The evolving role of long noncoding RNA HIF1A-AS2 in diabetic retinopathy: a cross-link axis between hypoxia, oxidative stress and angiogenesis via MAPK/VEGF-dependent pathway

Figures & data

Table 1. Demographic and clinical data of the studied groups.

Variable	Control	Diabetic without retinopathy	Nonproliferative diabetic retinopathy	Proliferative diabetic retinopathy	P value
Gender
Male	8(53.33%)	7 (46.67%)	8(53.33%)	9(60.00%)
Female	7 (46.67%)	8(53.33%)	7 (46.67%)	6(40.00%)
Age (years)	50.47 + 8.32	52.53 + 9.91	50.67 + 10.66	54.02 + 9.13	0.840
Duration (years)	–	2.60 + 1.24 *^▴◊	7.47 + 2.33*^# ◊	13.60 + 5.41*^#▴	<0.001*
BMI (kg/m^2)	28.35 + 2.30	33.82 + 3.37*	35.12 + 3.54*	34.45  + 3.04*	<0.001*
FBG (mg/dl)	91.33 + 2.74	171.47 + 27.11*^▴◊	205.80 + 27.69*^#	225.20 + 40.87*^#	<0.001*
Pp. BG (mg/dl)	115.27 + 12.03	228.53 + 39.78*^▴◊	275.33 + 35.10*^#	290.60 + 61.68*^#	<0.001*
HbA1c (%)	4.98 + 0.23	7.5 + 0.70*^▴◊	8.95 + 0.98 *^# ◊	9.75 + 1.06*^#▴	<0.001*
TC (mg/dl)	189.47 + 14.95	216.73 + 24.02^▴◊	256.87 + 41.07*^#	264.47 + 44.83*^#	<0.001*
TG (mg/dl)	133.17 + 6.88	187.33 + 11.52*^▴◊	233.27 + 29.93*^# ◊	273.40 + 31.69*^#▴	<0.001*
HDL-c (mg/dl)	46.67 + 2.38	42.60 + 3.52* ^◊	41.73 + 4.48* ^◊	39.33 + 3.38*^#▴	<0.001*
LDL-c (mg/dl)	116.17 + 0.37	136.66 + 0.49*^▴◊	168.49 + 0.52*^# ◊	170.48 + 0.71*^#▴	<0.001*
UACR (mg/g)	23.47 + 2.80	201.27 +  31.52*^▴◊	286.47 + 35.33*^# ◊	361.0 + 81.41*^#▴	<0.001*

Abbreviation: BMI: body mass index; FBG: fasting blood glucose; Pp. BG: 2h-post prandial blood glucose; HbA1c: glycated hemoglobin; TC: total cholesterol; TG: triacylglycerol; HDL-c: high-density lipoprotein–cholesterol; LDL-c: low-density lipoprotein–cholesterol; UACR: urinary albumin-to-creatinine ratio.

Statistical study is achieved using one-way ANOVA with Tukey’s post hoc test, SPSS computer program.

* Significant difference vs. control group (P < 0.05).

# Significant difference vs. diabetic group without retinopathy (P < 0.05).

▴Significant difference vs. nonproliferative diabetic retinopathy group (P < 0.05).

◊ Significant difference vs. proliferative diabetic retinopathy group (P < 0.05).

Table 2. Biochemical parameters of the studied groups.

Variable	Control	Diabetic without retinopathy	Nonproliferative diabetic retinopathy	Proliferative diabetic retinopathy	P value
HIF-1α level (pg/ml)	87.60 ± 9.53	226.40 ± 42.25*^▴◊	393.99 ± 57.42*^# ◊	712.65 ± 84.74*^#▴	<0.001*
VEGF level (pg/ml)	98.51 ± 17.55	154.36 ± 22.40*^▴◊	223.58 ± 24.33 *^#	244.16 ± 40.87*^#	<0.001*
Endoglin level (ng/ml)	0.58 ± 0.17	2.12 ± 0.43*^▴◊	3.34 ± 0.45 *^# ◊	4.69 ± 0.75 *^#▴	<0.001*
MAPK level (pg/ml)	42.54 ± 6.62	69.18 ± 8.64*^▴◊	85.58 ± 10.19*^# ◊	99.40 ± 20.79*^#▴	<0.001*
AGEs (U/ml)	34.60 ± 6.98	74.20 ± 9.99*^▴◊	93.60 ± 10.53*^# ◊	119.10 ± 12.81*^#▴	<0.001*
MDA level (nmol/L)	61.87 ± 9.46	97.51 ± 10.49*^▴◊	126.05 ± 11.56*^# ◊	173.44 ± 24.43*^#▴	<0.001*
TAC level (mmol/L)	1.65 ± 0.15	1.03 ± 0.21*^▴◊	0.91 ± 0.23*^# ◊	0.87 ± 0.36*^#▴	<0.001*
Total thiols level (μmol/ml)	271.41± 12.60	219.57 ± 14.70*^▴◊	192.28 ± 13.35*^# ◊	173.39 ± 29.88*^#▴	<0.001*
NO level (µmol/L)	31.77 ± 5.97	44.15± 4.28*^▴◊	51.52 ± 5.35*^# ◊	59.11 ± 9.84*^#▴	<0.001*
ONOO^− level (µmol/L)	3.91 ± 0.41	6.63 ± 0.55*^▴◊	8.24 ± 0.61*^#	8.92 ± 1.67 *^#	<0.001*

Abbreviation: AGEs: advanced glycation end products; HIF-1α: hypoxia inducible factor 1-alpha; MAPK: mitogen activated protein kinase; MDA: malondialdehyde; NO: nitric oxide; ONOO−: peroxynitrite; TAC: total antioxidant capacity; VEGF: vascular endothelial growth factor.

Statistical study is achieved using one-way ANOVA with Tukey’s post hoc test, SPSS computer program.

* Significant difference vs. control group (P < 0.05).

# Significant difference vs. diabetic group without retinopathy (P < 0.05).

▴Significant difference vs. nonproliferative diabetic retinopathy group (P < 0.05).

◊ Significant difference vs. proliferative diabetic retinopathy group (P < 0.05).

Figure 1. Photomicrographs representation of DNA damage in peripheral blood mononuclear cells using comet assay in control group (A), diabetic group without retinopathy (B), nonproliferative diabetic retinopathy group (C), proliferative diabetic retinopathy group (D). (The scale bar is 40 µm, 5 fields were assessed to provide this representative image).

Table 3. Comet assay parameters obtained by image analysis in peripheral blood mononuclear cells of the studied groups.

Variable	Control	Diabetic without retinopathy	Nonproliferative diabetic retinopathy	Proliferative diabetic retinopathy	P value
Tails length (µm)	1.36 ± 0.06	2.35 ± 0.13*^▴◊	4.58 ± 0.42*^# ◊	9.75 ± 0.37*^#▴	<0.001*
Tail DNA%	1.46 ± 0.04	2.69 ± 0.06*^▴◊	3.67± 0.08*^# ◊	8.66 ± 1.01*^#▴	<0.001*
Tail moment	1.99± 0.08	6.32 ± 0.21*^▴◊	16.81 ± 1.45*^# ◊	84.60 ± 6.75*^#▴	<0.001*

Note: Statistical study is achieved using one-way ANOVA with Tukey’s post hoc test, SPSS computer program.

* Significant difference vs. control group (P < 0.05).

# Significant difference vs. diabetic group without retinopathy (P < 0.05).

▴Significant difference vs. nonproliferative diabetic retinopathy group (P < 0.05).

◊ Significant difference vs. proliferative diabetic retinopathy group (P < 0.05).

Figure 2. HIF1A–AS2 relative expression in the studied groups. Data are represented as mean ± SD. * Significant difference vs. control group (P < 0.05). # Significant difference vs. diabetic group without retinopathy (P < 0.05). ▴Significant difference vs. nonproliferative diabetic retinopathy group (P < 0.05). ◊ Significant difference vs. proliferative diabetic retinopathy group (P < 0.05).

Table 4. Correlations between the studied parameters in diabetic retinopathy patients.

Variable		HIF1A–AS2	HIF-1α	VEGF	MAPK	Endoglin	MAPK	AGEs
HIF1A–AS2	r
	P
HIF-1α	r	0.719
	P	<0.001*
VEGF	r	0.487	0.613
	P	<0.001*	<0.001*
MAPK	r	0.460	0.644	0.813
	P	<0.001*	<0.001*	<0.001*
Endoglin	r	0.713	0.901	0.669	0.665
	P	<0.001*	<0.001*	<0.001*	<0.001*
AGEs	r	0.888	0.902	0.821	0.835	0.893
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
MDA	r	0.578	0.800	0.834	0.829	0.785	0.829	0.871
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
TAC	r	0.264-	0.335-	0.626-	0.641-	0.477-	0.641-	0.783-
	P	<0.05*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
Total thiols	r	0.448-	0.619-	0.827-	0.885-	0.675-	0.885-	0.863-
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
NO	r	0.459	0.672	0.770	0.828	0.710	0.828	0.841
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
ONOO^−	r	0.416	0.607	0.825	0.804	0.628	0.804	0.840
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
Tail DNA%	r	0.740	0.936	0.568	0.674	0.837	0.674	0.848
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
Tail moment	r	0.746	0.931	0.839-	0.648	0.815	0.648	0.779
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*
HbA1c %	r	0.470	0.673	0.395	0.401	0.624	0.401	0.859
	P	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*	<0.001*

Abbreviation: AGEs: advanced glycation end products; HbA1c: glycated hemoglobin; HIF-1α: hypoxia inducible factor 1-alpha; HIF1A–AS2: HIF1A antisense RNA 2; MAPK, Mitogen activated protein kinase; MDA, Malondialdehyde; NO, nitric oxide; ONOO−, Peroxynitrite; TAC, Total antioxidant capacity; VEGF, Vascular endothelial growth factor. P < 0.05 is significant. r, Pearson’s correlation coefficient.

Table 5. Multiple linear regression analysis for potential predictors of proliferative diabetic retinopathy.

	Unstandardized coefficients		Standardized coefficients
Variable	B	Standard error	Beta	t	P value
HIF1A–AS2	0.460	0.161	0.407	2.863	<0.001*
HIF-1α	0.001-	0.002	0.139-	0.614-	0.542
VEGF	0.002-	0.003	0.074-	0.530-	0.599
MAPK	0.025-	0.015	0.309-	1.927-	0.090
Endoglin	0.404	0.207	0.338	1.992	<0.05 *
AGEs	0.020	0.010	0.343	1.997	<0.05 *
MDA	0.014-	0.009	0.319-	1.562-	0.125
TAC	0.587-	0.754	0.110-	0.779-	0.440
Total thiols	0.014-	0.010	0.281-	1.323-	0.792
NO	0.020	0.015	0.161	1.336	0.188
ONOO^−	0.255	0.140	0.263	1.816	0.076
Dependent variable: HbA1c %

Abbreviation: AGEs: advanced glycation end products; HbA1c: glycated hemoglobin; HIF-1α: hypoxia inducible factor 1-alpha; HIF1A–AS2: HIF1A antisense RNA 2; MAPK: mitogen activated protein kinase; MDA: malondialdehyde; NO: nitric oxide; ONOO−: peroxynitrite; TAC: total antioxidant capacity; VEGF: vascular endothelial growth factor. P < 0.05 is significant. r: Pearson’s correlation coefficient.

Data availability statement

Research data are not publically shared.