In the past several decades, diabetes mellitus has become one of the most common chronic diseases worldwide. While the United States spends hundreds of billions of dollars on treatment for this disease, it continues to rank among the top 10 causes of death. One of its main complications, diabetic retinopathy, is the foremost cause of blindness for individuals younger than 65 years-old. In this condition, retinal blood vessels become more porous, allowing hemorrhages to form within the neural retina. The blood-retina barrier system, formed by endothelial cells lining retinal capillaries in the inner retina and retinal pigment epithelial (RPE) cells located between the outer photoreceptor layer and the choroid vasculature, loses its integrity, leading to vascular fluid leakage. This fluid accumulation within the retina, known as diabetic macular edema (DME), leads to decreased vision. Minority populations, such as people of African or Asian ancestry, are more likely to develop diabetes and diabetic retinopathy, including DME, than Caucasians.
The landmark Diabetes Control and Complications Trial (DCCT) showed that an intensive insulin regimen decreased the incidence and progression of diabetic retinopathy and other microvascular complications. Based on this work, tight, systemic control of blood glucose through insulin and other medications, such as metformin, remains a core element of treating diabetic retinopathy. In the past, retinopathy unable to be managed by strict glycemic control could only be treated with laser to photocoagulate portions of the retina. More recently, intravitreal injections of medications that act against vascular endothelial growth factor (VEGF) have decreased the need for photocoagulation and have allowed patients to retain more of their vision and neural retina. However, at least 40% of patients with diabetic retinopathy do not respond to anti-VEGF treatments, stressing the need for further research into other therapeutic targets.In addition to the benefits described in the DCCT, longitudinal studies of diabetic patients enrolled in this trial found that individuals who were given early intensive insulin therapy were significantly less likely than individuals initially treated with a single daily insulin injection to develop diabetic retinopathy or have worsening retinopathy for over 10 years after the initial change in treatment. Other investigations in Europe found similar results, showing that early glycemic control reduces morbidity and mortality associated with diabetes. Overall, these studies suggested that the cellular microenvironment of diabetes induces a metabolic memory in a patient’s cells that remains long after cells are returned to a more normal, euglycemic microenvironment. By clarifying how this memory is formed, researchers and clinicians may use this knowledge to develop additional therapies to treat diabetes and its complications.
While there has been extensive research examining how the nuclear genome contributes to the development of diabetes, there has been much less focus on the role of the mitochondria and its DNA in this process. Our laboratory employs a cytoplasmic hybrid (cybrid) system, in which a parent RPE cell line is chemically-treated to remove mitochondrial DNA (mtDNA) and then fused with patient platelets containing mitochondria but no nuclear DNA. Our resulting cybrids have patient-specific mtDNA, but all cell lines have the same nuclear genome, allowing us to investigate the influence of mtDNA on cellular health and functions. Using cybrid cell lines, we have shown that mtDNA associated with certain maternal ancestries (e.g. African versus European) or from patients with age-related macular degeneration differentially-alter levels of reactive oxygen species, pro-inflammatory genes, complement, and other factors.
The work in this dissertation uses our cybrid system to investigate how mtDNA affects RPE cell health and function. In Chapter 1, we examined how ancestral mtDNA background and mtDNA from diabetic versus unaffected patients may protect RPE cells against stresses seen in diabetes, such as high glucose or hypoxia. In Chapter 2, we investigated the mechanisms of this protective effect using RNA-seq and how it may alter cellular functions.
