Vitamin D Treatment Attenuates Heart Apoptosis After Coronary Artery Bypass Surgery: A Double-Blind,Randomized,Placebo-Controlled Clinical Trial

Abstract

Background:

Vitamin D plays an important role in immune system and in the regulation of inflammatory cytokines. Coronary artery bypass graft (CABG) with cardiopulmonary bypass (CPB) is associated with an extensive inflammatory response. The aim of this study is to examine the effect of vitamin D treatment on the apoptosis and inflammatory changes developed after CABG.

Methods:

This trial was conducted on 70 patients undergoing CABG with CPB. Patients were randomly administered either in placebo or in the group of orally consuming 150 000 IU vitamin D daily for 3 consecutive days before surgery. The right atrium sample was taken to assess caspases 2, 3, and 7 activity using immunohistochemistry method. The serum level of interleukin-10 (IL-10) and insulin-like growth factor 1 (IGF-1) were compared at intervals.

Results:

The average number of positive cells for caspases 2 and 3 were less in vitamin D group (P = .006 and P < .001, respectively). There was an increase in serum levels of IL-10 after 3 days from vitamin D treatment before surgery (vitamin D group = 4.4 ± 4.9 ng/mL and control group = 1 ± 0.5 ng/mL, P = .001). After operation, IL-10 increased in both groups, higher level in vitamin D group (P < .001). The comparison of serum IGF-1 showed significant difference after 3 days (P = .006) and remained higher in vitamin D group after CPB (P < .001).

Conclusions:

These findings suggest the apoptosis rate after CPB can be reduced by vitamin D. Vitamin D treatment may improve the inflammatory status before and after surgery. Further studies are needed to confirm the antiapoptotic property of vitamin D and clinical implication.

Keywords

vitamin D apoptosis insulin-like growth factor 1 coronary artery disease cardiopulmonary bypass interleukin-10

Introduction

A growing number of evidence indicated that vitamin D in addition to its traditional metabolic function has immunomodulatory capacity by upregulating anti-inflammatory markers and attenuating the inflammation, oxidative stress, and excessive immune responses (with desirable and undesirable effects as well).^1

-7 Vitamin D has a direct impact on heart and can independently modify the cardiovascular risks.^5,8,9 Many studies have shown its link as a protective factor against the cardiovascular disorders through attenuation of oxidative stress, prevention from apoptosis, and improvement in cardiac metabolism.^3

-10 The antiapoptotic role of vitamin D was demonstrated in the experimental models of ischemic–reperfusion injury through reduction in the expression of caspase; however, it has not been appraised in the clinical trials for cardiac disease.^3,11
-13

Both cardiopulmonary bypass (CPB) machine and obligatory heart ischemia/reperfusion through cardioplegic arrest are associated with an extensive inflammatory response and also with ischemic reperfusion injury in cardiac surgical patients.^14
-16 Apoptosis occurs after ischemic reperfusion injury, and the extent of cardiac apoptosis has a direct effect on postoperative cardiac function.^14

-17 Previous studies have reported an association among caspases 2, 3, and 7 and the ischemic reperfusion injury. Oxidative stress, which has been proved as the main activator of caspase 2, is the most important cause of cellular damage in ischemia–reperfusion injury.¹⁸ In preclinical studies, increased infarct size after infarction–reperfusion injury has been associated with the overexpression of caspase 3 and higher risk of cellular death.¹⁹ The activity of caspases 3 and 7 has been found as the main reasons of the proteolytic cleavage of cellular proteins in cardiovascular system.²⁰

The aim of this study is to determine the efficacy of oral administration of vitamin D before coronary artery bypass graft (CABG) on the degree of postoperative heart apoptosis (assessment of heart caspase activity) and the level of serum anti-inflammatory biomarkers.

Methods

Trial Design

This randomized, double-blind, placebo-controlled study was conducted for 70 patients undergoing coronary surgery with CPB. The study was approved by the local Ethical Committee and registered in www.isrctn.com (ISRCTN44896820). The informed written consent was obtained from the patients prior to enrollment.

Enrollment was started in December 2018 and was accomplished in April 2019. The inclusion criteria were as follows: the patients who were referred for elective and isolated CABG using CPB with vitamin D deficiency (defined as 25-hydroxyvitamin D < 20 ng/mL) and normal kidney function (creatinine < 1.5 mg/dL). Vitamin D was measured by an immunoluminometric assay. The exclusion criteria were as follows: recent myocardial infarction, urgent CABG, nonisolated coronary surgery, redo surgery, malignant disease, the presence of acute or chronic inflammatory diseases, history of vitamin D treatment within previous 6 months, or unwillingness to participate.

Intervention

Following the informed consent, eligible study participants were randomly assigned (using a computer-generated random code) in a 1:1 ratio to receive either placebo or a total of 450 000 international units (IU) vitamin D3 (three 50 000 IU of vitamin D3 tablet daily for 3 days) before operation. The placebo group received 3 inactive medication tablets similar to those for other group, daily at the same time point (Figure 1). Except the pharmacists, all the investigators, patients, and the medical team were blinded to the group allocation.

Figure 1.

Study flowchart.

Coronary artery bypass was performed in the culprit lesions for both groups by one surgical team. The standard protocols for general anesthesia, surgical, and CPB management were performed for all patients and have already been described in detail.²¹

Outcome Measures

The primary outcome was the degree of heart apoptosis by measuring the caspases 2, 3, and 7 activity from right atrial specimen with immunohistochemistry staining, the serum level of anti-inflammatory interleukin-10 (IL-10) and insulin-like growth factor 1 (IGF-1), and N-terminal (NT)-pro-brain natriuretic peptide (pro-BNP). The biopsy from right atrial appendage was taken at the end of surgery after removing the venous cannula in a nontraumatic manner and then was kept in formalin and parafinized in less than 24 hours. Blood samples were collected firstly at the baseline (by passing 24 hours from hospital admission; T1), before anesthesia induction (T2), at the end of surgery after protamine reversal (T3), and also at the first postoperative day (T4) in order to measure the serum level of IGF-1, IL-10, and pro-BNP. The blood samples were centrifuged at 2500 rpm for 15 minutes within 1 hour after blood sampling, and the serum was stored at −20°C until assayed.

Enzyme-Linked Immunosorbent Assay

The concentration of IL-10 was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit (Ref: BE53101, IBL International GmbH, Hamburg, Germany). The concentration of the IGF-1 was also measured by a quantitative ELISA kit (Ref: MD58011, IBL International GmbH). The pro-BNP measurement was done using a commercially available 2-site chemiluminescent immunometric assay (IMMULITE 2000 NT-pro-BNP, Siemens Healthcare, Mannheim, Germany). Serum vitamin D was detected using the high-performance liquid chromatography method (Agilent 1100 series HPLC, Germany).

Immunohistochemistry Studies

Immunohistochemical staining was performed on 5-µm thick sections. The slides were incubated at 37°C for 24 hours and then deparaffinized in preheated xylene and rehydrated through descending grades of alcohol, and after that they were washed in distilled water. Heat-induced antigen retrieval was done by microwave oven with citrate buffer (pH 6.0) for anti-caspase 7 and Tris–EDTA (pH = 8) for anticaspases 2 and 3. Endoperoxidase blocking was done by adding hydrogen peroxide onto the sections. Then, the protein block was added for 5 minutes, and slides were washed in Tris-buffered saline (TBS). The primary antibody as anti-caspase 2 antibody, rabbit monoclonal (clone EPR16790, abcam), anti-caspase 3 antibody, rabbit monoclonal (clone EPR16888, abcam), anti-caspase 7 antibody, and mouse monoclonal (clone 7-1-11, abcam) antibody were added and kept for 30 minutes, then they were washed in TBS. Mouse and Rabbit Specific HRP/DAB IHC Detection Micro-polymer Kit (ab80436) were used and incubated for 15 minutes, and then were washed with TBS. 3,3′-Diaminobenzidine (DAB) chromogen was added and kept for 5 minutes. Slides were washed in distilled water, and counter was stained with hematoxylin. Sections containing lymph node tissue were used as positive control. Negative control included primary antibody replaced with phosphate-buffered saline. Immunostained sections were reviewed for cytoplasmic expression of anti-caspase 2, anti-caspase 3, and anti-caspase 7. The number of immunoreactive cells was counted per high-power field (HPF; ×400). To fulfill that, at least 10 HPFs were assessed, and the average of positive cells was recorded.

Sample Size and Statistical Methods

The patients’ number (35 patients per group) was determined based on the previous trials investigating the caspase activity in the CABG setting.^15,22,23 Categorical variables were reported as numbers and percentages, whereas mean ± standard deviation was expressed for continuous variables. Repeated measures of analysis of variance and multiple comparisons were applied using the Bonferroni correction (type I error correction), to evaluate the change of measured inflammatory markers between the groups by over time. The Kolmogorov-Smirnov test was performed for normality. Continuous variables and categorical variables were compared between groups using Student t test (or Mann-Whitney test for those meeting abnormal distribution) and χ², respectively. All the statistical analyses were performed using SPSS version 23 (SPSS, Chicago, Illinois). A P value <.05 was considered as significant.

Results

Patients’ Characteristics

Of the 88 patients screened for inclusions/exclusion criteria, a total of 70 patients were eligible and randomized. The baseline demographic, clinical characteristics, and medication regimens were comparable between the groups (Table 1). The perioperative management, duration of surgery (aortic clamp time/pump time), administration of blood products, and vasoactive drugs were comparable between the groups. There were no operative complication and hospital mortality. The groups have no differences in terms of ventilation time, chest tube drainage, and postoperative atrial fibrillation. There was reported no 30-day in both groups.

Table 1.

Baseline Characteristics and Perioperative Clinical Data.^a

	Vitamin D Group, n = 35	Control Group, n = 35	P Value
Male gender, n (%)	27 (77)	28 (80)	0.29
Age, years	59 ± 6	58 ± 9	0.63
BMI, kg/m²	27 ± 3	27 ± 3	0.95
25(OH)D, ng/mL	12.8 ± 6	12.1 ± 5	0.68
EF, %	47 ± 10	48 ± 9	0.86
ESR, mm/h	13 ± 9	14 ± 8	0.71
LDL cholesterol, mg/dL	87 ± 28	80 ± 27	0.32
Cardiac risk factors
Hyperlipidemia, n (%)	9 (26)	16 (45)	0.21
Diabetes mellitus, n (%)	12 (34)	19 (54)	0.46
Hypertension, n (%)	18 (51)	22 (62)	1
Smokers, n (%)	15 (42)	21 (60)	0.62
Family history, n (%)	9 (25)	8 (22)	0.77
MI, n (%)	19 (54)	15 (42)	0.13
Medications
ACEI/ARB, n (%)	25 (71)	35 (100)	0.22
β-blocker, n (%)	26 (74)	33 (94)	0.69
Statin, n (%)	30 (85)	35 (100)	0.64
Insulin, n (%)	7 (20)	13 (37)	0.48
Aspirin, n (%)	29 (82)	35 (100)	0.44
Operative data
Clamp time, minutes	50 ± 7	53 ± 10	0.19
Pump time, minutes	99 ± 16	103 ± 19	0.53
Graft number	3.5 ± 0.5	3.5 ± 0.5	0.78
PRBC, U	1 ± 1	1 ± 1	0.51
Postoperative data
Ventilation time, hours	6.8 ± 2.3	8.3 ± 4.4	0.12
AKI, n (%)	7 (20)	7 (20)	0.76
Bleeding 6 hours, mL	216 ± 209	228 ± 194	0.85
Bleeding 12 hours, mL	360 ± 245	404 ± 245	0.45
PRBC, U	0.5 ± 0.7	0.8 ± 0.9	0.13
Total PRBC, U	46	59	0.07

Abbreviations: 25(OH)D, 25-hydroxyvitamin D; ACEI, angiotensin-converting enzyme inhibitor; AKI, acute kidney injury; ARB, angiotensin II receptor blocker; BMI, body mass index; EF, ejection fraction; ESR, erythrocyte sedimentation rate; IABP, intra-aortic balloon pump; LDL, low-density lipoprotein; MI, myocardial infarction; PRBC, packed red blood cell.

^a Values are mean ± standard deviation or number of patients (%).

Caspase Score

Cardiac immunostaining for active caspases 2, 3, and 7 are shown in Figure 2. The average intensity score of the immunohistochemistry reaction for caspase 7 was not statistically different between the groups (P = .07; 95% confidence interval [CI] for mean change, −1.07 to 0.05). However, the average number of positive cells with strong staining of caspases 2 and 3 were significantly lower in vitamin D group compared to the control group (P = .006; 95% CI for mean change, −1.20 to −0.20 and P < .001; 95% CI for mean change, −1.20 to −0.38, respectively).

Figure 2.

Immunohistochemistry staining for the activity of caspase 2 (A), caspase 3 (B), and caspase 7 (C) in right atrial biopsy specimens after cardiopulmonary bypass in control group (section 1) and vitamin D group (section 2). The positive control (section 3) and negative control (section 4) were taken from human lymph node tissue. Compared to the control group, the number of positive cardiomyocytes for caspases 2 and 3 are less significant in the vitamin D group. Positivity (arrows) for active caspase is shown by cytoplasmic staining (original magnification ×400).

Measurement of IL-10

There was a significant change in serum level of IL-10 over time (P < .001; Figure 3). The baseline serum level of IL-10 was similar between the groups (P = .3; 95% CI for mean change, −0.06 to 0.19). There was a significant increase in IL-10 serum level by passing 3 days from the treatment with vitamin D before surgery (vitamin D group = 4.4 ± 4.9 ng/mL and control group = 1 ± 0.5 ng/mL, P = .001; 95% CI for mean change, 1.59-5.22). The IL-10 serum level reached to a peak level just after operation following an intense increase in both groups, which was higher in vitamin D group (P < .001; 95% CI for mean change, 52.43-145.98). Although in the first day after operation, its level dramatically decreased, the mean level remained higher in vitamin D group (P = .03; 95% CI for mean change, 0.36-6.80).

Figure 3.

The serum level of biomarkers over time. (A) IL-10: Interleukin-10, (B) IGF-I: Insulin-like growth factor 1, (C) pro-BNP: N-terminal pro-brain natriuretic peptide. Vitamin D group, n = 35; control group, n = 35. There were the significant time effects for IL-10 and IGF-I. Time points: (T1) before intervention, (T2) before anesthesia induction, (T3) at the end of the operation, and (T4) at the first day after operation. Data are expressed as mean ± standard deviation. Solid line: Vitamin D group, dashed line: Control group.

Measurement of IGF-1

Repeated-measure test showed that the serum level of IGF-1 had a significant difference between the groups (P = .002; Figure 3). The baseline level of IGF-1was comparable between the 2 groups (P = .1; 95% CI for mean change, −5.21 to 24.84); however, the measurement after 3 days (vitamin D administration) exhibited a significant difference (P = .006; 95% CI for mean change, 10.71-58.91). Cardiopulmonary bypass induced an increase level in both groups, with persistency in difference between the 2 groups (P < .001; 95% CI for mean change, 32.78-86.94). The day after operation, the IGF-1 decreased; nevertheless, it stayed higher in vitamin D group (P = .003; 95% CI for mean change, 15.45-62.97).

Measurement of Pro-BNP

There was no significant change in serum levels of pro-BNP over time (P = .5; Figure 3). The vitamin D supplementation had no significant effect on the level of pro-BNP before operation. The pro-BNP serum level reached a peak, by passing 1 day from operation in both groups with no significant difference.

Discussion

The result of the present study demonstrated that oral high-dose vitamin D treatment could decrease the rate of heart apoptosis that was induced during coronary bypass surgery with CPB and would also increase the serum level of IL-10 and IGF-1, as anti-inflammatory biomarkers in the perioperative period.

The cardiac surgery and CPB with aortic clamp and cardioplegic arrest can induce the activation of the inflammatory cascade and is associated with ischemic reperfusion injury, release of reactive oxygen species (ROS), and triggering apoptotic pathways.^14
-16 Apoptosis may happen in response to ischemic reperfusion injury through either caspase activation or mitochondrial dysfunction.^17,22 Apoptosis plays a key role in the development of heart failure. Activations of caspases 2, 3, and 7 in cardiac myocytes of CABG patients were proved, and the number of apoptotic cells has a reverse correlation with cardiac function and long-term survival.^15,18
-20,22

In addition to well-known traditional function of bone health, the pleiotropic effects of vitamin D have been assessed in nonskeletal tissues as immunomodulatory (with both positive and adverse effect), antiapoptotic, and cardioprotective.^{1

-5,7

-13,24} In clinical studies, vitamin D could reverse the left ventricular remodeling and improve the clinical symptoms and cardiac function even in heart failure patients.^10,25 The antiapoptotic role of vitamin D was shown in neonates with sepsis that could inhibit immune response and improved prognosis.¹¹ This defined biologic pathway could support our finding of less apoptotic cardiac cells in vitamin D group.

There are plenty of experimental studies showing that vitamin D reduced the rate of apoptotic cells and preserved organ structure during ischemic reperfusion state in different organs such as the heart.^6,7,11
-13 There is no consensus about the association of vitamin D concentration with cardiovascular outcomes in clinical studies. Some randomized clinical trials (RCTs) and meta-analyses reported no beneficial effects of vitamin D effects on cardiovascular outcomes.^26,27 However, numerous studies indicated the association of vitamin D deficiency with increased major cardiovascular events, low cardiac output, and worse postsurgical outcomes in cardiac surgical patients.^25,28

-31 Although the involved mechanisms are not fully understood, vitamin D maintains mitochondrial function, attenuates inflammatory response, and prevents from caspase activation.^3,5,11 It may be due to a pharmacological effect, which was induced by intermittently high-dose vitamin D administration.

However, there are very few studies on the effect of perioperative vitamin D administration in cardiac surgery patients. Tsutsumi et al³² showed the reverse association between vitamin D level and predicted operative mortality in patients’ undergoing cardiac surgery. Also, in another study, short-term consumption of high-dose vitamin D in ventilated intensive care patients could decrease the hospital stay.³³ Some studies have shown adverse effect of intermittent high-dose vitamin D and suggest daily and low-dose supplemental vitamin D.^1,34,35 Although vitamin D deficiency is identified as a potential cardiovascular risk factor and mortality, the role of vitamin D supplementation to improve cardiovascular end points is still a matter of debated.^24,35

-38 The inconclusive results could be explained in terms of heterogeneity between the design of studies, different study populations, vitamin D doses, and dosage intervals.^1,2

Besides its metabolic action, IGF-1 has also beneficial cardiovascular effects. The low level of IGF-1 is a negative prognostic factor and is associated with increased major adverse cardiac events. The IGF-1 reduces the endothelial dysfunction and inflammation, limits ischemic reperfusion injury, and interferes with apoptosis.^39
-41 Recent studies suggested the reciprocal association and interaction between vitamin D and IGF-1, and positive regulatory mechanism with ROS.^42
-44 This could explain the increased serum level of IGF-1 after vitamin D supplementation in our study, either before operation or in response to probably increased ROS level induced by CPB after operation.

The increased level of anti-inflammatory IL-10 immediately after cardiac surgery created the balance among inflammatory markers induced by CPB and surgery. Accordingly, it is atheroprotective and has an inhibitory effect on apoptosis in experimental studies, and its dominancy is associated with uneventful recovery after operation.^45,46 In agreement with our study, the heart failure patients treated with vitamin D showed significant increase in serum level of IL-10 with no significant change in BNP level.^47,48 The increased level of IL-10 following vitamin D treatment before operation, and even more increase after CPB in comparison with control group, could be explained at least in part by its immunomodulatory property.

Although this single-center study had a relatively small sample size and was limited to cardiac surgery patients, the study design was RCT with homogenous patients.

Conclusion

To the best of our knowledge, this is the first investigation to assess the antiapoptotic role of vitamin D in clinical cardiac setting. The present study suggests oral high-dose vitamin D treatment in CABG patients could reduce the heart cells apoptosis rate. Vitamin D may be protective by reducing apoptosis and increasing anti-inflammatory biomarkers. Probably, the biochemical/cellular effects of vitamin D cannot be translated into improved clinical outcome. Additional studies are needed to define the putative mechanisms underlying such benefits and the potential prognostic value in a long-term follow-up.

Footnotes

Authors’ Note

This article has been extracted from the thesis written by Dr. Tasdighi (Registration No: 210).

Acknowledgments

The authors thank Professor Ali Dabbagh for his thoughtful review and valuable comments.

Author Contributions

ET, PT and MF made substantial contributions to the conception and design of the work. ET, FP and MF collected data and data analysis and interpretation of data. PT performed Immunohistochemistry study of samples. ET, MH, MB, RB, SMS, PT, FP and FM made drafting the work. ET and FM revised it for important intellectual content and final approval.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The present study was supported by grants from the Research Department of the School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran (no. 13312).

ORCID iD

Mahnoosh Foroughi

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Zittermann

Tenderich

Berthold

Stehle

Koerfer

. Vitamin D supplementation improves cytokine profiles in patients with congestive heart failure: a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr. 2006;83(4):754–759.