and properly manage this metabolic abnormality. Moreover, the EuroSIDA cohort study has shown a significant association between vitamin D deficiency and faster progression to acquired immune deficiency syndrome (AIDS) with increased mortality.Citation1
It has been suggested that vitamin D could protect against endothelial dysfunction, atherosclerosis, and vascular calcifications, and some cohort studies have demonstrated a significant correlation between vitamin D deficiency and higher risk of premature atherosclerosis in the general population, particularly in patients with type 2 diabetes mellitus.Citation6–8 However, to date clinical studies have yielded conflicting results about the association of vitamin D insufficiency to atherosclerotic disease in both HIV-uninfected and HIV-infected people.
A retrospective, cross-sectional study was performed to investigate the potential correlation between subclinical atherosclerosis and vitamin D insufficiency or other risk factors in adult HIV-infected patients on stable combination antiretroviral therapy (cART).
Methods
We performed a retrospective, cross-sectional study of HIV-1-infected patients followed at our Clinic of Infectious Diseases, aged from 40 to 60 years, receiving a stable cART, and with at least one vitamin D measurement and one extracranial carotid ultrasonography performed between April 1 and September 31, 2017, or between April 1 and September 31, 2018.
Exclusion criteria were age <40 and >60 years, previous diagnosis of diabetes mellitus or atherosclerotic cardiovascular disease (coronary artery disease, myocardial infarction, or cerebrovascular disease), change in current cART in the last 6 months, active opportunistic diseases or severe infectious diseases, acute or chronic inflammatory diseases, acute or chronic renal failure, acute hepatitis, liver cirrhosis, alcoholism, current intravenous drug dependence, pregnancy, or an underlying treatment with vitamin D. Current alcohol use and intravenous drug dependence were defined as a daily alcohol consumption >30 g and ≥1 intravenous drug use within 6 months before the enrollment.
The ultrasound investigation of the extracranial carotid arteries (common, internal and external arteries) was performed in our Hospital by the same physician using a Philips HDI 5000 power color-Doppler with 7.5-MHz probes (Koninklijke Philips Electronics, Eindhoven, the Netherlands). In each ecographic evaluation, the patients were placed in a supine position after at least 10 min of acclimatization in a comfortable room. The common carotid, the bifurcation and at least the first two cm of the internal carotid arteries were evaluated in the short and long axis during the tele-diastolic phase. During the investigation, the head of the patient was hyper-extended and extra-rotated from the opposite side. The morphological investigation of the carotid lesions was performed using both ultrasonography and the ultrasound power color-Doppler to better characterize the profile of the lesion and the intima-media thickness (IMT). Subclinical atherosclerosis was defined as an IMT ≥0.9 mm at any site, and presence of carotid plaque was defined as an IMT ≥1.2 mm at any site, in conformity with the guidelines of the European Society of Cardiology.Citation9
The 25-hydroxyvitamin D (vitamin D) concentration was measured using an Automated Chemiluminescence Immunoassay (CLIA) from the IDS-ISYS System (IDS Immunodiagnostic Systems Holdings, Tine and Wear, United Kingdom), and the inferior detection limit was 2.6 ng/mL. The vitamin D insufficiency was defined as a serum concentration <30 ng/mL, and vitamin D deficiency as a serum concentration <20 ng/mL.Citation10 Patients were enrolled into the study if they had at least two vitamin D measurements within the enrolment period and only if they had all the measurements below 30 ng/mL or all the measurements above or equal to 30 ng/mL. For these subjects only the last measurement was considered.
For each patient the following demographic, clinical, and laboratory data were collected: sex, age, race, mode of acquisition of HIV infection, stage of HIV disease, duration of HIV infection, current cART and antiretroviral class exposure, physical examination, smoking habits, body mass index (BMI), waist circumference, arterial pressure, medical history and current clinical manifestations, current and past medications, spot urinalysis and blood tests. Blood tests included triglycerides, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, glucose, complete liver and kidney function tests, CD4+ and CD8+ T lymphocyte count, HIV RNA, parathyroid hormone (PTH), calcium and phosphorus. All patients were fasting for at least 8 h prior to the blood tests.
All the plasma samples were analyzed for HIV-RNA level using the automated COBAS AmpliPrep Instrument for specimen processing and the COBAS TaqMan Analyzer for amplification and detection (Roche CobasAmpliPrep/Cobas TaqMan HIV-1 tests version 2.0; Roche Diagnostics, Mannheim, Germany) and the limit of quantification was <20 copies/mL.
Arterial hypertension was defined as an arterial pressure ≥140/90 mmHg on at least two prior visits, or by a physician diagnosis, or by current use of antihypertensive drugs. Metabolic syndrome was diagnosed when at least 3 of the following 5 criteria were present: waist circumference >102 cm for men and >88 cm for women; fasting triglycerides ≥150 mg/dL; HDL cholesterol <40 mg/dL for men and <50 mg/dL for women; fasting glucose ≥110 mg/dL; blood pressure ≥130/85 mmHg.Citation11 The estimated glomerular filtration rate (eGFR) was calculated using the 2009 Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation.Citation12
Variables were described by using proportions for categorical variables, mean and standard deviation (SD) for continuous variables with normal distribution, while comparisons between groups were performed using a Student t test or a Wilcoxon test for normally and non-normally distributed continuous variables, respectively. The Gaussian distribution of continuous variables was previously assessed by the Anderson–Darling normality test. Group comparisons were made using the Fisher exact test for categorical variables. Univariate and multivariate analyses were performed to assess variables independently associated with subclinical atherosclerosis. All variables with a p-value <0.10 on univariate analysis were included in the maximum model and underwent stepwise selection during multivariate analyses. A p-value <0.05 was considered statistically significant. Statistical analysis was performed by XLSTAT 2015.1 Microsoft Excel Statistical Software (Microsoft, Redmond, WA, USA).
The study was approved by the Ethics Committee of the S.Orsola-Malpighi Hospital in Bologna and all the enrolled patients provided a signed written consent before participating in the study.
Results
From 1 April 2017 to 31 September 2017, and from 1 April 2018 to 31 September 2018, 188 patients were enrolled into the study, and their characteristics are summarized in . The mean age was 49.1 years, 86.2% were male, 92.5% were Caucasian, 21.8% were current or previous injecting drug users, 46.3% were homosexuals or bisexuals, 51.1% were smokers. Hypertension was present in 29.2% of patients, metabolic syndrome in 27.7%; 10.1% had a concomitant chronic hepatitis C and 5.3% had a chronic hepatitis B. The mean BMI was 25.9 kg/m2 and 67 (35.6%) subjects had a BMI >28 kg/m2; the mean waist circumference was 91.4 cm and 43 men (26.5%) had a waist circumference >102 cm.
Table 1. Patient characteristics according to presence of subclinical atherosclerosis.
The mean vitamin D concentration was 35.2 ng/mL, 84 (44.6%) patients had a vitamin D insufficiency, and 29 (15.4%) a vitamin D deficiency. With regard to metabolic parameters, 91 (48.4%) patients had total cholesterol >200 mg/dL, 84 (44.7%) had LDL cholesterol >150 mg/dL, and 96 (51.1%) had triglycerides >200 mg/dL.
The mean duration of HIV infection was 13.4 years, and 8.5% had a previous diagnosis of AIDS. The plasma HIV viral load was below 20 copies/mL in 176 (93.6%) subjects, the mean current and nadir CD4 lymphocyte counts were 567 and 238 cells/mm3, respectively.
Current cART and cumulative exposure to antiretroviral drugs are listed in . The most common antiretroviral combinations included tenofovir disoproxil fumarate (TDF)/emtricitabine (in 39.9% of patients), abacavir/lamivudine (33.5%), rilpivirine (20.7%), raltegravir (19.1%), dolutegravir (14.9%), and ritonavir- or cobicistat-boosted darunavir (13.8%). The mean cumulative exposure to antiretroviral classes was 6.6 years for non-nucleoside reverse transcriptase inhibitors (NNRTIs), 6.1 years for protease inhibitors (PIs), and 3.5 years for strand transfer integrase inhibitors (INSTIs).
Table 2. Current antiretroviral therapies and past exposure to antiretroviral drugs of the study cohort.
Subclinical atherosclerosis was diagnosed in 105 patients (corresponding to 55.8% of the study cohort), and carotid plaques were detected in 53 subjects (28.2%). The mean values of IMT in common carotid arteries, internal carotid arteries, and carotid bifurcations of the enrolled patients are summarized in .
Table 3. Mean values of carotid intima-media thickness of the 188 enrolled patients.
The factors significantly (p < 0.10) associated with subclinical atherosclerosis in the univariate models are shown in . Patients with subclinical atherosclerosis compared with those without subclinical atherosclerosis were more likely to have vitamin D insufficiency and deficiency, age >50 years, smoking habitus, hypertension, BMI >25 kg/m2, waist circumference >90 cm, metabolic syndrome, duration of HIV infection >10 years, current CD4 T lymphocyte count >500 cells/mm3, nadir CD4 T lymphocyte count <200 cells/mm3, total cholesterol >200 mg/dL, LDL cholesterol >150 mg/dL, triglycerides >150 mg/dL, and a cumulative exposure to PIs >5 years. A cumulative exposure to NNRTIs >5 years was associated with a reduced risk of atherosclerosis.
Table 4. Analysis by univariate and multivariate logistic regression of factors associated with subclinical atherosclerosis in the study cohort.
A multivariate logistic regression model was built including all variables showing a statistically significant association with subclinical atherosclerosis by the univariate analysis (p < 0.10). Vitamin D insufficiency and deficiency, age >50 years, smoking, hypertension, BMI >25 kg/m2, metabolic syndrome, duration of HIV infection >10 years, nadir CD4 T lymphocyte count <200 cells/mm3, LDL cholesterol >150 mg/dL, triglycerides >150 mg/dL and a cumulative exposure to PIs >5 years remained significantly associated with an increased risk of subclinical atherosclerosis ().
Discussion
Besides the well-known effects of vitamin D on bone metabolism, numerous studies have also demonstrated an association between vitamin D deficiency and nonskeletal chronic diseases, especially cardiovascular diseases and cancers. Indeed the valuable physiological functions of vitamin D include many anti-inflammatory properties, several modulatory effects on peripheral cellular immunity, and also a significant cardiovascular protective action. Vitamin D seems to slow down the atherosclerosis progression and preserve cardiovascular health by its anti-inflammatory effects, suppression of proatherogenic T lymphocytes, inhibition of vascular smooth muscle cells proliferation, protection against advanced glycation products, and maintenance of normal endothelial function.Citation13–15
Some clinical studies in the general population have detected a correlation between a poor vitamin D status and an increased risk of accelerated atherosclerosis and cardiovascular disease mortality. Increasing evidence documented higher carotid intima-media thickness (IMT) and prevalence of carotid plaques (which are reliable surrogate markers of subclinical atherosclerosis and predictors of cardiovascular events) in patients with vitamin D deficiency. Moreover, low vitamin D serum levels are frequently associated with traditional cardiovascular risk factors, such as diabetes mellitus, hypertension, obesity, sedentary lifestyle, older age, hypercholesterolemia, and smoking, and no studies have demonstrated a causative link between hypovitaminosis D and cardiovascular diseases. However, largest studies and trials investigating the effect on vitamin D supplementation on cardiovascular risk factors and atherosclerosis progression have led to inconclusive results.Citation16,Citation17 In the VITAL randomized, placebo-controlled trial the effect of a supplementation with vitamin D (2000 UI daily) and omega-3 fatty acids (1 g daily) for the prevention of cancer and cardiovascular disease was evaluated in 25,871 adult patients. In this trial, the vitamin D supplementation did not result in a lower incidence of cardiovascular events and cancer than placebo.Citation18 In the ViDA randomized, placebo-controlled study 5110 adult patients were treated with vitamin D (100,000 UI monthly) or placebo for a median follow-up of 3.3 years. In this study too, there was no effect of vitamin D supplementation on the main study outcomes, including cardiovascular diseases.Citation19
A systematic meta-analysis including 11 studies and 16434 patients showed that the serum vitamin D concentration was negatively associated with carotid atherosclerosis. Even though a great heterogeneity among the individual studies, the pooled analysis indicated that a high serum concentration of vitamin D was a protective factor against increased carotid IMT and atherosclerotic plaques.Citation20 Another meta-analysis evaluated 21 studies (8569 patients) with data on carotid IMT and 6 studies (4772 patients) investigating the prevalence of carotid plaques. In this review too patients with vitamin D deficiency showed a significantly higher carotid IMT and an increased prevalence of carotid plaques compared to those with normal vitamin D concentration. A significant difference in carotid IMT was also confirmed when comparing patients with vitamin D insufficiency with controls.Citation21
A possible correlation between low serum levels of vitamin D and higher carotid IMT has been investigated also among HIV-infected persons, but to date clinical data are still limited.
The first study reporting an association between vitamin D status and atherosclerotic disease measured by carotid IMT in HIV-infected people was that of Choi AI and Colleagues.Citation22 This cross-sectional study evaluated 139 HIV-positive patients with a mean age of 45 years, 84% men, 76% on antiretroviral therapy (with HIV RNA <1000 copies/mL in 75% of cases), and with a median CD4 T lymphocyte count of 336 cells/mm3. The prevalence of vitamin D insufficiency was 52% and in multivariate linear regression models vitamin D insufficiency was associated with higher mean carotid IMT. This graded relationship between vitamin D concentration and carotid IMT was also confirmed after adjustment for traditional cardiovascular risk factors and HIV-related factors.Citation22
A case-control study assessed the relationship between vitamin D status and markers of immune reconstitution and cardiovascular disease in 149 HIV-positive individuals and 34 HIV-negative controls. In multivariable linear regression analysis, the serum concentration of vitamin D was positively associated with CD4 T cell restoration after the beginning of cART, and odds of having higher carotid IMT was significantly increased in patients with lower vitamin D levels.Citation23 In a prospective cohort study of 169 HIV-infected African-American cocaine users with low cardiovascular risk at baseline, vitamin D deficiency was independently associated with development of subclinical coronary artery disease on cardiac computed tomography.Citation24
The relationship between vitamin D status and progression of carotid atherosclerosis was investigated in the Canadian HIV Vascular Study, involving 128 HIV-positive persons with a mean age of 46.5 years (89% men). In linear regression analyses, baseline vitamin D concentration was inversely associated with carotid IMT increase in both univariate and multivariate models, showing that patients with vitamin D deficiency had a faster progression of carotid atherosclerosis.Citation25
However, clinical data are not concordant. The Hawaii Aging HIV-Cardiovascular Cohort Study enrolled 100 HIV-positive adults with a median age of 52 years (86% males), a median CD4 T lymphocyte count of 510 cells/mm3, and on stable cART, to investigate the correlation between vitamin D and three markers of arterial dysfunction. In this study vitamin D concentration was found to be associated only with brachial artery flow-mediated dilation, but not with carotid IMT and with coronary artery calcium scores.Citation26
A cross-sectional study evaluated 89 HIV-infected men with a mean age of 42 years, 75% on cART, and with a median CD4 T lymphocyte count of 467 cells/mm3. Vitamin D insufficiency was detected in 80.9% of patients and was significantly associated with a higher carotid IMT in the univariate analysis, but the multivariate linear regression model did not confirm an independent association between carotid IMT and vitamin D insufficiency.Citation27
Markers of inflammation and immune activation have been associated with lower vitamin D levels among HIV-positive people, suggesting a possible biological explanation for the increased cardiovascular risk reported in subjects with vitamin D insufficiency. HIV-infected patients with low vitamin D concentration, when compared to those with normal vitamin D status, had significantly increased levels of interleukin-6, tumor necrosis factor-α, D-dimer and markers of monocyte activation.Citation28–30 Moreover, data in the general population show that vitamin D receptor is expressed within human carotid plaques and correlates with pro-inflammatory M1 macrophages. Therefore, a low intraplaque expression of this receptor predicts major adverse cardiovascular events in patients with carotid stenosis probably by favoring an increased number of M1 macrophages within atherosclerotic plaques.Citation31
In our study, we have found a high prevalence of subclinical atherosclerosis (55.8%), in addition to a significant prevalence of vitamin D insufficiency and deficiency (44.6% and 15.4%, respectively). In conformity with other observational studies involving HIV-positive people,Citation22–25 mean vitamin D serum levels were significantly lower among subjects with subclinical atherosclerosis compared to those with normal carotid IMT values, and the multivariate linear regression analysis confirmed and independent association of subclinical atherosclerosis with vitamin D insufficiency, such as with other traditional risk factors (older age, smoking, hypertension, metabolic syndrome, higher BMI, higher LDL cholesterol, and higher triglycerides). With regard to specific HIV-related factors, subclinical atherosclerosis was found to be associated with longer duration of HIV infection, lower nadir CD4 T cell count, and longer exposure to protease inhibitors. So our study seems to confirm an increased risk of subclinical atherosclerosis in HIV-infected subjects with vitamin D insufficiency, such as in those with a longer duration of HIV disease, a worse immunological status, and a greater exposure to specific antiretroviral drugs with well-known proatherogenic properties (protease inhibitors).
Our study has obviously several potential limitations, mostly given the retrospective, cross-sectional design that precludes definitive conclusions regarding the causal relationships. Data were abstracted from the medical records and they can be occasionally incomplete or inaccurate. We have not included a control group without HIV infection. Less than 14% of participants were women and more than 92% were Caucasian, so the results could be influenced by sex and ethnicity of the study population. The evaluation of solar exposure and vitamin D intake from food sources was not performed, but the observation period included only the spring and summer months, so the solar exposure was probably sufficient, and stable for all patients.
In conclusion, our study confirms the association between hypovitaminosis D and accelerated atherosclerotic disease previously showed in other reports involving HIV-positive people. Moreover, it suggests longer duration of HIV infection, poorer immunological status, and exposure to a specific antiretroviral class (protease inhibitors) as potential risk factors for subclinical atherosclerosis. Further enlarged studies evaluating the potential relationship between vitamin D status and cardiovascular disease in HIV-positive people are needed in order to better understand and properly manage this metabolic complication associated with several HIV-related comorbidities.
Authors’ contribution
L.C. coordinated the study, performed research, analyzed and interpreted the data, and wrote the manuscript. V.C. and M.B. contributed to the design of the study, the analysis and the interpretation of the data. G.D.P., B.G., P.M., and I.B. contributed to collection and statistical analysis of the data. M.C.R. and P.V. contributed to the interpretation of the data, and the writing of the manuscript.
Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The local Medical Ethics Committee of the S.Orsola-Malpighi Hospital approved the study (approval no. 197/2017/O/Oss). All patients provided written informed consent in accordance with the Declaration of Helsinki.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Notes on contributors
Leonardo Calza
Leonardo Calza, MD is Associate Professor of Medicine in the Department of Medical and Surgical Sciences, University of Bologna, Italy. He acts as a primary care provider for HIV-infected persons and as a teacher for medical students, residents, and others on the fundamentals of HIV medicine and clinical research. Prof Calza has been involved in the care of HIV-infected patients for more than 20 years, providing care for more than 2500 HIV-infected persons. He is also a member of the expert panel of the Italian Society of Infectious Diseases for the Italian Antiretroviral Therapy Guidelines. Prof Calza received his medical degree from the University of Bologna School of Medicine and completed his residency in Infectious Diseases at the S.Orsola University Hospital in Bologna, Italy.
Marco Borderi
Marco Borderi, MD, is a medical physician acting as a care provider for HIV-infected persons in the Infectious Diseases Unit at the S.Orsola University Hospital in Bologna, Italy. His scientific activities include coordinating and/or principal investigation of several clinical studies, especially on HIV/AIDS. He has served as chairman of numerous scientific national conferences on HIV/AIDS and as an advisor for national and international boards. Dr Borderi received his medical degree from the University of Bologna School of Medicine.
Bianca Granozzi
Bianca Granozzi, MD, is a medical physician and researcher acting as a care provider in the Infectious Diseases Unit at the S.Orsola University Hospital in Bologna, Italy. Her research activities have focused on HIV infection and mycobacterial infections.
Pietro Malosso
Pietro Malosso, MD, is a medical physician and researcher acting as a care provider in the Infectious Diseases Unit at the S.Orsola University Hospital in Bologna, Italy. His research activities have focused on HIV infection and bacterial infections.
Livia Pancaldi
Livia Pancaldi, MD, is a medical physician and researcher acting as a care provider in the Infectious Diseases Unit at the S.Orsola University Hospital in Bologna, Italy. Her research activities have focused on HIV infection and bacterial infections.
Isabella Bon
Isabella Bon, PhD, is a researcher specialist in Virology, in charge of the Retrovirus Laboratory of the Operative Unit of Microbiology, S.Orsola University Hospital in Bologna. Her research activities have focused on viral pathogenetic mechanisms and diagnosis of infection mostly human immunodeficiency virus type 1-2 and other retroviruses (HTLV-I/II). Dr Bon received her medical degree from the University of Bologna School of Medicine.
Maria Carla Re
Maria Carla Re, PhD, is Professor of Microbiology at the University of Bologna School of Medicine in Bologna, Italy. She is specialist in Virology, in charge of the Retrovirus Laboratory of the Operative Unit of Microbiology, S.Orsola University Hospital in Bologna, where she is Director of Operative Unit of Microbiology. Her research activities have focused on viral pathogenetic mechanisms and diagnosis of infection mostly human immunodeficiency virus type 1-2 and other retroviruses (HTLV-I/II).
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