Potential Protective Function of Adiponectin in Diabetic Retinopathy

In 1995, a 30-kDa adipocyte complement-associated protein (Acrp30), also known as APN, was identified as a novel adipocytokine that is exclusively produced by adipocytes [21]. Subsequent research revealed APN to be a protein encoded by the ADIPOQ gene (Adipose Most Abundant Gene Transcript 1 [APM1]) [22]. Its N- and C-termini of APN both contain a globular area, and the protein is structurally a member of the complement 1q (C1q) protein family. The collagen structural domain regulates APN multimerization, and disulfide bonds hold the APN oligomers together, which can structures ranging from trimers and hexamers to high-molecular-weight multimers [23‐25]. Nearly all APN present in plasma appear to be full-length APN, but earlier research detected small amounts of globular APN (gAPN) in human plasma [26], possibly resulting from cleavage of APN by leukocyte elastase secreted by activated monocytes and/or neutrophils [27].

Mainly through the actions of AdipoR1 and AdipoR2 on muscle and liver, APN is involved in the regulation of glucose, lipid, and energy metabolism as well as having anti-inflammatory effects [12]. Administration of recombinant APN in mice increases glucose uptake and increases lipid oxidation in muscle, reduces fatty acid uptake and hepatic gluconeogenesis in the liver, enhances insulin sensitivity [26, 28] and glucose tolerance [29], improves systemic insulin resistance [30], and may increase free fatty acid oxidation in muscle [26, 31]. This insulin-sensitizing effect of APN appears to be mediated, at least in part, via activation of AMP-activated protein kinase (AMPK) [32‐34]and through peroxisome proliferator-activated receptors (PPARs) [35, 36] to promote fatty acid oxidation [37]. Dysregulation of APN is therefore thought to be related with diseases associated with metabolic X syndrome, such as insulin resistance [38], obesity [39], T2DM [40], and cardiovascular disease [41]. Additionally, AdipoR1 and AdipoR2 are progestin and adipoQ (PAQ) receptors and may work to improve ceramidase activity, lower serum ceramide levels, and increase sphingosine-1-phosphate (S1P) levels, which shield cardiomyocytes and shield pancreatic-cells from apoptosis. Additionally, AdipoR1 and AdipoR2 are PAQ receptors and may work to improve ceramidase activity, lower serum ceramide levels, and increase S1P levels, which protect cardiomyocytes and prevent pancreatic cells from apoptosis [42].

In diabetes patients, APN regulates glucolipid homeostasis and improves insulin resistance. In T2DM, hypoadiponectinemia boosts the levels of plasma glucose by limiting fatty acid oxidation, ramping up free fatty acids, slowing glucose absorption by skeletal muscle cells, and promoting gluconeogenesis in hepatic cells [59]. By encouraging lipid oxidative expansion and benign storage, APN improves the lipid profile. Additionally, it prevents inflammation, fibrosis, and steatosis while maintaining functional β cells to promote glucose homeostasis [60]. Furthermore, by binding to AdipoR1 and AdipoR2, APN increases ceramidase activity, resulting in increased ceramide catabolism and formation of its anti-apoptotic metabolite, S1P, which counteracts apoptosis in pancreatic β cells, improves hepatocyte insulin sensitivity [61], and promotes glycogen synthesis in skeletal muscle in T2DM [62]. Importantly, APN may also improve insulin resistance via the AMPK pathway by improving the catabolic state of the branched-chain amino acids (BCAAs) in the diabetic state (BCAAs are closely associated with insulin resistance) [63].

In hyperglycemic human microvascular retinal endothelial cells (HMRECs), APN was found to dramatically decrease the rate of reactive oxidative stress (ROS) and reactive nitrogen species (RNS) formation, elevate the expression of the antioxidant enzyme superoxide dismutase 2 (SOD2), and improve oxidative stress and apoptosis rates [64]. In one study, the level of oxidative stress-responsive apoptosis-inducing protein ORAIP, which acts as a pro-apoptotic ligand to promote apoptosis and damage the retina, was considerably higher in the vitreous humor of patients with PDR than in controls when exposed to oxidative stress. Oxidative stress downregulates microRNA (miRNA) levels, which encourages vascular endothelial growth factor (VEGF) expression and aids the progression of DR [65]. APN is associated with increased overall antioxidant status and decreased oxidative load cells [66]. Low APN concentration is significantly associated with elevated superoxide anion produced by NADPH oxidase in human artery wall arteries in T2DM [56]. Meanwhile, APN elevates nitric oxide (NO) bioavailability and lowers oxidative stress in the aorta via activating the AMPK/eNOS and cAMP/PKA signaling pathways [67]. By enhancing the PI3K/AKT/mTOR pathway, APN may inhibit rhesus choroid endothelial (RF-6A) cell autophagy [68]. However, more research is required to determine whether the aforementioned routes exist in the retinal microvasculature.

One study identified ceramide as a key factor in TOLL-like receptor 4-mediated insulin antagonism, and the anti-inflammatory effects of APN may be directly related to ceramide depletion [61]. Ceramide intracellular accumulation contributes to the development of diabetic microvascular complications through lipotoxic mechanisms, such as cell death, oxidative stress, and inflammation [69]. T-calmodulin mediates the function of APN in promoting exosome biogenesis and secretion, which can promote ceramide efflux and reduce cytosolic ceramide content in cultured endothelial cells and serve as a preventative measure against vascular injury [70] (Fig. 1).

The concentrations of several intraocular cytokines in the aqueous humor and serum APN are well correlated [71]. APN levels in the brain and aqueous humor are significantly lower than those in the blood, pointing to a possible role for the blood–brain barrier and blood-retinal barrier (BRB) in maintaining APN homeostasis. AdipoR1 actively transports APN into the retina through the BRB, or via BRB catabolism, or it may be stimulated in DR to express endogenous or local APN [11, 47, 72].

APN can stabilize the BRB by inhibiting the increase in inflammatory factors, thereby ultimately delaying the DR process. Inflammation is inextricably linked to the development of DR: individuals with DR have considerably higher levels of inflammatory biomarkers than those without DR in their serum, aqueous humor, vitreous humor, and retina [45, 73‐76]. Diabetic rat models were found to have significantly higher levels of vitreous APN, TNF-α, interferon-gamma (INF-γ), matrix metalloproteinase (MMP)-2, and MMP-9 than the controls [77]. There is evidence that every unit increase in TNF-α level in T2DM increases the risk of retinopathy by 4085-fold [48]. Inflammatory cytokines, such as IFN-γ and TNF-α, increase VEGF-A and -C secretion in retinal pigment epithelium (RPE) cells and choroidal fibroblasts. VEGF-A causes BRB disruption and increases vascular permeability, activates a number of molecules that activate the kallikrein-kinin System (KKS) pathway, produces significant amounts of bradykinin binding to bradykinin receptor 1 (inflammatory response) and bradykinin receptor 2 (retinal vasodilatation) in the retina, promotes aggregation of neutrophils and microglia, and may increase vascular permeability and vascular edema, affecting inflammatory and angiogenic functions [45, 65]. BRB disruption results in cellular hypoxia, increased expression of hypoxia-inducible factor 1 (HIF-1), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and oxygen-regulated protein 150 (ORP150), increased transcriptional translation of VEGF genes, binding of VEGF to ORP150 at the endoplasmic reticulum, secretion of cells to bind to the VEGF receptor (VEGFR) in target cells, and activation of downstream PI3K. Through the FAK pathway, VEGFR also activates paxillin, which causes cytoskeletal reorganization and cell migration [65]. The bioavailability of PDGF, FGF, and epidermal growth factor can all be decreased by the formation of complexes with APN [78]. APN inhibits TNF-α-induced activation of IKB-NF-κB (inhibitor of κB-nuclear factor kappa B) signaling via the cAMP pathway, decreases vascular cell adhesion molecule 1 (VCAM-1), prevents retinal leukocytes from adhering to vessel walls with inflammatory lesions, and inhibits endothelial cell migration and engraftment. These actions prevent BRB breakdown [70] and the development of pathological microvessels in the retina [79]. APN may inhibit myelomonocyte proliferation, phagocytic activity, and macrophage production of inflammatory mediators such as TNF-α [80]. The lack of APN-dependent anti-inflammatory and anti-MMP actions may hasten the degradation of retinal Claudin-5 and significantly enhance vascular permeability [70].

By preventing a high glucose-induced reduction in cell viability, cell migration, and tube formation, APN interferes with retinal angiogenesis [68]. Inhibition of basal tube formation and blood vessel migration by APN expression in human umbilical vein macrovascular endothelial cells (HUVEC) and human retinal microvascular endothelial cells (hREC) is consistent with the action of intravitreal bevacizumab (IVB). APN is able to suppress retinal neovascularization by limiting the migration and proliferation of recombinant VEGF and PDR vitreous-induced hREC, as well as by inhibiting the proliferation and phosphorylation of ERK1/2 in recombinant VEGF-induced hREC [81]. APN upregulates the expression of the AdipoR1 and AdipoR2 genes, downregulates the expression of the TNF-α, interleukin-8 (IL-8), and interleukin-1beta (IL-1β) inflammatory proteins, adhesion molecules Intercellular adhesion molecule-1 (ICAM-1) and E-selectin, improves barrier dysfunction in hyperglycemic cells, inhibits leucocyte and endothelial cell migration, and improves basal tube formation. During hyperglycemia, APN enhances barrier function and decreases retinal endothelial cell permeability, consequently reducing inflammation and promoting angiogenesis. Molecular mechanisms of APN may directly affect inflammatory HMRECs and leukocytes, inhibit ROS, induce apoptosis, suppress NF-κB inflammatory signaling pathways, and downregulate TNF-α-mediated inflammatory responses. APN lessens the hyperglycemic effects of HMRECs by inhibiting endothelial cell adhesion and modulating extracellular matrix organization while improving barrier function (Fig. 2) [64].

APN is thought to be a regulator of vascular remodeling because of its pleiotropic effects on maintaining vascular integrity. Based on research on the human retinal pigment epithelial cell line ARPE-19, which revealed a negative association between APN and VEGF levels [82], APN may play a preventative role in disorders associated with angiogenesis. APN is a crucial modulator of pathological neovascularization and inflammation [83]. Endothelial dysfunction resulting in poor endothelial cell repair and neovascularization are the primary symptoms of DR. Endothelial cell destruction, blood vessel loss, an increase in ROS production, an increase in inflammatory cells and cytokines, leukocyte leakage, and an increase in vascular leakage are all caused by hyperglycemia. Subsequent cellular hypoxia and ischemia will result in compensatory neovascularization.

APN can slow down or even inhibit the development of DR by improving hemodynamics. APN activates AMPK activity-dependent guanylyl cyclase, which in turn activates eNOS, causing NO production by endothelial cells, regulating vascular diameter, altering blood flow, and causing diastole of small porcine retinal arteries in vitro [84]. In men with T2DM and no or mild NPDR, circulating APN levels have been found to be positively linked with retinal blood flow. Retinal blood flow and APN concentrations are raised by increased blood flow velocity, expanded blood tube diameter, and decreased resistance [85]. In the early stages of DR, blood flow is reduced. It is possible that early use of APN may be able to prevent or even suppress the development of DR by increasing blood flow and delaying the onset of hypoxia.

Laser photocoagulation, vitreous injection of anti-VEGF medication, and vitreous surgery are currently used to treat DR. Lasers, however, can harm retinal cells permanently since they are destructive. There are also many shortcomings in the use of anti-VEGF drugs, including long-term resistance, high treatment costs, potential risk of retinal detachment due to intraocular injections, unresponsiveness in some patients, and poor patient compliance [86, 87]. It is urgently necessary to find a safe and effective target with minimal side effects in terms of treating DR and enhancing patient quality of life (Table 1).

APN may become a target therapeutic site for DR in the future. Leukemia inhibitory factor-human umbilical cord mesenchymal stem cells (LIF-hUCMSCs) protect the retina of diabetic rats by upregulating APN and neurotrophin-4 (NT-4) expression and downregulating high-sensitivity C-reactive protein (hs-CRP) expression in the retina [88]. Recombinant adeno-associated virus-adiponectin (rAAV2/1-ACrp30) downregulates NF-κBp65 and IκBα expression to improve impaired glucolipid metabolism, diabetes and its liver disease in diabetic rats, inhibits TNF-α secretion by macrophages, induces IκB degradation, blocks NF-κB activation, and reduces inflammatory responses. Clinical trials for recombinant APN therapy are not yet underway, however APN is a promising therapeutic target for the prevention and treatment of T2DM and its complications [89]. Higher concentrations of APN do not induce cytotoxicity in human endothelial cells and can be safely used as a therapeutic anti-angiogenic target for retinal angiogenesis. APN, which has anti-angiogenic, anti-proliferative, and anti-migratory activities, may be a possible therapeutic target for the therapy of posterior segment angiogenic processes (PDR and choroidal neovascular) in the eye. APN can be used in conjunction with IVB for the treatment of ocular angiogenesis based on the results of in vitro experiments demonstrating that APN and IVB have a comparable ability to inhibit tube formation and that the combination of APN and IVB significantly prevents the formation of VEGF and PDR vitreous-induced tubes in HUVEC [81]. IVB has also been linked to cases of herpetic epithelial keratitis that turned into meta-herpetic ulcers [90]. APN may be utilized in the future to treat patients who have not responded to IVB treatment or even as an alternative to IVB treatment. More research might be done in the meantime to show whether the combination of APN and IVB protects DR more effectively and safely than APN alone and whether the combination could lessen some of the negative effects of IVB. In contrast, other research suggests that the anti-APN antibody may be able to improve retinal edema and ischemia by decreasing the expression of VEGF-related factors and tight junction-related proteins in the retina. Retinal neovascularization was also reduced [86]. These various results could be the outcome of different experimental models, treatments, and observation periods. One study showed that higher levels of physical activity were associated with a lower incidence of glycoplasia [91] and that better behavioral tests and electroretinogram (ERG) results were obtained after exercise in a glucose rat model [92, 93]. Therefore, the authors hypothesized that exercise may have its protective effect on the eye by modulating the expression of APN and AdipoR in the plasma and retinal regions [94], pending further studies.

APN may have varied effects on the retina depending on the stage of DR, providing protection in the early stages and causing fibrosis in the later stages by inhibiting blood vessels. In one study, an increase in APN among various inflammatory molecules associated with retinal fibrosis in patients with proliferative vitreoretinopathy (PVR) and in patients with PDR led the authors to hypothesize that APN may regulate the switch to vascular fibrosis and drive the formation of fibrovascular membranes in the late stages of PVR and PDR, promoting the progression of retinal fibrosis [95]. According to these authors, APN may be a candidate for treating angiogenesis early on in PDR and promoting posterior segment fibrosis at a later stage. Additionally, novel therapeutic modalities may be created to specifically target APN for the treatment of posterior segment fibrosis in vitreoretinal disease [95].

The APN receptor (APNR), certain drugs, and APN homologs may have beneficial effects on DR. APN and its receptors are expressed in the murine neural retina and RPE choroid. Orthox endothelium angiogenic cells are activated by APN-AdipoR1-AMPK to protect and repair endothelial damage. APN-AdipoR2-PPARα inhibits inflammation and oxidative stress-induced insulin resistance [11]. Animals with knockouts of ADIPOR1 display early visual system abnormalities that ultimately result in retinal degeneration, elevated interphotoreceptor retinoid-binding protein (IRBP) levels, and spatial distribution anomalies. As a result, it is possible that APNR1 functions independently of APN and contributes more to visual biology than glucose metabolism [96]. In muscle and liver, AdipoRon had very similar effects to APN. Oral AdipoR agonists are a promising treatment for T2DM, even if only APN shows the most benefits, including reducing insulin resistance and impaired glucose tolerance and lengthening the shorter life expectancy of patients on a high-fat diet [37]. Some medications may protect against DR via the APN pathway. Through APN-mediated regulation of TNF-α, thiazolidinediones (TZDs) reduce pathological retinal microangiogenesis in ischemic retinal disease [97]. The fibroblast growth factor 21 (FGF21)-APN-ceramide axis regulates energy expenditure and insulin action in mice. FGF21 is heavily reliant on APN and can rapidly and potently stimulate APN secretion while reducing ceramide accumulation in obese animals [98], improving the lipid profile and insulin resistance, and preventing kidney damage [99]. However, it has also been suggested that FGF21 prevents early DR independently of downstream APN [100]. FGF21/APN can be used as a biomarker of glycemic deterioration and a stand-alone predictor of prodromal diabetes and diabetes mellitus since its level increases gradually with decreasing glycemic control in T2DM [101]. A long-acting FGF21 analog (PF-05231023) with protective effects on DR in streptozotocin-induced diabetic mice was shown to restore retinal function and photoreceptor cell morphology, inhibit oxidative stress-induced photoreceptor cell inflammation, and reduce retinal inflammation [100]. The closest paralog to APN is an adipocytokine called C1q/TNF-related protein-9 (CTRP9) [102], which plays a beneficial role in glucose metabolism and vascular protection, lowering blood glucose in diabetic mice [103]. CTRP9 inhibits the expression of IL-1β, TNF-α, monocyte chemoattractant protein-1 (MCP-1), and adhesion molecules in the retina of db/db mice, inhibits the activation of NF-κB, balances the expression of pigment epithelium-derived factor (PEDF) and VEGF, and downregulates tight junction protein, thereby inhibiting the breakdown of BRB and alleviating early vascular leakage in the diabetic retina, and ultimately achieving protection against DR [104]. It may be that in the future, APNR, drugs that protect against DR via the APN pathway, and the relationship between homologs of APN and DR and the specific mechanisms can be used in conjunction with APN to diagnose DR and co-administer drugs, among other possibilities.

APN may be a potential marker in the future as a target therapeutic site for DR and as a predictor of DR. APN may be utilized as a predictor and marker of T2DM and its prognostic analysis [105], and as a predictor and marker for complications in type 1 diabetes mellitus [106]. Serum APN correlates with intraocular cytokine (including VEGF) concentrations in atrial fluid and can be used as a clinical predictive marker for intraocular inflammation associated with aqueous humor APN, aqueous humor VEGF, and serum VEGF [71].