Diabetic Retinopathy as a Ceramide Disease Reversible by Anti-Ceramide Immunotherapy

Background

Diabetic Retinopathy (DR) is one of the most common metabolic disorders worldwide. Diabetes not only leads to chronic complications in macrovascular and microvascular systems but also imposes significant socio-economic burdens. As a microvascular complication, DR is a leading cause of blindness among working-age adults. The late stage of DR is characterized by vision loss and fluid accumulation in the macular region (referred to as diabetic macular edema, DME) or uncontrolled retinal neovascularization (known as proliferative diabetic retinopathy, PDR). Although controlling dyslipidemia can slow the progression of diabetic vascular complications, the underlying mechanisms in the retina are not fully understood.

Currently, the first-line treatment for patients with PDR or DME is anti-vascular endothelial growth factor (VEGF) therapy. However, this therapy is ineffective or limited in efficacy for approximately 40% of patients. Additionally, laser photocoagulation therapy mainly targets late-stage DR and cannot completely reverse the disease progression. Therefore, it is crucial to find new treatment strategies for early non-proliferative diabetic retinopathy (NPDR), where pathological changes are still reversible.

In recent years, ceramide, a bioactive sphingolipid signaling molecule, has become increasingly recognized for its role in various metabolic diseases, particularly the imbalance between long-chain (C16) and very long-chain (C26) ceramides in DR progression. This study demonstrates that metabolic abnormalities induced by diabetes lead to the formation of pathological ceramide-rich platforms (CRPs) on the surface of retinal endothelial cells, and targeting these platforms with anti-ceramide antibodies can inhibit diabetes-induced endothelial cell apoptosis, dysfunction, and increased permeability.

Research Source

This research was conducted by Tim F. Dorweiler, Arjun Singh, Aditya Ganju, Todd A. Lydic, Louis C. Glazer, Richard N. Kolesnick, and Julia V. Busik, and the findings were published in the July 2, 2024, issue of the journal Cell Metabolism (Volume 36, pages 1521-1533). The authors are affiliated with Michigan State University, Boston Children’s Hospital, Harvard Medical School, Memorial Sloan Kettering Cancer Center, and the University of Oklahoma Health Sciences Center in the United States.

Detailed Research Process

Overall Design and Process of the Study

a) The study was divided into two main parts: clinical sample analysis and in vitro experiments, validated by animal models. The research included the following steps and methods: 1. Clinical Sample Analysis: Mass spectrometry (nano-electrospray high-resolution mass spectrometry) was used to analyze the levels of various ceramides in the vitreous humor of PDR patients and non-diabetic control subjects. 2. Cellular Experiments: Retinal endothelial cells (RECs) were used in vitro, with CRP formation induced by tumor necrosis factor-alpha (TNFα) and interleukin-1 beta (IL-1β). The effects of these ceramide-rich platforms on endothelial cell apoptosis were observed. 3. Animal Model Validation: The effects of anti-ceramide antibodies in preventing retinal endothelial cell apoptosis and increased vascular permeability were evaluated in ischemia/reperfusion (I/R) and streptozotocin (STZ)-induced diabetic retinopathy models in mice and rats.

b) Specific experimental procedures included: 1. Clinical Sample Analysis: - A small-scale clinical trial was conducted, and the levels of ceramides in the vitreous humor of PDR patients and non-diabetic controls were measured using nano-electrospray mass spectrometry (ESI-MS/MS). - Results showed significantly elevated levels of C16 ceramide and reduced levels of C26 ceramide in PDR patients, supporting the ceramide imbalance hypothesis.

1. Cellular Experiments:

   • Bovine retinal endothelial cells (BRECs) were used as a standard model for studying diabetic retinopathy.
   • The formation of CRPs induced by TNFα and IL-1β and the resulting apoptosis were observed using 2D and 3D imaging techniques. These experiments were the first to use 3D confocal imaging technology, generating detailed CRP “movies” with Imaris software, showing extensive formation of CRPs on the endothelial surface leading to apoptosis.

2. Animal Model Validation:

   • In the I/R model, a single intravitreal injection of 2μg anti-ceramide antibody significantly reduced the expression levels of retinal inflammatory markers (TNFα, IL-1β, IL-6, ICAM-1) and decreased vascular permeability.
   • In the STZ-induced diabetic rat model, a single intravitreal injection of anti-ceramide antibody also significantly reduced retinal inflammation and vascular permeability, validating its efficacy under more physiologically relevant conditions.

Interpretation of Research Results

Main Findings of the Study

1. Manifestation of Ceramide Imbalance in PDR Patients:

   • The vitreous humor of PDR patients showed significantly increased C16 ceramide levels and decreased C26 ceramide levels. This imbalance is closely related to the pathological state of the retina, supporting the critical role of CRPs in DR progression.

2. CRP Formation and Apoptosis Induced by TNFα and IL-1β:

   • In the BRECs cell experiment, TNFα and IL-1β induced CRP formation, leading to rapid endothelial cell apoptosis. This process was effectively blocked by the anti-ceramide antibody 6B5 single-chain variable fragment (scFv), confirming the importance of CRP formation in cellular signaling.

3. Efficacy of Anti-Ceramide Therapy in Animal Models:

   • In the I/R mouse model, anti-ceramide antibody significantly reduced retinal inflammation and increased vascular permeability, demonstrating its efficacy in acute ischemic injury.
   • In the STZ-induced diabetic rat model, anti-ceramide antibody significantly reduced retinal inflammatory markers and vascular permeability, further validating its efficacy under diabetic conditions.

Logical Relationships of the Results

The study revealed that ceramide imbalance is a key factor in diabetic retinopathy, leading to retinal vascular damage and apoptosis through CRP formation. Anti-ceramide therapy effectively reduced retinal inflammation and pathological progression by blocking CRP formation, providing a viable therapeutic strategy.

Conclusions of the Study

Scientific and Practical Value of the Study

This study highlights the importance of ceramide imbalance in diabetic retinopathy and demonstrates the efficacy of anti-ceramide therapy in preventing disease progression. As a novel therapeutic approach, anti-ceramide immunotherapy holds promise for early intervention in diabetic retinopathy, preventing its progression to the proliferative stage, and offers new avenues for treating other metabolic diseases.

Study Highlights

• The discovery and demonstration of the critical role of ceramide imbalance in diabetic retinopathy, particularly the ratio changes of C16 and C26 ceramides.
• The first use of 3D imaging technology to showcase the CRP formation process induced by TNFα and IL-1β, revealing its central role in retinal endothelial cell apoptosis.
• Experimental results showed that anti-ceramide antibodies could effectively block the pathological progression of diabetic-induced retinal vascular damage, offering new targets and strategies for treating DR.

Other Valuable Information

The research also suggests that anti-ceramide antibodies have good prospects for preventing acute ischemic injury (such as GI-ARS death) and are currently being developed in collaboration with the NIH and the Department of Defense for emergency prevention and treatment strategies in nuclear disasters.

Limitations and Constraints of the Study

Although the study provided important findings, there are some limitations: 1. The clinical sample analysis had a small sample size, requiring larger studies to determine if total ceramide levels are affected by diabetes. 2. The animal models did not fully replicate the proliferative stage of human DR, necessitating further models to study retinal neovascularization.

This study offers new insights into the treatment of diabetic retinopathy, suggesting that CRP and its regulatory mechanisms could be important targets for future DR therapies. Through early intervention, more effective treatment and prevention of diabetic retinopathy may be achieved in the future.