Elevated serum OxLDL is associated with progression of type 2 Diabetes Mellitus to diabetic retinopathy

Accepted Manuscript Elevated serum OxLDL is associated with progression of type 2 Diabetes Mellitus to diabetic retinopathy Kaviarasan Kuppan, Jithu Mohanlal, Arif Mulla Mohammad, Narasimha Das Undurti, Angayarkanni Narayanasamy PII:

S0014-4835(18)30619-5

DOI:

https://doi.org/10.1016/j.exer.2019.05.008

Reference:

YEXER 7668

To appear in:

Experimental Eye Research

Received Date: 19 August 2018 Revised Date:

30 March 2019

Accepted Date: 13 May 2019

Please cite this article as: Kuppan, K., Mohanlal, J., Mohammad, A.M., Undurti, N.D., Narayanasamy, A., Elevated serum OxLDL is associated with progression of type 2 Diabetes Mellitus to diabetic retinopathy, Experimental Eye Research (2019), doi: https://doi.org/10.1016/j.exer.2019.05.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Elevated serum OxLDL is associated with progression of type 2 Diabetes Mellitus to Diabetic Retinopathy Kaviarasan Kuppana, Jithu Mohanlala, Arif Mulla Mohammadb, Narasimha Das Undurtic, Angayarkanni Narayanasamya*

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Running title: OxLDL-Ab and diabetic retinopathy a

Biochemistry and Cell Biology Department, Vision Research Foundation, Chennai-600 006 Shri Bhagwan Mahavir Vitreo Retinal Services, Medical Research Foundation, Chennai-600 006 c School of Biotechnology, Jawaharlal Nehru Technological University, Kakinada-533 003

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b

Address all communications to: *Corresponding Author

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Dr. N. Angayarkanni, PhD Professor and Head R.S. Mehta Jain Department of Biochemistry and Cell Biology Vision Research Foundation, Chennai-600 006 India Phone: +91-44-42271500 Fax: +91-44-28254180 E-mail: [email protected]

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Type 2 Diabetes Mellitus Type 1 Diabetes Mellitus Paraoxonase 1 Reactive oxygen species Malondialdehyde Advanced Glycation End products Oxidized LDL antibody Glycosylated Hemoglobin Nonproliferative diabetic retinopathy Proliferative diabetic retinopathy Diabetic retinopathy High density lipoprotein cholesterol Low density lipoprotein cholesterol Very low density lipoprotein cholesterol Thiobarbituric acid reactive substances

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T2DM T1DM PON1 ROS MDA AGE oxLDL-Ab HbA1c NPDR PDR DR HDL-C LDL-C VLDL-C TBARS

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Abbreviations

ACCEPTED MANUSCRIPT 3 ABSTRACT Hyperlipidemia is associated with the progression of diabetic retinopathy (DR). Paraoxonase 1 (PON1), an esterase is known to prevent systemic LDL oxidation. This study assessed if serum

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oxLDL is associated with the progression of Type 2 DM to DR. This study is part of a three-year hospital based prospective study where 87 subjects were recruited. This included T2DM without DR (n=22); Non-Proliferative (NPDR) (n=21) and Proliferative DR (PDR) (n=22) along with

esterase

activity

and

plasma

Malondialdehyde

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age/sex matched controls (n=22). Serum oxLDL-Ab was estimated by ELISA. Serum PON (MDA)

level

were

estimated

by

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spectrophotometry and the serum Advanced Glycation End products (AGE) level by spectroflourimetry. The systemic levels of oxLDL, AGE and MDA were increased with the progression of T2DM without DR to DR as seen by ANOVA (P>0.05). Serum oxLDL-Ab levels showed a positive correlation to total cholesterol (P=0.04) as evaluated in the DR group. Statin

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intake was found to lower PON esterase activity (P>0.05) as well as the levels of serum oxLDL and MDA. Based on this pilot study, it is proposed that elevated serum oxLDL is a potential risk factor for the progression of type 2 DM to DR and that measuring oxLDL-Ab can have a

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prognostic value.

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Keywords: oxLDL; AGE; PON; MDA; T2DM; Diabetic Retinopathy.

ACCEPTED MANUSCRIPT 4 1. Introduction Diabetic retinopathy (DR), a retinal vascular complication of Diabetes Mellitus (DM) occurs due to prolonged hyperglycemia. Established risk factors for diabetic retinopathy include

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long duration of DM, poor glycemic control, hypertension and dyslipidemia.(Lee et al., 2015) Nearly all the type 1 DM cases and 75% of type 2 DM (T2DM) develop DR over 15 years of disease duration as shown in earlier epidemiological studies.(LeCaire et al., 2013) Metabolic

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syndrome associated with DM leads to diabetic complication such as the retinopathy.(Fox et al., 2015; Gao et al., 2016) Therefore, it is important to identify risk factors, prognostic markers and

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mechanism for the onset and/or progression of DR, which can pave way for new preventive therapeutic strategies.(Klein et al., 1984) However, discrepancies in the study outcome on relating metabolic syndrome with retinopathy changes in non-diabetic subjects(Gao et al., 2016) indicate that factors other than hyperglycemia can play a catalyst role. Studies demonstrate that

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oxLDL plays a key in the process of atherosclerosis.(Holvoet et al., 1999; Holvoet et al., 1998) Elevated levels of ox-LDL-Ab have been found to be markers or predictors of accelerated atherosclerotic process.(Holvoet et al., 1999) Antibodies against circulating oxLDL also seem to

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play a significant role in retinopathy progression and oxidative modification of LDL which plays pathogenic roles in the disease complications of diabetes, including retinal changes.(Yu et al.,

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2016) Levels of Advance Glycation end products (AGE), which can glycate LDL (Stirban et al., 2014) and Malondialdelyde (MDA) are indicative of oxidative stress that promotes oxidation of LDL,(Ho et al., 2013) while PON 1, seen in the apoB of LDL, regenerates LDL form oxLDL based on its esterase activity.(Mehdi and Rizvi, 2012) Therefore, this study was aimed to determine if the levels of serum oxLDL is a risk factor for DR development in DM cases.

ACCEPTED MANUSCRIPT 5 2. Methods 2.1. Patients recruitment As a part of a three-year prospective study T2DM cases and control subjects were recruited

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based on inclusion criteria as per Institutional ethics board approval. A total of 87 individuals were included in this study. Of these, 22 were identified with T2DM without DR (mean age ± SD: 51 ± 13 years; 14M/8F), 21 with Non-Proliferative Diabetic Retinopathy (NPDR) (mean age

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± SD: 58 ± 10 years; 15M/6F) and 22 with Proliferative Diabetic Retinopathy (PDR) (mean age ± SD: 52 ± 7 years; 19M/3F). Age and sex matched controls (n = 22, mean age ± SD: 44 ± 7

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years; 13M/9F) were included in the study. In the T2DM without DR group, there were no statin takers. In the DR group, 15 were statin takers (35%) and 28 (65%) were statin non-takers. Patients were recruited based on the diagnosis criteria set by the American Diabetes Association’s (ADA) for the T2DM diagnosis, with glycosylated hemoglobin (HbA1c) level ≥

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6.4 % and/or a fasting blood sugar (FBS) ≥ 126 mg/dL. Patient undergoing any medical treatment other than T2DM and metabolic syndrome were excluded. Detailed anthropometric measures, medical history, lifestyle, biochemical investigations data and ophthalmic status were

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documented at the time of recruitment of the study subjects. For laboratory investigations blood and urine samples were drawn after 12 hour overnight fast. Plasma/serum was separated by

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centrifugation at 2500 rpm for 5 min at RT, aliquoted and was stored at -80 °C until analysis. The routine laboratory investigations including HbA1C (%), fasting and post prandial plasma glucose, serum lipid profile were assayed using clinical chemistry analyzer (Dade Behring, Minnesota, USA) and microalbumin in urine (mg/L) was measured according to the protocol described in the kit (Nycocard Reader II /Nycocard kits, Axis-Shield, Oslo, Norway). The DR patients were further sub-grouped as NPDR and PDR based on International Clinical Diabetic

ACCEPTED MANUSCRIPT 6 Retinopathy Disease Severity Scale.(2002) Patients with any other ophthalmic diseases other than DR were excluded. 2.2. OxLDL-Ab, PON aryl esterase and AGE analysis

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Serum oxLDL-Ab was assayed using ImmuLisa oxidized LDL antibody ELISA kit (IMMCO Diagnostics) as previously described(Witztum and Steinberg, 1991). Serum PONAREase activity was done according to the method of Cabana et al.(Cabana et al., 2003)

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Enzyme activity was expressed as micromoles phenyl acetate hydrolyzed per milliliter per minute. Serum AGE was measured by the method of Bhatwadekar et al.(Bhatwadekar and

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Ghole, 2005) The results were expressed in terms of µg/mg protein. 2.2.1. MDA analysis as oxidative stress marker

Malondialdehyde reacts with thiobarbituric acid in an acidic condition to generate a pink colour chromophore which was spectrophotometrically read at 535 nm(Devasagayam and

3. Statistical analysis

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Tarachand, 1987), based on which plasma MDA was estimated.

Statistical analyses were performed by independent-sample t-test; Pearson’s χ2

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correlation for correlation between variables. McNemar test has been used where sample size was too less and the distribution was skewed as these factors voids the homogeneity of variance

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assumption. All the statistical analyses were done using SPSS V.14.0 (IBM, USA). Data are expressed as Mean ± SD. P< 0.05 was considered significant. 4. Results

Biochemical parameters such as plasma glucose, HbA1c, µ-albumin and serum lipid

levels were measured in all the four groups namely non-diabetic control, T2DM with no DR, T2DM with NPDR and T2DM with PDR in the study. Among all the parameters examined,

ACCEPTED MANUSCRIPT 7 HbA1c, triglycerides, TC/HDL-C ratio, and Urine µ-albumin were increased in the T2DM cases with and without DR. Disease progression was indicated by fasting glucose, HbA1c and microalbumin (Table 1).

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There was a significant increase in the levels of serum oxLDL-Ab, AGE and plasma MDA with a decrease in serum PON arylesterase activity in the DR cases compared to that of control (Table-2). Fig. 1(a-c) shows the distribution chart where there is shift in the median with

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disease progression. ANOVA revealed that the progressive increase in these parameters is associated with the disease progression. Amongst the parameters studied in the various groups,

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serum oxLDL seems to be a potential early marker, as it significantly increased in T2DM without DR cases (Table 2). Amongst various correlations studied, oxLDL-Ab was found to correlate positively with the levels of total cholesterol (r=0.43, P=0.04) as well as to the TC/HDL ratio (r=0.43, P=0.04) (Fig. 2), thus showing that oxLDL-Ab is yet another potential marker of

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metabolic syndrome. In DR group, after classifying them as statin and non-statin takers, the statin takers showed lowered oxidative stress as seen by 22.45% (Table 3) decrease in the levels of serum oxLDL-Ab and 24.37% (Table 3) decrease in plasma MDA as compared to statin non-

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takers.

McNemar test was done to identify the risk factors that can cause DR in diabetics, based

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on the serum/plasma parameters studied. Accordingly, oxLDL-Ab with an OR of 2.629 (95% CI=0.773 to 8.938, P=0.002) was found to be the only significant risk factor for DR in statin non-takers.

5. Discussion

T2DM being a global problem increasing at an alarming rate, the most common complication is retinopathy, where DM disease duration is reportedly a predictor of disease progression.(Raman

ACCEPTED MANUSCRIPT 8 et al., 2014; Rema and Pradeepa, 2007) Studies show that tight regulation of blood glucose as well as hypertension can prevent progression to DR. (Park and Roh, 2016; Rudnisky et al., 2012) importantly, hyperlipidemia, which influences the blood pressure, is a major factor that warrants

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attention.(Kowluru et al., 2016) As per the UK DRIVE cross-sectional study, ethnic variation is another factor that influences DR prevalence amongst diabetics.(Sivaprasad et al., 2012a; Sivaprasad et al., 2012b) Though interventions such as laser treatment, early vitrectomy,

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intravitreal injection of steroids help in the management of DR, anti-VEGF or lipid lowering

progression.(Simo et al., 2015)

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therapy has not always been successful or strongly supported by facts preventing the disease

In this cross-sectional study, both NPDR and PDR cases had diabetic duration of more than 10 years. A progressive increase in the levels of fasting glucose, HbA1c, triglycerides and µ-albumin in diabetic groups indicate disease progression from diabetes to DR. In this

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background, our results based on ANOVA show a progressive increase in oxidative stress as seen by plasma MDA levels apart from serum AGE levels. Lipid peroxidation is an important source of ROS formation. Moreover AGE can induce ROS generation.24 AGE-RAGE-Oxidative

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stress axis is associated with vascular complications of diabetes.25 Reports have shown that AGEs are involved in the pathological process of DR by inducing TNF-α production from the

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microenvironment that triggers leukocyte infiltration to the site of vascular injury thereby causing vascular inflammation.(Krady et al., 2005),(Zong et al., 2010) HDL-associated enzyme, Paraoxonase 1 (PON1) that acts as an esterase is responsible for

its ability to prevent the accumulation of lipid peroxides on LDL.(Kumar and Rizvi, 2014; Mackness et al., 1993) A significant decrease in the levels of PON arylesterase activity was observed in the NPDR and PDR cases compared to controls. Serum PON1 arylesterase activity is

ACCEPTED MANUSCRIPT 9 decreased in subjects with myocardial infarction (Maturu et al., 2013) and in subjects with type I or type II diabetes.(Ikeda et al., 1998; Kota et al., 2013) Besides, streptozotocin-induced diabetes resulted in a progressive decrease in serum levels of PON1 arylesterase.(Patel et al., 1990)

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Decreased PON1 arylesterase acitivty is reported in vascular complications of DR which has been associated with lipid peroxidation.(Ikeda et al., 1998) PON/C-reactive protein ratio was proposed as inflammation marker seen in DR(Nowak et al., 2010). In recent years, growing

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evidence indicate inflammation as an important event in the pathogenesis of PDR. oxLDL has been shown to trigger proinflammatory events through the activation of pathways associated

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with innate immunity.(Binder et al., 2002) oxLDL and AGE-LDL IgG antibodies also induce immune complexs that are pro-inflammatory in the microvasculature of the retina.(Fu et al., 2014) In vitro work has shown that oxLDL in association with such immune complexes may cause damage through pericyte loss, one of the initial steps in the development of retinopathy.(Fu

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et al., 2014)

There was a report showing that hypercholesterolemia patients treated with statin (hydroxymethylglutaryl

coenzyme

A

reductase

inhibitors)

showed

increased

PON1

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activity.(Deakin et al., 2003) Lowered plasma PON1 activity observed in T2DM with and without DR as reported in studies can also be attributed to increased enzyme

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glycation.(Bharathidevi et al., 2017), impaired antioxidant defense and dietary factors.(Deakin and James, 2004) These data add evidence to the observations that lowering of PON1 activity, particularly in T2DM increases the risk of atherosclerosis. Studies suggest that statins may help in preventing the progression of DR. In randomized

controlled trials (Gupta et al., 2004; Sen et al., 2002) on DR patients with hypercholesterolemia, statins were found to retard the progression of hypercholesterolemia and reduce the severity of

ACCEPTED MANUSCRIPT 10 hard exudates, clinically significant macular edema, and dyslipidemia. A relative decrease in PON1 activity seen in statin takers can be an indirect effect of lowered oxidative stress in DR cases where there is lowering both of MDA levels and oxLDL levels which is beneficial. PON1

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activity enhancing effects of statins without increase in protein is however reported in cardiovascular diseases based on meta-analysis(Ferretti et al., 2015). However, PON regulation in association with statin intake in the DR cases needs to be probed further. The positive

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correlation between TC and oxLDL-Ab in statin non-takers is the further evidence that oxLDLAb levels are highly associated with the total cholesterol levels and is lowered in statin-takers by

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around 24%. Larger studies are required to validate the beneficial effect of statins in lowering serum oxLDL in DR cases. Studies have shown that circulating levels of oxLDL-Ab were decreased in patients with statin treatment.(Nou et al., 2016; Zhang et al., 2005) It is recently reported that increase in AGE-LDL and oxLDL is associated with

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progression in retinopathy as studied in type 1 DM patients.(Lopes-Virella et al., 2012) Consistent with this report, based on ANOVA this study showed that oxLDL measured in terms of oxLDL-Ab is potentially associated with progression of DR in T2DM. A larger sample size

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shall validate this observation further. Yet, cross tabulation analysis followed by McNemar test showed that oxLDL-Ab was a significant risk factor for predicting DR in statin non-takers alone.

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In summary, this study shows that oxLDL-Ab is a risk factor associated with the

progression of DR. It is therefore proposed that serum oxLDL can be a prognostic marker for DR in the T2DM cases. Statin therapy appears to be potentially beneficial in lowering the levels of oxLDL-Ab and oxidative stress in these cases.

ACCEPTED MANUSCRIPT 11 Acknowledgement This study was funded by the Department of Biotechnology, Government of India

Disclosure

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The authors declare no other conflict of interest.

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(DBT No. BT/PR11627/MED/30/157/2010), New Delhi, India.

ACCEPTED MANUSCRIPT 12 References

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Figure legends

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Fig. 1a. Distribution graph for oxLDL-Ab levels in control and diabetic groups The median levels were found to be 24.25, 33.16, 25.95 EU/mL in diabetic groups and 18.61 EU/mL in control Fig. 1b. Distribution graph for PON arylesterase activity in control and diabetic groups

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Fig. 1c. Distribution graph for AGE levels in control and diabetic groups

Fig. 2. Relationship between TC vs oxLDL-Ab and TC/HDL vs oxLDL-Ab in statin non-

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takers in DR

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Table 1 Biochemical Parameters in the study recruits Diabetic (n=22) 6.28 ±5.44

NPDR (n=21) 14.48 ± 9.11

PDR (n=22) 14.3 ±7.38

Fasting glucose (mg/dL)

96.95 ± 7.59

161.81 ± 58.76*

144.10 ± 53.51*

142..91 ± 61.78*

0.001

HBA1c (%)

5.65 ± 0.44

7.48 ± 1.72*

7.26 ± 1.26*

8.09 ± 1.77*

0.001

Total cholesterol(mg/dL)

184.77 ± 31.90

178.55 ± 42.71

172.38 ± 52.65

181.1± 65.47

NS

Triglyceride (mg/dL)

103.86 ± 40.12

127.7 ± 60.78

115.26 ± 49.90

187.4±194.58*

0.057

HDL-C(mg/dL)

47.55 ± 6.76

46.18 ± 7.60

42.76 ± 6.71

42.3±10.31

NS

LDL-C(mg/dL)

116.45 ± 27.45

106.99 ± 39.84

106.47 ± 44.89

99.6±53.43

NS

VLDL-C(mg/dL)

20.77 ± 8.02

25.55 ± 12.16

23.15 ±9.28

36.1±38.51

NS

Urine µ-albumin (mg/L)

6.29 ± 4.46

58.16 ± 77.06*

86.16 ± 85.73*

143.37 ± 83.30*

0.0001

TC/HDL ratio

3.94 ± 0.79

4.07 ± 1.21

4.71 ± 1.53*

0.081

Values are means ± S.D.

*p < 0.05; control vs respective group

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Duration of diabetes

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Control (n=22) -

3.91 ± 0.97*

ANOVA p-value NS

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Diabetic (n=22)

NPDR (n=21)

AGE (µg/mg protein)

13.81 ± 7.17

17.14 ± 7.33

19.69 ± 8.43*

TBARS (nmol/mL)

47.88 ± 32.74

72.37 ± 44.15

98.79 ± 53.99*

Paraoxonase – PON1-Aryl esterase (U/mg protein)

74.2 ± 15.32

71.72 ± 24.17

18.31 ± 8.69

26.73 ± 12.86*

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OxLDL-Ab (EU/mL)

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Values are means ± S.D.

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Table 2 Serum AGE, PON1 & plasma TBARS levels in control T2DM and DR groups

PDR (n=22)

ANOVA p-value

25.03 ± 14.56*

0.005

115.34 ± 114.18*

0.014

61.03 ± 16.20*

62.01 ± 19.20*

34.47 ± 21.67*

28.44± 13.45* (S+: 11; S-: 11)

0.059

0.019

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Table 3 Serum OxLDL-Ab, AGE, PON & plasma TBARS levels in statin takers and non-takers in DR

Statin non takers (n=28)DR

Statin takers (n=15)DR

Age

55.5 ±10.26

53.4 ±7.34

Total cholesterol (mg/dL)

239.08±38.88

141.88±48.67*

LDL-C (mg/dL)

138.38±51.84

83.13±38.21*

0.978

OxLDL-Ab (EU/mL)

33.98 ± 19.05

26.35 ± 15.52 22.45 % decrease

0.255

AGE (µg/mg protein)

22.92 ± 13.89

21.24 ± 7.47

0.068

PON1 (U/mg protein)

62.44 ± 16.47

30.28 ± 23.40* 51.5% decrease

0.999

TBARS (nmol/mL)

117.51 ± 102.68

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88.87 ± 58.78 24.37 % decrease

Power analysis for sample size, >0.70 is considered as statistically significant Values are means ± S.D.

*p < 0.05; respective group vs control

Power analysis

0.163

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Manuscript Title

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Elevated serum OxLDL is associated with progression of type 2 Diabetes Mellitus to Diabetic Retinopathy

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Highlights

This study highlights the relevance of oxLDL measured as oxLDL-Ab as a prognostic

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marker for the progression of Type 2 Diabetes Mellitus to Diabetic Retinopathy.