Aqueous humor levels of vascular endothelial growth factor and adiponectin in
patients with type 2 diabetes before and after intravitreal bevacizumab injection
Q2Ciro Costagliola
a,*, Aurora Daniele
b, Roberto dell
’Omo
a, Mario R. Romano
a, Fabiana Aceto
a,
Luca Agni
fili
c, Francesco Semeraro
d, Antonio Porcellini
eaDpt di Medicina e di Scienze per la Salute, Università degli Studi del Molise, Via F. De Sanctis, snc, 86100 Campobasso, Italy bDpt di Scienze Ambientali, Seconda Università degli Studi di Napoli, Caserta, Italy
cDpt di Medicina e Scienze dell’Invecchiamento, Università G. D’Annunzio, Chieti, Italy dDpt di Chirurgia e Medicina Legale, Università di Brescia, Brescia, Italy
eDpt di Biologia Strutturale e Funzionale, Università degli Studi“Federico II”, Napoli, Italy
a r t i c l e i n f o
Article history: Received 16 July 2012
Accepted in revised form 5 February 2013 Available online xxx
Keywords:
proliferative diabetic retinopathy diabetic macular edema vascular endothelial growth factor adiponectin
aqueous humor
a b s t r a c t
To determine the levels of vascular endothelial growth factor (VEGF) and adiponectin (APN) in the aqueous humor of patients with type 2 diabetes before and after injection of bevacizumab (IVB). Twenty eyes of twenty consecutive patients with type 2 diabetes with PDR and clinically significant macular edema were enrolled in this study. Aqueous samples were collected at baseline and one month after IVB to evaluate VEGF and APN levels. Twenty age-matched patients undergoing cataract surgery were used as control. Best-corrected visual acuity (BCVA) and foveal thickness (FT) changes after IVB were also measured. Safety was assessed by recording the incidence of ocular and non-ocular adverse events. At baseline APN and VEGF levels were significantly lower in controls than in PDR patients (APN: 3.6 1.1 vs 18.7 4.5 ng/ml; VEGF: 22.6 16.1 vs 146.2 38.71 pg/ml). After IVB, both compounds significantly decreased. FT and BCVA at baseline were significantly different between controls and patients (FT: 215.6 34.8 vs 532.7 112.4mm; BCVA: 23.6 4.2 vs 18.4 7.3 letters). After IVB a significant decrease of FT with a concomitant improvement of BCVA occurred. Neither ocular nor systemic adverse events were reported. Ourfindings demonstrate that patients with type 2 diabetes, PDR and macular edema show VEGF and APN levels in aqueous humor higher than those found in control subjects. IVB signi fi-cantly reduced the levels of both compounds, which remained anyway at concentrations higher than those recorded in control subjects.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Diabetic retinopathy (DR), the most frequent diabetic micro-vascular complication, affects 30%e50% of all diabetic patients and represents the main cause of legal blindness in developed countries (Semeraro et al., 2011). It is caused by changes in the retina microvasculature and hyperglycemia itself seems to be the key factor in the etiology of DR although, recently, focus has been directed to the molecular basis of the disease, and several biochemical factors other than hyperglycemia, have been
consid-ered (Cai and Boulton, 2002). These mechanisms act affecting
cellular metabolites and inducing release of cytokines (Caldwell et al., 2003); among these, vascular endothelial growth factor (VEGF) is the most representative, and its role in angiogenesis and
microvascular permeability is well known (Aiello et al., 1994;
Caldwell et al., 2003).
Current evidence indicates that VEGF plays a central role in the development of choroidal neovascularisation (CNV). In fact, vitre-ous levels of VEGF were found to be significantly higher in patients with CNV compared to those found in healthy controls (Aiello et al., 1994;Kvanta et al., 1996;Wells et al., 1996), as well as intravitreous injection of VEGF is able to induce proliferation of choroidal endothelial cells in experimental animal models (Tolentino et al.,
1996). Since VEGF plays a key role in the pathogenesis of CNV,
targeting VEGF has been an attractive strategy in the treatment of CNV, initiating extensive research in recent years (Ferrara et al.,
2007). Anti-VEGF therapy can arrest choroidal angiogenesis and
also reduce vascular permeability, frequently the main cause of visual acuity deterioration. Thesefindings provide the rationale for anti-VEGF therapy in retinal vascular diseases associated with new vessel formation such as diabetic retinopathy (Sawada et al., 2007;
Matsuyama et al., 2009).
* Corresponding author. Tel.: þ39 (0)8744041.
E-mail address:ciro.costagliola@unimol.it(C. Costagliola).
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Experimental Eye Research
j o u r n a l h o m e p a g e : w w w . e l s ev i e r . c o m / l o c a t e / y e x e r
0014-4835/$e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.exer.2013.02.004 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
Adiponectin (APN) is a polypeptide hormone produced exclu-sively in adipocytes that circulates at very high levels in the bloodstream. In experimental studies, APN has been shown to exert anti-inflammatory and anti-atherosclerotic effects, and to inhibit neo-intimal thickening and vascular smooth muscle cell prolifera-tion in mechanically injured arteries (Kubota et al., 2002). Plasma APN concentrations are decreased in obesity, insulin resistance, type 2 diabetes, coronary disease and hypertension (Frystyk et al., 2005). Several studies have indicated that APN possesses anti-in-flammatory properties and thus may negatively modulate the process of atherogenesis (Goldstein and Scalia, 2004). The role of APN in the development of microvascular disease (such as diabetic retinopathy and nephropathy) is largely unknown. Clinically, pa-tients with type 2 diabetes suffering from diabetic retinopathy (proliferative as well as non-proliferative) are reported to have lower levels of APN than matched patients without retinopathy (Yilmaz et al., 2004).
The aim of this study was to determine the level of VEGF and APN in the aqueous humor of patients with diabetic proliferative retinopathy (DPR) and to evaluate the effects of intravitreal bev-acizumab (IVB) on the concentration of these compounds. 2. Materials and methods
2.1. Subjects
Twenty eyes of twenty consecutive patients with type 2 diabetes
mellitus (DM), PDR and clinically significant macular edema were
enrolled in this study. Diabetes mellitus was diagnosed based on the
American Diabetes Association criteria (Expert Committee on the
Diagnosis and Classification of Diabetes Mellitus, 2003). Diagnosis
of PDR was made based on an international standard (Watkins,
2003). Patients in the PDR group had been diagnosed with type 2
DM an average of 15 (2.5) years earlier. Clinically significant
macular edema was defined according to the EDTRS criteria (ETDRS report no. 19. Early Treatment Diabetic Retinopathy Study Research Group, 1995). Twenty age-matched patients undergoing cataract surgery constituted the control group. The procedures used in this study were conformed to the tenets of the Declaration of Helsinki and were performed after receiving institutional review board approval. Informed consent was obtained from all patients. 2.2. Diagnostic procedures
At baseline all the patients underwent BCVA measurement us-ing an early treatment diabetic retinopathy study (ETDRS) chart at
4 m, fundus biomicroscopy, tonometry, fluorescein and
indoc-yanine green angiographies (FA and ICGA), and spectral domains optical coherence tomography (SD-OCT). All examinations, with the exception of angiographic tests, were repeated one month after bevacizumab intravitreal injection (IVB). Angiographic tests and OCT scans were recorded using Spectralis SD-OCT (Heidelberg En-gineering, Heidelberg, Germany).
2.3. Inclusion and exclusion criteria
In the PDR group patients were included if they presented one or more of the following abnormalities: new vessels on the disc and new vessels elsewhere. Eyes with vitreous hemorrhage obscuring retina details or evidence of tractional retinal detachment on OCT
were excluded from the study group. All diagnoses were confirmed
by at least 2 doctors independently at the time of admission (C.C. and R.d.O.).
Subjects were excluded if they had type 1 diabetes, were younger than 18, or were older than 90 years of age. To minimize
the compounding complications of data interpretation from other risk factors, we excluded all subjects and patients with hyperten-sion, hyperlipidemia, nephropathy, coronary heart disease, heart failure or renal failure and those who had ocular surgery within 3 months preceding inclusion.
In the control group exclusion criteria were age younger than 18 and older than 90 years, arterial hypertension, hyperlipidemia, nephropathy, coronary heart disease, heart failure or renal failure; any type of retinal disease, glaucoma, previous vitrectomy, laser coagulation, diabetes mellitus, use of immunosuppressive drugs, malignant tumors at any location, and participation in any study of investigational drugs within 3 months before recruitment. 2.4. Aqueous sampling and bevacizumab injections
All patients with DPR received intravitreal injections of 1.25 mg/ 0.05 mL of bevacizumab (Avastin; Genentech Inc, South San Fran-cisco, California, USA). Immediately before the intravitreal injec-tion, aqueous samples were obtained by aspirating 0.05e0.1 mL of aqueous using a 30-gage needle connected to a tuberculin syringe at the temporal limbus. IVB injection was then performed using a
30-gage needle in the inferotemporal quadrant at 3.5e4 mm
posterior to the limbus. The undiluted aqueous samples were transferred into sterile containers and immediately stored in
a80C freezer until analysis. The same procedure was performed
one month later, when PDR patients underwent to the second bevacizumab intravitreal injection.
In controls aqueous humor samples were collected in the same fashion described above for eyes with PDR, before performing surgery. The undiluted aqueous samples were transferred into sterile containers and immediately stored in a80C freezer until analysis.
2.5. Vascular endothelial growth factor and adiponectin assay Collected samples were gradually equilibrated to room tem-perature before beginning the assay and diluted up to 500
m
L with the sample diluent provided by the manufacturer and the dilution factor calculated for each sample. The VEGF content wasdeter-mined in 50
m
L of diluted sample with a human VEGF ELISA kit(EHVEGF, Pierce Biotechnology, Rockford, Illinois, USA) according to the manufacturer’s instruction. VEGF concentration in the AH was assessed against an x-point standard curve, extending from 3.750 to 500 pg/mL. The minimum detectable level was 3.5 pg/mL. Values inferior to 3.5 pg/mL were considered equal to 1 for statistical analysis. VEGF was measured in triplicate.
APN concentrations were measured with an ELISA using a polyclonal antibody produced in-house vs a human APN amino
acid fragment (H2N-ETTTQGPGVLLPLPKG-COOH) as previously
described in detail (Daniele et al., 2008). APN was measured in triplicate.
2.6. Statistical analysis
According to sample size calculation a sample size of 20 patient-control pairs had 80% power at the 5% significance level to detect a 10% difference in selected parameters between control subjects and patients. Twenty eyes of 20 patients (8 male and 12 female) with bilateral PDR and 20 eyes of 20 patients (12 male and 8 female) undergoing cataract surgery were included in this study.
All statistical analyzes were carried out using SPSS 12.0 for Windows (SPSS, Inc, Chicago, Illinois, USA). The aqueous levels of VEGF and APN were expressed as a mean with standard deviation (SD). To analyze the statistical differences, the Wilcoxon signed rank test was used between pre-injection and post-injection 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
clinical data, and the ManneWhitney U test was used both between control and PDR groups. The relationship among clinical and lab-oratory variables was analyzed using nonparametric methods (Spearman
r
correlations). A p value< 0.05 was judged as statis-tically significant.3. Results
The demographics and clinical characteristics of patients with PDR and controls at study entry are summarized inTable 1. 3.1. Changes in APN and VEGF level
At baseline the mean SD aqueous concentration of APN was
3.6 1.1 ng/ml in controls and 18.7 4.5 ng/ml in PDR patients
(p < 0.005, ManneWhitney U test), whereas VEGF levels were
22.6 16.1 pg/ml in controls and 146.2 38.7 pg/ml in PDR
pa-tients (p< 0.005 ManneWhitney U test). After IVB injection, the
mean SD aqueous concentrations of both APN and VEGF
signifi-cantly decreased (p< 0.001 and p < 0.01, respectively, Wilcoxon signed rank test) (Fig. 1).
3.2. Changes in FT
Values (mean SD) of FT at baseline were significantly different in controls and in patients (215.6 34.8
m
m and 532.7 112.4m
m, respectively; p< 0.005, ManneWhitney U test) (Table 1).Intra-vitreal bevacizumab induced a significant decrease of FT to
258.7 103.6
m
m (p< 0.001; Wilcoxon signed rank test). No dif-ferences in FT between controls and PDR patients were recorded after IVB (ManneWhitney U test) (Fig. 1).3.3. Changes in BCVA
Mean BCVA (letters SD) at baseline was 23.6 4.2 in controls and 18.4 7.3 in PDR patients (p < 0.01; ManneWhitney U test) (Table 1). One month later, a significant improvement secondary to IVB was recorded (22.1 2.7 letters; p < 0.01; Wilcoxon signed rank test). IVB makes the differences in BCVA between controls and patients not significant (ManneWhitney U test) (Fig. 1).
3.4. Correlation analysis (Spearman’s rho)
In controls no correlation among the considered variables was
recorded. In PDR patients a significant correlation was always
documented both before and after IVB. The correlation was positive
between APN and BCVA (rho¼ 0.41, p < 0.05; and rho ¼ 0.39,
p< 0.01) and VEGF and FT (rho ¼ 0.49, p < 0.01; and rho ¼ 0.40, p< 0.01). Conversely, APN and VEGF (rho ¼ 0.43, p < 0.01; and
rho¼ 0.38, p ¼ 0.05), APN and FT (rho ¼ 0.46, p < 0.01; and rho¼ 0.40, p < 0.01), VEGF and BCVA (rho ¼ 0.39, p < 0.05 in both
cases) and BCVA and FT (rho ¼ 0.44, p < 0.01; and rho ¼ 0.41,
p< 0.01) were negatively correlated. These data are displayed in
Table 2. Moreover, following IVB, no correlation was found either between the percentage of decrease and the baseline levels of both VEGF and adiponectin or functional parameters.
Table 1
Demographics and clinical characteristics of diabetic patients and controls at study entry.
Characteristics Controls Diabetic patients p
20 20
Gender
Male 12 8 e
Female 8 12 e
Mean age SD (years) 64.2 4.7 63.3 6.5 n.s.
Median age 64 63 n.s.
Type of diabetes None Type 2 e Mean BCVA (letters SD) 23.6 4.2 18.4 7.3 0.01 Foveal thicknessmm (mean SD) 215.6 34.8 532.7 112.4 0.005 IOP mmHg (mean SD) 15.7 1.9 14.6 2.3 n.s. n.s.¼ not significant; ManneWhitney U test.
Fig. 1. Legend: Biological (Adiponectin and VEGF) and functional (BCVA and FT) parameters before and after IVP in diabetic patients. *p< 0.01; **p ¼ 0.005; PDR pa-tients versus controls (ManneWhitney U test)p< 0.01;p< 0.001; PDR patients
(Wilcoxon signed rank test).
Table 2
Correlation (Spearman rho) between laboratory and clinical variables in controls and diabetic patients.
Controls Diabetic patients before IVB
Diabetic patients after IVB
rho p rho p rho p
APNeVEGF 0.11 n.s. 0.43 < 0.01 0.38 0.05 APNeBCVA 0.15 n.s. 0.41 < 0.05 0.39 < 0.01 APNeFT 0.09 n.s. 0.46 < 0.01 0.40 < 0.01 VEGFeBCVA 0.16 n.s. 0.39 < 0.05 0.39 < 0.05 VEGFeFT 0.08 n.s. 0.49 < 0.01 0.40 < 0.01 BCVAeFT 0.12 n.s. 0.44 < 0.01 0.41 < 0.01 APN¼ adiponectin; VEGF ¼ vascular endothelial growth factor; BCVA ¼ best-corrected visual acuity; FT¼ foveal thickness; PDR ¼ proliferative diabetic reti-nopathy; n.s.¼ not significant.
241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370
3.5. Safety assessment
Safety was assessed by the incidences of ocular and non-ocular adverse events and serious adverse events, including those potentially related to VEGF inhibition. During the study, neither severe decrease of vision immediately after the injection nor sys-temic adverse events were reported. Ocular and non-ocular adverse events are summarized inTable 3.
4. Discussion
Intravitreal anti-VEGFs represent a promising treatment for PDR, minimizing the risk for exudative complications, progression of retinal neovascularization, vitreous hemorrhage, and decreased
vision caused by macular edema (Salam et al., 2011). Increased
levels of VEGF were detected in ocularfluids from eyes with PDR.
This, together with the efficacy of treatment with the anti-VEGF
agents, indicates that VEGF contributes to the pathogenesis of PDR and reflects successful translational research (Aiello et al., 1994;Arevalo et al., 2009;Cheung et al., 2012;Han et al., 2012). In the current study, IVB significantly reduced macular edema, but the aqueous humor levels of VEGF still remained about three times higher than those recorded in control subjects (Fig. 1). It is possible that bevacizumab also modulates mediators other than VEGF involved in the pathogenesis of macular edema.
Adiponectin is an adipocyte-derived secretory protein that im-proves systemic glucose tolerance and protects the vasculature from atherosclerosis. Circulating levels of adiponectin decrease both in obesity and in patients with type 2 DM. Additionally, hypo-adiponectinemia is an independent risk factor for developing type 2 DM and cardiovascular disease (Misu et al., 2012). The role of this molecule has been highlighted in the pathogenesis of obesity-related illnesses, including type 2 DM, because it plays an impor-tant role in the regulation of insulin sensitivity, as well as in vascular endothelial function (Kougias et al., 2005). Serum adipo-nectin levels were found to be positively correlated with the severity of retinopathy in type 2 DM; high adiponectin level in serum is thought to be a response to endothelium damage (Goldberg, 2009;Kato et al., 2008;Mao et al., 2012).
In our PDR patients APN levels in aqueous humor were signi
fi-cantly higher than those recorded both in control subjects and after IVB. Thesefindings agree with those recently reported byMao et al. (2012), who have found that the averaged APN concentration in
aqueous humor from PDR patients was significantly higher than
that found in control subjects. A possible explanation of thisfinding may be due to the increased blood retinal barrier permeability
documented in PDR patients (Cunha-Vaz et al., 1975). Another
possible explanation could be due to a local reparative response to endothelial dysfunction; in fact, APN induces endothelial nitric oxide production in vitro (Chen et al., 2003). Moreover, in animal
models, APN deficiency is associated with increased inflammatory
responses under conditions of stresses (Shibata et al., 2005), whereas in humans, circulating APN levels well correlate with blood inflammatory marker levels, being highest in the presence of chronic inflammatory diseases. This effect is mediated by a down regulation of a TNF-
a
, whose levels are chronically increased in Type 2 DM (Higuchi et al., 2009;Woo et al., 2012). In the current study, IVB significantly reduces APN levels. This finding could be due to the effect of VEGF inhibition on adipocytes differentiation. In fact, in vivo the inhibition of VEGFR2 affects adipocytes differenti-ation, with a consequent decrease of adipokines secretion. Thus, it is not surprising that the bevacizumab treatment, through a VEGF inhibition, also reduces the levels of adiponectin (Fukumura et al.,2003). It is possible that the elevated levels of both VEGF and
APN recorded in the aqueous humor were secondary to iris vessels leakage, therefore independent from vitreous levels. This discrep-ancy between vitreous and aqueous levels might also justify why the morphologic improvement did not correspond to a normali-zation of biochemical parameters. Although, none of the studied patient showed clinical evidence of iris neovascularization, further studies comparing simultaneously vitreous and aqueous levels of VEGF and APN in patients with and without iris neovascularization are needed to clarify the influence of iris leakage on the concen-tration of these compounds in the aqueous humor.
The efficacy of treatment with the anti-VEGF agents indicates
that VEGF contributes to the pathogenesis of PDR. However, the mechanism that underlies the increase of VEGF and APN produc-tion is uncertain. Since the inhibiproduc-tion of VEGF is not associated with total regression of retinal neovascularization secondary to PDR (Avery et al., 2006), it is important to realize that other factors might play a role in retinal neovascularization processes in PDR patients. Insulin-like growth factor, angiopoietins, stromal derived factor-1, basicfibroblast growth factor-2, hepatocyte growth factor, tumor necrosis factor, interleukin-6, erythropoietin (EPO) and pigmented epithelium-derived factor (PEDF) are those identified as novel factors in the DR pathogenesis. Watanabe and associates (Watanabe et al., 2005) reported that EPO is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal angiogenesis in PDR. It is up-regulated by ischemia, advanced glycation end products, and insulin-like growth factor, and it seems to be more closely associated with PDR than VEGF (Holekamp et al., 2002). PEDF, a member of the serine protease inhibitor family, has been recently shown to be a highly effective inhibitor of angiogenesis in animal and cell culture models. Pro-duction of PEDF is decreased by hypoxia, and hyperglycemia
may influence the expression of PEDF in the eye. VEGF and PEDF
act as pro-angiogenic and anti-angiogenic factors, respectively (Holekamp et al., 2002; Mohan et al., 2012). When the balance between angiogenic and anti-angiogenic factors is lopsided the progression of PDR easily occurs. The mechanism by which the diabetic risk factor initiates the vascular disruption in retinopathy remains unclear, and several pathways involving many factors beyond VEGF have been implicated. VEGF is the factor that has been extensively studied in the pathogenesis of DR, and the pharmaco-logic approach is predominantly targeted versus the VEGF mole-cule, due to the few number of ocular and systemic adverse effects (Table 3). Furthermore, the anti-VEGF therapies appear to be of transient benefit in PDR, as the edema recurs within a few weeks, and repeated injections are necessary (Rangasamy et al., 2012;
Rinaldi et al., 2012). Moreover, as demonstrated in a recent study performed in macaque, the half-life of the intravitreally injected
bevacizumab was shorter in vitrectomized eyes (Kakinoki et al.,
2012). Therefore, in PDR vitrectomized patients the number of
IVB should be proportionally increased, making this approach not eligible. The VEGF inhibition itself may not achieve neutralization
of other inflammatory molecules involved in the cascade of the
Table 3
Incidence of ocular and non-ocular adverse events.
No. of patients, n (%) Ocular adverse event
Eye pain 10 (50.0)
Ocular hyperemia 15 (75.0) Conjunctival hemorrhage 3 (15.0)
Retinal hemorrhage 1 (5)
Increased intraocular pressure 1 (5) Reduced visual acuity 1 (5) Non-ocular AEs Hypertension 1 (5) Nausea/Vomiting 1 (5) 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500
breakdown of blood retinal barrier. Thus, the future approach to DR must be multifactorial, i.e. acting toward molecules like APN, EPO and TNF-
a
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