Researchers at Columbia University Irving Medical Center have developed eye drops that could prevent vision loss after retinal vein occlusion, a major cause of blindness for millions of adults worldwide.
A study, in mice, suggests that the experimental therapy -which targets a common cause of neurodegeneration and vascular leakage in the eye – could have broader therapeutic effects than existing drugs.
The study was published in Nature Communications.
What is retinal vein occlusion?
Retinal vein occlusion occurs when a major vein that drains blood from the retina is blocked, usually due to a blood clot.
As a result, blood and other fluids leak into the retina, damaging specialized light-sensing neurons called photoreceptors.
Standard treatment for the condition currently relies on drugs that reduce fluid leakage from blood vessels and abnormal blood vessel growth.
But there are significant drawbacks. These therapies require repeated injections directly into the eye, and for the patients who brave this daunting prospect, the treatment ultimately fails to prevent vision loss in the majority of cases.
The new treatment targets an enzyme called caspase-9, says Carol M. Troy, MD, Ph.D., professor of pathology & cell biology and of neurology in the Taub Institute for Research on Alzheimer’s Disease and the Aging Brain at Columbia University Vagelos College of Physicians and Surgeons, who led the studies.
Under normal conditions, caspase-9 is believed to be primarily involved in programmed cell death, a tightly regulated mechanism for naturally eliminating damaged or excess cells.
However, in studies of mice, the Troy lab discovered that when blood vessels are injured by retinal vein occlusion, the caspase-9 becomes uncontrollably activated, triggering processes that can damage the retina.
Eye drops prevent retinal injury
The Troy lab found that a highly selective caspase-9 inhibitor, delivered in the form of eye drops, improved a variety of clinical measures of retinal function in a mouse model of the condition.
Most importantly, the treatment reduced swelling, improved blood flow, and decreased neuronal damage in the retina.
“We believe these eye drops may offer several advantages over existing therapies,” says Troy. “Patients could administer the drug themselves and wouldn’t have to get a series of injections. Also, our eye drops target a different pathway of retinal injury and thus may help patients who do not respond to the current therapy.”
Next steps
The researchers are preparing to test the eye drops in people with retinal vein occlusion during a phase I clinical trial.
Moving forward, the Troy lab will also study whether caspase-9 inhibitors can be used to treat other vascular injuries caused by overactivation of the enzyme, including diabetic macular edema (another common cause of blindness) and stroke.
“Vascular dysfunction is at the heart of many chronic neurological and retinal disorders, because high energy demands in the brain and eye render these tissues exceptionally vulnerable to disruption in blood supply,” says the study’s first author, Maria Avrutsky, Ph.D., postdoctoral research scientist in pathology & cell biology at Columbia University Vagelos College of Physicians and Surgeons.
The central nervous system (CNS) is one of the most metabolically active tissues in the body, rendering it exquisitely sensitive to vascular dysfunction; decreases in blood flow lead to hypoxia and ischemia which induce both neuronal loss and an increase in vascular permeability.
Breakdown in endothelial barrier function allows fluid leakage into the parenchyma, leading to edema – a devastating and potentially lethal consequence in the CNS1. Dysregulation of the barrier has been implicated in aging-related cognitive decline2 and in acute and chronic neurologic disorders, including stroke, multiple sclerosis, Alzheimer’s disease, and diseases that disrupt retinal blood supply such as retinal vein occlusion (RVO) and diabetic macular edema3.
RVO affects between 1 and 2% of persons over the age of 40 and is the second leading cause of new blindness in working age adults4. Obstruction of blood flow stimulates increased expression of vascular endothelial growth factor (VEGF)5,6, a key component of retinal hypoxia response.
Müller cells secrete VEGF7, which acts on endothelial cells to promote vasodilation and increase vascular permeability8. In patients with RVO, the initial retinal occlusions may recanalize by 2 weeks9, while edema and microvascular dysfunction can persist for months or years.
Current treatment strategies focus on attenuating retinal edema either by vascular stabilization via anti-VEGF agents or broad-spectrum suppression of inflammation via corticosteroids4.
The pivotal studies on anti-VEGF treatment in RVO (BRAVO and CRUISE trials) demonstrated significant reduction of retinal swelling and improved visual acuity in eyes receiving intravitreal ranibizumab, a VEGF-neutralizing antibody10,11.
However, levels of intraocular VEGF vary widely across eyes with RVO12, and many treated patients continue to experience vision decline13. In follow-up studies of patients receiving anti-VEGF treatment, refractory edema persisted in 50% of branch RVO eyes and in 56% of central RVO eyes14, which suggests that additional, non-VEGF-mediated, pathways contribute to retinal edema and vision loss in RVO.
Furthermore, existing interventions do not address the extensive retinal degeneration which occurs even when edema has successfully been treated15, highlighting the need to identify signaling pathways which can promote neuronal survival in retinal disease.
In hypoxia–ischemia it has been proposed that neuronal injury is a consequence of a hypoxia-induced cascade of pathologic signaling, including excitotoxicity, release of free radicals, and mitochondrial dysfunction16.
The caspase family of proteases are activated in neurodegeneration, and hypoxia–ischemia increases expression of total and cleaved caspase-9 in CNS tissues17,18. Neuronal apoptosis in the ischemic brain is mediated by the caspase-9-dependent intrinsic (mitochondrial) death pathway, and inhibition of caspase signaling improves functional outcomes in animal models of cerebral ischemia18–21.
In contrast to the well-studied role of caspases in neuronal death, less is known about caspase signaling in ischemic vasculature and studies have offered conflicting conclusions regarding endothelial cell death in retinal hypoxia–ischemia injury22,23.
The neuroretina is an accessible model system for studying barrier function in a physiologic setting24,25. Laser-induced RVO offers a robust model of neuronal dysfunction and loss of vascular integrity in the CNS5,26, and recent studies have demonstrated that mouse RVO is tractable for examining pathogenic mechanisms of hypoxia–ischemia via pharmacological approaches6,27–31.
Here, we report that endothelial caspase-9 activity creates a mechanistic link between vascular and neuronal injury in mouse RVO. Topical (eye-drop) application of a highly specific caspase-9 inhibitor reduces retinal ischemia and provides robust morphologic, cellular, and functional neuroprotection; genetic deletion of caspase-9 from endothelial cells phenocopies the protective effects of pharmacological caspase-9 inhibition, identifying specifically endothelial caspase-9 activity as the proximal event in hypoxia/ischemia-induced neuronal injury.
In contrast to the canonical role of caspase-9 as an initiator of apoptosis, RVO-induced activation of endothelial caspase-9 is not associated with endothelial cell death. The unexpected link of endothelial caspase signaling to neuronal function provides insights into neurovascular biology and therapeutic approaches for treatment of neurovascular disease.
Discussion
Prior studies of CNS ischemic injury have demonstrated that hypoxia/ischemia induces temporal and spatial changes in neuronal function and vascular integrity, but it has not been elucidated whether these events are mechanistically integrated or separate pathways.
RVO triggers inflammatory and hypoxic responses, both of which contribute to breakdown of the blood-retina barrier1. We employed an established mouse model of RVO to characterize the contribution of retinal ischemia to pathological symptoms common in retinal vascular occlusive disorders.
Using clinically relevant in vivo imaging, we show close association between measures of obstructed blood flow, and subsequent functional and morphological retinal pathologies.
Vascular endothelium, which forms the blood brain/retinal barrier, is uniquely positioned as an active interface between neurons and inflammatory processes60.
We report that in RVO, nonapoptotic activation of endothelial caspase-9 orchestrates vascular dysfunction and neuronal injury, supporting a non-cell autonomous consequence of endothelial caspase-9 activity (Fig. 9).
RVO-induced caspase activation in neurons is driven by caspase-9 mediated endothelial dysfunction. We used inducible endothelial caspase-9 knockout mice to interrogate the endothelial cell specific role of caspase-9 in RVO pathology, and we also provide a tool, Pen1-XBir3, with which to investigate caspase-9 function via pharmacological approaches. Inhibition or genetic excision of caspase-9 from endothelial cells rescues vascular integrity and restores neuronal activity in an adult model of retinal hypoxia/ischemia.
Taken together, these data suggest that targeting endothelial cell pathways could provide effective therapies for attenuating ischemic neuronal injury, and identify a nonapoptotic role for caspase-9 and caspase-7 in vascular barrier dysfunction.
Model of caspase-9 activity in ischemic neurovascular injury.
Hypoxia–ischemia injury activates caspase-9 in endothelial cells, inducing nonapoptotic endothelial dysfunction. Caspase-9 promotes capillary ischemia and induces endothelial barrier breakdown and neuronal injury. Pen1-XBir3 protects CNS tissues from hypoxia–ischemia injury by inhibiting caspase-9.
Proteomics studies indicate that RVO elicits changes across a broad array of signaling pathways, highlighting the molecular complexity of hypoxia/ischemia injury61. The chronic nature of ischemia and microvascular dysfunction in human disease further adds to the complexity of retinal vascular disorders. Multiple causative pathways have been implicated in the pathogenesis of retinal edema, including VEGF signaling, the kinin/kallikrein system, and the Angiopoietin-2/Tie2 axis1.
Our results identify nonapoptotic endothelial caspase-9 signaling as a key mediator of ischemic injury, and underscore the importance of vascular endothelial dysfunction in CNS hypoxia–ischemia injury.
These results invite further exploration into the mechanisms regulating caspase signaling during chronic retinal ischemia, the downstream targets of endothelial caspase-9 that mediate vascular dysfunction, and the process by which these pathways intersect with other factors known to mediate retinal vascular disease.
Caspase-9 activity has been tightly associated with initiation of the intrinsic apoptosis pathway, and far less is known about caspase biology in non-apoptotic processes42. However, a growing body of literature has identified physiologic functions of caspase-9 in development and differentiation, which are independent of apoptosis: caspase-9 promotes muscle and cardiomyocyte differentiation and proliferation62, hematopoietic development63, and neuronal maturation and axonal pathfinding64,65.
Although less is known about potential nondeath roles of caspases in endothelial cells, recent work has suggested a potential barrier protective role for endothelial caspase-366. These studies highlight that caspases can be active in cells that are not dying, and that the physiological consequence of caspase activation depends on cellular context. However, we have limited understanding of the nonapoptotic effects of caspase-9 in disease because the activation of effector caspases in disease tissues is routinely interpreted as a hallmark of apoptotic cell death.
We see low basal levels of caspase activation present in uninjured retinas. Basal activation of caspase-3 in various cell types, caspase-6 in retinal astrocytes, and caspases-9/-7 in neuronal processes indicates cell-type specific regulation of physiological caspase activity in adult CNS tissues. We identify caspase-7 as the main effector caspase induced by caspase-9 in RVO, and the only effector caspase induced by RVO in endothelial cells.
While it is possible that caspase-3 induction and/or apoptosis in endothelial cells could be evoked by more extreme ischemic injury (such as complete abrogation of retinal blood flow), we did not detect induction of caspase-3 cleavage or endothelial cell death in our injury model.
Our findings also provide a mechanistic insight into the function of endothelial caspase-9 in systemic disorders with vascular barrier dysfunction. In vitro studies have indicated that pan-caspase inhibition may modulate endothelial secretion of prothrombotic and proinflammatory factors by regulating exocytosis of Weibel-Palade bodies, suggesting a mechanistic link between caspase activation and endothelial inflammation67.
While few studies have looked at caspase-9 expression specifically in patient endothelial cells, increased caspase-9 has been reported in atherosclerotic lesions68 and in Behcet’s disease, a multisystem disorder driven by endothelial dysfunction, which often includes ocular findings of retinal edema and atrophy69.
The distinction between cell-death and non-cell-death caspase signaling is important for identifying potential therapeutic targets in disease. Our findings invite re-examination of apoptotic vs nonapoptotic caspase-9 pathways in disease pathologies. Taken together, these data suggest that endothelial caspase-9 may play a direct role in mediating endothelial barrier dysfunction and neuronal injury, and that targeting endothelial cell pathways could provide effective therapies towards supporting neuronal health.
More information: Maria I. Avrutsky et al, Endothelial activation of caspase-9 promotes neurovascular injury in retinal vein occlusion, Nature Communications (2020). DOI: 10.1038/s41467-020-16902-5
