Molecular Vision 2017; 23:185-197 <>
Received 01 June 2016 | Accepted 29 March 2017 | Published 31 March 2017

Serum retinol-binding protein-induced endothelial inflammation is mediated through the activation of toll-like receptor 4

Mei Du,1,4 Ashley Martin,1,4 Franklin Hays,2,4 Jennifer Johnson,2 Rafal A. Farjo,3 Krysten M. Farjo1,4

1Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Department of Biochemistry, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 3EyeCRO LLC, Oklahoma City, OK; 4Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Correspondence to: Krysten M. Farjo, University of Oklahoma Health Sciences Center, 940 Stanton L Young Blvd, BMSB 617, Oklahoma City, OK 73104; Phone: (405) 271-8001 ext. 48446; FAX: (405) 271-3181; email:


Purpose: Elevation of serum retinol-binding protein 4 (RBP4) induces inflammation in primary human retinal microvascular endothelial cells (HRECs) via a retinol-independent mechanism; thus, it may play a causative role in the development and progression of vascular lesions in diabetic retinopathy (DR). Since HRECs do not express the classical RBP4 receptor, stimulated by retinoic acid gene 6 (STRA6), this study focuses on identifying the endothelial cell receptor and signaling that mediate RBP4-induced inflammation.

Methods: HRECs were treated with a toll-like receptor 4 (TLR4) small molecule inhibitor (Cli95, also known as TAK-242), TLR4 neutralizing antibody, or mitogen-activated protein kinase (MAPK) inhibitors before treatment with purified recombinant RBP4. The HREC inflammatory response was quantified by in vitro leukostasis assays, western blotting, and enzyme-linked immunosorbent assay (ELISA). To understand how the serum binding partner for RBP4, transthyretin (TTR), may affect RBP4 activity, we also measured RBP4 and TTR levels in serum and retinal lysates from RBP4-Tg and wild-type mice.

Results: TLR4 inhibition significantly reduced RBP4-induced expression of pro-inflammatory proteins and in vitro leukostasis. RBP4 treatment significantly increased phosphoactivation of p38 and c-Jun N-terminal protein kinase (JNK). The p38 inhibitor (SB203580) attenuated RBP4-stimulated vascular cell adhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1), monocyte chemoattractant protein (MCP-1), and interleukin 6 (IL-6) production, while the JNK inhibitor (SP600125) reduced RBP4-stimulated sICAM-1, endothelial cell selectin (E-selectin), and MCP-1 production. The MAPK inhibitors only showed partial (50–70%) suppression of the RBP4-stimulated proinflammatory response. Moreover, TLR4 inhibition did not decrease RBP4-induced MAPK phosphoactivation, suggesting that RBP4-mediated MAPK activation is TLR4 independent and occurs through a secondary unknown receptor. We also found that the RBP4/TTR molar ratio was exceptionally high in the retina of RBP4-Tg mice, indicating an abundance of TTR-free RBP4.

Conclusions: RBP4-induced inflammation is largely mediated by TLR4, and in part, through JNK and p38 MAPK signaling. The high TTR/RBP4 molar ratio in serum likely protects the endothelium from the proinflammatory effects of RBP4 in vivo, whereas elevation of serum RBP4 causes a significant increase in TTR-free RBP4 in retinal tissue. This offers insight into how RBP4-Tg mice can develop retinal neurodegeneration without coincident retinal microvascular pathology.


Retinol-binding protein 4 (RBP4) is a novel adipokine (adipose-derived cytokine) that is clinically associated with obesity, insulin resistance, type 2 diabetes (T2DM), and cardiovascular disease [1-11]. In addition, patients with proliferative diabetic retinopathy (DR) have increased serum RBP4 levels compared to diabetic patients with mild or no retinopathy [12,13], which raises the possibility that RBP4 is somehow involved in the pathogenesis of DR. We demonstrated previously that RBP4 elevation induces inflammation in primary human retinal microvascular endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) by increasing the expression of proinflammatory cytokines, chemokines, and adhesion molecules, including interleukin 6 (IL-6), monocyte chemoattractant protein (MCP-1), endothelial cell selectin (E-selectin), vascular cell adhesion molecule 1 (VCAM-1), and intracellular adhesion molecule 1 (ICAM-1) [14]. We have also shown that RBP4-induced endothelial inflammation is retinol independent and involves activation of nuclear factor κB (NF-κB) [14]. In the present study, we used HRECs as a model system to learn more about the cell receptor and signaling pathways that modulate the proinflammatory activity of RBP4.

The upstream mechanisms of RBP4-induced endothelial (HREC) inflammation are unclear. We have shown that the primary RBP4 receptor, stimulated by retinoic acid gene 6 (STRA6), is not expressed in HRECs or HUVECs [14]. Therefore, RBP4 must activate an alternative receptor signaling pathway to induce endothelial inflammation. Others have shown that RBP4 impairs insulin signaling in adipocytes indirectly by inducing proinflammatory cytokines in macrophages through retinol-independent, toll-like receptor 4 (TLR4)- and c-Jun N-terminal protein kinase (JNK)-dependent signaling pathways [15]. Moreover, in mice overexpressing RBP4 (Gene ID: 5950, OMIM 180250; RBP4-Tg), RBP4 triggers adipose tissue inflammation and insulin resistance via the activation of antigen-presenting cells in adipose tissue through a JNK-dependent pathway that does not involve STRA6 [16]. Thus, the present study investigated whether TLR4 mediates RBP4-induced endothelial inflammation by using a selective TLR4 antagonist small molecule inhibitor (Cli95, also known as TAK-242), TLR4 neutralizing antibody, and specific inhibitors targeting the p38, JNK, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) to evaluate the effect of blocking TLR4 and its downstream signaling on RBP4-induced endothelial inflammation.


Purified RBP4

Recombinant human histidine (His)-tagged RBP4 was expressed in Escherichia coli and purified as described previously [14], with some modifications to improve protein yield and quality. The cDNA encoding human RBP4 was subcloned into a pBAD-His expression vector (Invitrogen, ThermoFisher Scientific, Waltham, MA) to ensure tight induction control of RBP4 expression. E. coli strain BL21-A1 cells, transformed with the pBAD-His-RBP4 expression vector, were induced by addition of 0.2% (w/v) arabinose in working volumes of 2–4 l and grown overnight. His-tagged RBP4 was purified as described previously [14]. Briefly, cells were harvested by centrifugation at 4,000 ×g. Cell pellets were resuspended in lysis buffer (50 mM Tris, pH 7.5, 2 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 0.1% Triton X-100). The cell suspension was sonicated 5 times for 20 seconds, freeze-thawed twice and centrifuged at 10,000 ×g for 25 min to pellet the insoluble fraction. The insoluble pellet fraction was solubilized in 5 M guanidine hydrochloride containing 10 mM dithiothreitol with vigorous stirring at room temperature (RT) for 6 h and then batch bound overnight at 4 °C to nickel resin. Following purification of His-RBP4 by nickel affinity chromatography, the protein underwent oxidative refolding, as previously described [14]. For purification of retinol-bound (holo) RBP4 (holo-RBP4), an approximately 10-fold molar excess of retinol was included in the refolding buffer, and all steps after the addition of retinol were performed in a dark room lit by dim red light. After refolding, the protein was exchanged through dialysis into PBS (1.8 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl) and purified by size exclusion chromatography (SEC) to select fractions of properly folded His-RBP4, which constituted approximately 80% of the nickel-purified protein. Samples were concentrated before SEC using a 50-ml stirred cell (Amcion catalog no. 5122, Millipore; Billerica, MA) with an Ultracel 10 kDa Ultrafiltration Disk (Millipore catalog no. 13622, Millipore), to a final volume of approximately 10 ml under 30 psi N2. All SEC was performed on a Hitachi Chromaster system using a SuperDex 200 10/300GL column (GE Healthcare catalog no. 17517501, GE Healthcare Life Sciences, Marlborough, MA) and following ultraviolet (UV; 260 nm, 280 nm, and 330 nm) and refractive index signals. Retinol-free (apo) His-tagged RBP4 (His-apo-RBP4) was used for the studies illustrated in Figure 1, Figure 2, and Figure 3 to ensure that all RBP4 activity was retinoid independent, although similar results were obtained with retinol-bound His-holo-RBP4 protein. As in our previous study, we tested the level of endotoxin contamination in purified RBP4 protein and found that it was below the level of endotoxin present in the PBS solvent used for dissolving RBP4 and negative control protein bovine serum albumin (BSA).

HREC culture

Primary HRECs were obtained from the Cell Systems Corporation (Kirkland, WA) and cultured in EGM-MV (Promocell, Germany) at 37 °C in 5% CO2 and 95% air. The cells were grown and maintained on collagen I–coated tissue culture plates from Corning Life Sciences (Kennebunk, ME). For the experiments, HREC confluent monolayers were incubated in endothelial cell basal media with 2% (v/v) fetal bovine serum (FBS) for 8–16 h to induce quiescence. All experiments were conducted using HRECs at passages 4 to 11, as we confirmed that cells maintained similar viability up to passage 11 (data not shown). TLR4 receptor inhibitor and MAPK inhibitor studies were conducted by preincubating cells with the inhibitors for 2 h before the addition of RBP4 at 100 µg/ml. TLR4 chemical inhibitor Cli95 (aka TAK-242), TLR4 neutralizing antibody TLR4 neutralizing monoclonal antibody (mAb), and control immunoglobulin G (IgG) were purchased from InvivoGen (San Diego, CA). The MAPK inhibitors SP600125, SB203580, and U0126 were purchased from Cell Signaling Technology Inc. (Danvers, MA).

Culture of THP-1 cells and in vitro leukostasis assay

The human monocytic cell line (THP-1) was obtained from the American Type Culture Collection (Manassas, VA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% (v/v) FBS and 0.05 mM β-mercaptoethanol. Leukocyte adhesion assay was performed as previously described [14]. Briefly, HRECs were grown to confluence in six-well plates and treated in serum-starved 2% (v/v) FBS medium with BSA or RBP4 for 20 h with or without a 2 h preincubation with TLR4 inhibitors. THP-1 monocytes were seeded at 1.25 million cells/well and co-cultured with HRECs for 3 h. Unbound THP-1 monocytes were thoroughly removed by gently washing 3X with PBS, and cells were fixed in 4% (v/v) paraformaldehyde for 10 min. Adherent monocytes were photographed using phase-contrast microscopy at a magnification of 20×, and they were counted/scored in at least four random fields for each treatment.


HREC culture media were collected after the indicated treatments, centrifuged at 10,000 ×g for 2 min to remove cell debris, and stored at −80 °C. Expression of proinflammatory molecules in cell culture supernatant was determined by sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. ELISA kits from R&D Systems, Inc. (Minneapolis, MN) were used to measure soluble extracellular levels of VCAM-1 (catalog no. DY809), ICAM-1 (catalog no. DY720), MCP-1 (catalog no. DY279), IL-6 (catalog no. DY206), and E-selectin (catalog no. DY724).

Immunoprecipitation and western blot analysis

Cell extracts were prepared by homogenization in lysis buffer containing 50 mM Tris, pH 7.4, 40 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM Na4P2O7, 10 mM sodium β-glycerol phosphate, 0.5% (v/v) Triton X‐100, and 100× phosphatase and protease inhibitor cocktail, as described previously [17]. Cell debris was removed by centrifugation, the supernatant was collected, and protein concentration was determined by Bradford assay. A total of 1 mg of protein was incubated with the MyD88 antibody (catalog no. 4283, Cell Signaling Technology; Danvers, MA). Immunoprecipitates were washed 5X with lysis buffer, boiled in Laemmli buffer (100 mM Tris, pH 6.8 RT, 200 mM dithiothreitol, 4% (w/v) sodium dodecyl sulfate [SDS], 0.2% (w/v) bromophenol blue, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 20% (v/v) glycerol) for 5 min, and analyzed by western blotting.

For western blot analysis, cells or tissues were lysed in Laemmli buffer, resolved by SDS–polyacrylamide gel electrophoresis (PAGE), and subjected to immunoblotting analysis with the indicated antibodies. The following antibodies were used: from from Santa Cruz Biotechnology (Dallas, TX), TLR4 (catalog no. sc-293072), β-actin (catalog no. sc-47778), and VCAM-1 (catalog no. SC-8304); from Cell Signaling Technology, JNK (catalog no. 9258), (P) JNK (phospho-Thr183/Tyr185; catalog no. 4668), p38 (catalog no. 8690), (P) p38 (phosphor-Thr180/Tyr182; catalog no. 4511), ERK, (catalog no. 4695), and (P) ERK (phosphor-Thr202/Tyr204; catalog no. 4370); and from Novus Biologicals (Littleton, CO), GAPDH (catalog no. NB300–221). Band intensities were normalized to β-actin or GAPDH and quantified using UVP analyzing software (BioSpectrum® Imaging System, UVP, Upland, CA).


RBP4-Tg mice, which overexpress human RBP4 under control of the mouse muscle creatine kinase (MCK) promoter on a C57BL/6J background, have been described previously [18], and the animals were kindly provided by Loredana Quadro. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center (OUHSC) and adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were fed a standard chow diet (Labdiet 5001) ad libitum. This diet contained a sufficient amount of vitamin A (15 IU g−1). All studies were performed on age-, sex-, and strain-matched wild-type controls. Both sexes were used in all studies, and no sex-dependent differences in phenotype were observed.

Mouse serum was collected by superficial temporal vein puncture from live mice that had fasted for 6 h. Retinal tissue was dissected after mice were euthanized by CO2 asphyxiation and quickly perfused with PBS at a flow rate of 8 ml/min for 3 min to remove blood cells. Retinas were rapidly collected using Winkler’s method, snap frozen, and stored at −80 °C. The retinas were suspended in lysis buffer (50 mM Tris-HCl, pH 7.8, 0.1 M NaCl, 5 mM EDTA, 0.1% [w/v] SDS, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.1% (w/v) Triton X-100, 2.5% [v/v] glycerol) and sonicated on ice at a 20% intensity for two rounds of 5 s each. Insoluble material was removed by centrifugation, and the protein concentration was determined by Bradford colorimetric assay. ELISA kits for mouse TTR (catalog no IRKTAH1161; Innovative Research, Novi, MI), mouse RBP4 (catalog no MRB400; R&D Systems, Minneapolis, MN), and human RBP4 (catalog no DRB400; R&D Systems) were used to quantify target molecules in serum and retina lysates. RBP4 and TTR levels in retina lysates were normalized according to the total retinal protein content in each sample.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using Prism 4 software (La Jolla, CA) with one-way analysis of variance (ANOVA) using Tukey’s post hoc analysis to compare differences among three or more groups. A value of p<0.05 was accepted as significant.


RBP4-induced endothelial inflammation is TLR4-dependent

We first examined TLR4 protein expression in HRECs by western blot. We observed abundant expression of TLR4 in HRECs, and RBP4 treatment did not change the expression level of TLR4 protein (Figure 1A). Next, we sought to determine whether RBP4 activates TLR4 signaling in HRECs. TLR4 is known to signal via binding interaction with an adaptor protein, MyD88 [19,20]. Therefore, we performed coimmunoprecipitation (co-IP) to assess whether RBP4 treatment promotes MyD88 binding to TLR4. We observed increased interaction between TLR4 and MyD88 in HREC lysate at 30 min after RBP4 treatment (Figure 1B), suggesting that RBP4 activates MyD88-dependent TLR4 signaling in HRECs. To further address whether TLR4 is the membrane receptor that mediates RBP4-induced inflammation in HRECs, we applied a specific chemical inhibitor of TLR4, Cli95, which is a small molecule that binds to the intracellular domain of TLR4 at cysteine-747 and inhibits TLR4 adaptor protein–protein interaction and signaling [21,22]. Cli95 significantly decreased RBP4-mediated induction of cellular VCAM-1 protein (Figure 1C,D) and reduced extracellular soluble protein levels of VCAM-1, ICAM-1, E-selectin, MCP-1, and IL-6 (Figure 1E-I). Similarly, pretreatment with TLR4 neutralizing antibody, but not control IgG, significantly blocked RBP4-mediated induction of proinflammatory factors (Figure 2A-E).

We previously showed by in vitro leukostasis assays that RBP4-induced expression of proinflammatory genes leads to increased monocyte adherence on HREC monolayers [14]. Herein, we performed in vitro leukostasis assays to quantify the net effect of TLR4 inhibition on RBP4-induced inflammation. Consistent with our previous findings, HRECs treated with RBP4 (100 µg/ml), or lipopolysaccharide (LPS) as a positive control, showed significantly increased leukostasis compared to negative controls (Figure 1J,K; p<0.001; n=4). RBP4-induced leukostasis was significantly inhibited in HRECs pretreated with TLR4 chemical inhibitor (Figure 1J,K) or TLR4 neutralizing antibody (Figure 2F,G), demonstrating that RBP4-induced HREC inflammation is largely TLR4-dependent.

RBP4 induces proinflammatory molecules through the activation of JNK and P38 signaling

Previous studies have shown that stimulation of TLR4 can initiate phosphorylation of three MAPKs, namely JNK, p38, and ERK, which leads to subsequent cytokine secretion [23]. Other studies have revealed that RBP4 causes insulin resistance in adipocytes by inducing proinflammatory cytokines in macrophages through a JNK- and TLR4-dependent mechanism [15]. To determine whether RBP4 activates the MAPK signaling pathway to promote inflammation in HRECs, we measured the phosphoactivation of MAPKs by western blot. Within 30–120 min of RBP4 treatment, HRECs exhibited significantly increased phosphoactivation of p38 (Figure 3A,D) and JNK (Figure 3B,E). Phosphorylated ERK1/2 showed a trend toward increased expression at 10–30 min, but this did not reach statistical significance (Figure 3C,F).

To further investigate the role of MAPK signaling pathways in RBP4-mediated cytokine production, we employed specific inhibitors of p38, JNK, and ERK. The p38 inhibitor (SB203580) attenuated RBP4-stimulated sVCAM-1, sICAM-1, MCP-1, and IL-6 production, while the JNK inhibitor (SP600125) reduced RBP4-stimulated sICAM-1, E-selectin, and MCP-1 production (Figure 3G-K). The ERK inhibitor (U0126) only showed inhibitory effect on sICAM-1 production (Figure 3H). The MAPK inhibitors only showed partial (50–70%) suppression of the RBP4-stimulated proinflammatory response (Figure 3G-K). Moreover, TLR4 inhibition does not decrease RBP4-induced MAPK phosphoactivation (Figure 3L), suggesting that RBP4-mediated MAPK activation is TLR4 independent and occurs through a secondary unknown receptor. Taken together, these results suggest that RBP4-induced endothelial inflammation is mediated in part through the activation of the JNK and p38 pathways.

The RBP4 level exceeds the binding capacity of transthyretin in neuronal retinal tissue

Our previous study found no retinal microvascular pathology in RBP4-Tg mice aged up to 6 months [24], which is perplexing, as the data herein and in our previous study [14] clearly showed that RBP4 can induce endothelial inflammation in cultured HRECs. In plasma, RBP4 (95%) is mostly found in a complex with its carrier transthyretin (TTR), which increases its molecular mass, thereby preventing its removal by glomerular filtration [25]. Previous studies reported that association with TTR can stabilize RBP4 and block receptor binding and subsequent activation of cell signaling [26,27]. TTR is normally present in three- to fivefold molar excess to RBP4 in human [26,28] and rodent serum [24]. We speculated that the high molar ratio of TTR/RBP4 in serum, which is maintained even in RBP4-Tg mice (Figure 4B), facilitates TTR-mediated inhibition of RBP4 pro-inflammatory activity to protect endothelium, including the retinal microvasculature, in vivo.

Intriguingly, our previous study found that RBP4-Tg mice have early-onset progressive retinal neurodegeneration [24]. We have previously shown that RBP4-Tg mice have a ~7-fold increase in retinal RBP4 level relative to wild-type mice [24]. Thus, to address whether TTR-mediated inhibition of RBP4 proinflammatory activity is possible in the neuronal retina, we measured the level of TTR present in perfused retinal neuronal tissue lysates from wild-type and RBP4-Tg mice. The TTR levels in RBP4-Tg retinas were similar to those in wild-type mice (Figure 4A), meaning that retinal tissue has an extremely high RBP4/TTR molar ratio (about 70) in RBP4-Tg mice (Figure 4B). This suggests that the increased levels of RBP4 present in the retina of RBP4-Tg mice may promote retinal neurodegeneration due to the lack of TTR-mediated inhibition of RBP4.


Recent studies revealed that RBP4 is a proinflammatory adipokine associated with obesity [1,2], insulin resistance [1-6], cardiovascular disease [7-11], and T2DM and its microvascular complications [2,6,13,29]. While a proinflammatory effect of RBP4 has been implicated in these diseases, the underlying cellular mechanisms are not understood. TLR4 and other receptors involved in the innate immune response recognize damage-associated molecule patterns (DAMPs) from endogenous proteins that are elevated due to stress, inflammation, and cell death. Recent studies have shown that the retinol-independent proinflammatory activity of RBP4 in adipose tissue is mediated through TLR4 activation [15,16]. The data herein showed that TLR4 inhibition significantly blocked RBP4-induced expression of proinflammatory genes, including VCAM-1, ICAM-1, IL-6, MCP-1, and E-selectin, in HRECs. We also observed phosphoactivation of MAPK signaling in response to RBP4 treatment, and MAPK inhibitors significantly reduced RBP4-mediated expression of proinflammatory genes. Thus, this is the first study to show that RBP4-induced inflammation in HRECs is largely mediated by TLR4, and in part, through JNK and p38 MAPK signaling.

RBP4 induces phosphoactivation of p38 and JNK, and inhibitors of p38 and JNK activation can partially inhibit RBP4-induced inflammation in HRECs. However, p38 and JNK inhibitors were not as effective in reducing RBP4-induced production of proinflammatory molecules as TLR4 inhibitors were. Moreover, inhibition of TLR4 did not block RBP4-mediated phosphoactivation of p38 and JNK (Figure 3L). These data indicate that p38 and JNK phosphoactivation is mediated through an alternative receptor to TLR4, and that this signaling pathway acts secondarily to the TLR4-dependent pathway to promote inflammation in HRECs. The receptor for advanced glycation end-products (RAGE) is a likely candidate for this alternative RBP4 receptor in HRECs. RAGE is expressed in endothelial cells [30] and activates p38 and JNK signaling to induce inflammation [31-33]. Moreover, RAGE is known to interact and crosstalk with TLR4 (reviewed in [34]). Therefore, it may be that maximal RBP4 proinflammatory action is mediated by cooperative binding and activation of the RAGE/TLR4 complex. Indeed, this could explain why the chemical TLR4 inhibitor Cli95 was more effective at blocking RBP4-induced inflammation (Figure 1C-K) compared to the TLR4 neutralizing antibody (Figure 2), since Cli95 blocks cellular signaling activation [21,22], whereas the antibody can only block ligand-mediated activation of TLR4, but ligand activation of a TLR4/RAGE complex through binding to RAGE may still be intact.

A substantial body of research has shown that TLR4 is expressed in a variety of ocular tissues and plays an important role in protection against microbial infection [35-38]. In the retina, TLR4 is primarily expressed in microglial cells, but it can also be detected in photoreceptors and Müller cells [39-41]. TLR4 activation promotes retinal degeneration by increasing neuroinflammation, oxidative stress, and mitochondrial DNA damage [42-44]. Moreover, TLR4 deficiency can reduce retinal degeneration [45]. We have previously shown that RBP4-Tg mice have early-onset progressive retinal degeneration that features neuroinflammation [24]. Thus, RBP4 may be activating TLR4 in the retina to promote neurodegeneration. Most retinal cells also express RAGE, and RAGE is involved in the pathogenesis of DR, including retinal neurodegeneration [46-48]. Thus, future studies in RBP4-Tg mice should investigate whether TLR4 or RAGE contributes to RBP4-induced retinal neurodegeneration.

Interestingly, our previous study found no retinal vascular pathology in RBP4-Tg mice aged up to 6 months, despite significant retinal neurodegeneration [24]. Under normal physiological conditions, most RBP4 (95%) circulates in plasma in a complex with TTR at a 1:1 molar ratio, and only a small fraction can be found in serum as TTR-free RBP4 [26]. In addition, serum TTR levels exceed RBP4 levels, even in RBP4-Tg mice (Figure 4B). Thus, the high TTR/RBP4 molar ratio in serum likely inhibits RBP4 proinflammatory activity in the retinal vasculature. However, we showed that increased serum RBP4 levels in RBP4-Tg mice result in an increased level of TTR-free RBP4 in the neuronal retina (Figure 4B). Therefore, TTR-mediated inhibition of RBP4 activity is largely restricted to the vascular compartment, whereas in peripheral tissues, such as the retina, TTR-free RBP4 is likely more active. This may explain why RBP4-Tg mice have early-onset retinal neurodegeneration but no detectable retinal microvascular abnormalities up to 6 months of age.

It is well documented that retinal neurodegeneration precedes retinal microvascular disease in DR [49-74]; therefore, future studies in aged RBP4-Tg mice should determine whether retinal microvascular pathology occurs subsequent to retinal neurodegeneration during aging, since RBP4-mediated retinal neurodegeneration could act as an initial trigger to promote both neuronal and vascular damage in DR. Moreover, since elevated RBP4 levels have been clinically linked to proliferative DR [12,13], as well as cardiovascular disease [7-9,75], it may be that additional factors, such as hyperglycemia, dyslipidemia, or systemic inflammation, can enhance RBP4 proinflammatory action on the endothelium. Notably, RBP4-Tg mice have normal bodyweight, blood glucose, and triglyceride levels [24]. Thus, RBP4-Tg mice may develop more severe retinal microvascular disease than wild-type mice in response to a high-fat diet or genetically or experimentally induced diabetes, although this requires further investigation. Surprisingly, we found that neither db/db nor streptozotocin (STZ)-induced diabetic mice develop elevated serum RBP4 levels by 6 months of age (unpublished data, not shown). This finding reveals that elevation of RBP4 is not essential for the development of DR, since both db/db and STZ mice develop characteristics of DR before 6 months of age in the absence of RBP4 elevation [76-86]. However, it is unclear how an elevated serum RBP4 level, which is present in a large portion of diabetic patients based on clinical studies [1-6], may affect the progression of DR. Thus, further studies should utilize db/db and STZ mice in conjunction with RBP4-Tg mice to investigate how elevated serum RBP4 affects the progression and severity of DR.


We thank Loredana Quadro for providing RBP4-Tg mice. The research reported in this study was supported by an institutional development award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM104934 (K.M.F. received funding as a promising junior investigator on this grant), an American Heart Association beginning grant-in-aid (13BGIA16920097; principal investigator, K.M.F.), and a bridge grant from the Presbyterian Health Foundation (K.M.F.).


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