*9.2. Apoptosis*

The intrinsic (mitochondrial) pathway of apoptosis is initiated by increased permeabilization of the mitochondrial outer membrane and activation of the apoptotic signaling cascade. Notably, the intrinsic pathway is induced by increased oxidative stress [194], making this pathway highly relevant in DR, as virtually all cell types in the retina experience hyperglycemia-induced oxidative stress [116,123,164,195]. RGCs [133] and the retinal microvasculature in particular have been shown to undergo oxidative stress-induced apoptosis [114,160]. Several groups have reported that administration of exogenous antioxidants preserved mitochondrial integrity and prevent cell death in the diabetic retina [116,133]. These findings reiterate the concept that oxidative stress is an early event in the pathogenesis of DR, whereas cell death generally occurs as a later and secondary event.

## **10. Therapeutic Implications**

#### *10.1. Therapies Focused on Maintaining the Integrity of Retinal Mitochondria*

The Diabetes Control and Complications Trial showed that intensive insulin replacement therapy reduces the incidence and slows the progression of DR [5]. However, the incidence of DR remains high, and many patients still progress to PDR despite advances in diabetes care. Accordingly, second-line treatments are frequently necessary to manage the later complications of DR. These therapies are highly e ffective in managing the sight-threatening complications of DR. Thus, it is critical to better understand the early stages of DR and develop new therapies to prevent its progression. The need for new therapies in early stages of DR is further highlighted by the "metabolic memory" phenomenon (for a review, see [2]), which may be a consequence of persistent mitochondrial damage and oxidative stress [142]. This hypothesis is consistent with the benefit of mitochondrial targeted therapy to preserve mitochondrial integrity and vision in experimental DR.

An important antioxidant therapy is the Szeto–Schiller (SS) tetra-peptide, SS-31 (elamipretide), which is concentrated in the inner mitochondrial membrane and selectively stabilizes cardiolipin [196], a phospholipid critical for mitochondrial integrity and function, which is prone to oxidative damage in diabetes [197]. SS-31 enhances the interaction between cytochrome c and cardiolipin to facilitate better electron transfer from complex III to complex IV, and reduces mitochondrial oxidative stress [198,199]. Evidence from animal studies suggests that SS-31 could reduce the risk of vascular disease in diabetes, as administration of SS-31 alleviated the microvascular retinal disease in rodent models of DR [200,201], and increased SIRT1 while ameliorating retinal inflammation in rodent and human subjects with T2D [202]. However, human studies have shown limited e fficacy to date, and no studies have been performed in patients with DR.

Numerous studies have suggested that oxidative stress may be modulated by uncoupling proteins (UCPs), a family of proteins named for their ability to uncouple electron transport from ATP synthesis. These proteins decrease mitochondrial ROS production by decreasing the electrochemical gradient [203], a process called "mild uncoupling". The theory is based on the observation of Korshunov et al. [204] that ROS generation increases in an exponential manner when mitochondrial membrane potential exceeds a threshold that is higher than that corresponding to the mitochondrial energetic state in in vivo settings. The hypothesis is also based on the assumption that the diabetic milieu increases the availability of energetic substrates (glucose, FAs) to the retina, which are oxidized to increase mitochondrial membrane potential ("hyperpolarization"). This hypothesis is supported by the observation that *UCP2*-deficient mice exhibit increased ROS generation [205], whereas overexpression of the *UCP2* gene preserves mitochondrial function in human umbilical endothelial cells [206]. In the retina, UCP2 protein levels and activity are increased in retinal endothelial cells grown in high-glucose conditions [207], which may indicate a compensatory response to oxidative stress. However, administration of the uncoupling agen<sup>t</sup> niclosamide ethanolamine has shown no benefit in the treatment of diabetes or its complications in db/db mice [208] indicating the lack of benefit of uncouplers in in vivo settings. A potential explanation is that Oxphos is a process regulated by energy demand rather than substrate availability. In diabetes, increased substrate availability does not increase oxidative metabolism that exceeds ATP synthesis [109], a mechanism that would lead to "hyperpolarization". While UCPs may be an important endogenous defense mechanism in the setting of oxidative stress, their therapeutic utility is unclear.

Improving the e fficiency of electron flow within the defective mitochondrial ETC may be an optimal approach to relieve the increased electron density at specific ETC sites and eliminate the risk of oxygen univalent reduction and superoxide formation. Methylene blue, a redox compound that provides an alternative electron route between mitochondrial complex I and cytochrome c [209], preserves mitochondrial and photoreceptor integrity by preventing oxidative stress in a model of complex I defect [153] and experimental diabetes [34].

Idebenone, a synthetic benzoquinone that mediates electron transfer to complex III by bypassing complex I [210], is a free radical scavenger [211], reduces intracellular ROS and increases ATP production in complex I-defective cells [212–214], and promotes an increase in mitochondrial mass by regulating mitophagy [215]. Its mitochondrial protective e ffects have recently been investigated as a drug therapy for Leber's hereditary optic neuropathy, a rare genetic mitochondrial disease that causes rapid and progressive bilateral vision loss in young adults. A 24-week multi-center double-blind, randomized, placebo-controlled trial in patients with Leber's hereditary optic neuropathy show a mild benefit in visual acuity [216] that was confirmed when treatment started 5 years after the diagnosis [217]. The benefic outcomes are considered a result of idebenone to restore the bioenergetics in the remaining dysfunctional RGC. Evidence for e fficacy of idebenone in human patients with primary or acquired mitochondrial defects are still limited, and restrict its use in DR.

#### *10.2. Clinical Trials and the Role of Antioxidants in the Management of DR*

Despite the evidence that antioxidants can slow the progression of DR, clinical trials have had mixed results. This topic was very recently reviewed by Garcia-Medina et al. [218], but is summarized here for completeness. The most successful interventions have been those that use combined antioxidant therapy (CAT). The Diabetes Visual Function Supplement Study showed that CAT may improve visual acuity and contrast sensitivity among participants with T1D and T2D without clinically detectable retinopathy or with mild non-proliferative diabetic retinopathy (NPDR) [219]. This finding is supported by the prior work of Hu et al. [220], who showed that patients with NPDR have lower levels of lutein and zeaxanthin, and further demonstrated that supplementation with these antioxidants reduces oxidative stress and may improve visual function. Although the previous two studies sugges<sup>t</sup> a role of CAT in the managemen<sup>t</sup> of DR, the conclusions are limited by the short study duration (6 and 3 months, respectively) relative to the chronicity of DR. To date, the longest trial that has been conducted was a 5-year follow-up of patients taking a commercially available multi-vitamin showing that antioxidants may slow the progression of DR as detected by ophthalmic examination [221]. However, in contrast to the above studies, the investigators did not observe a significant improvement in visual acuity. These discrepancies highlight the need for additional studies to find better therapeutic strategies that decrease the mitochondrial ROS generation by preserving mitochondrial function rather than scavenging the already generated ROS.
