Iron chelation therapy to prevent poststroke cognitive impairments: role of diabetes and sex

Diabetes is a major risk factor for cerebrovascular and neurodegenerative diseases including ischemic stroke, poststroke cognitive impairment (PSCI), and Alzheimer’s disease and related dementias (1). Increased hemorrhagic transformation (HT) is a characteristic pathognomonic feature of stroke in diabetic animals and humans (2). Furthermore, the occurrence of HT has been found to be significantly enhanced by thrombolytic therapy (2). Extravasation of blood following HT and subsequent release of iron leads to iron overloading of cells in the neurovascular unit (NVU; 3). Elevated cellular labile iron ion levels promote the generation of cellular and mitochondrial superoxide and hydroxyl free radicals that set off the cascade of membrane phospholipid oxidation leading eventually to plasma membrane lysis and cell death referred to as ferroptosis (4). Unlike apoptosis or autophagy, ferroptosis is a type of regulated cell death inhibited by iron-chelating reagents (3, 4). In addition to the causative role of ferroptosis in cerebrovascular diseases such as stroke, it also plays a mechanistic role in the development of various neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases (3). Ferroptosis induces stroke injury and poor outcomes by multiple mechanisms including promotion of blood-brain barrier (BBB) breach (3, 5) and neuroinflammation by releasing proinflammatory cytokines after the stroke (3). Furthermore, elevated expression of iron transfer and storage proteins like transferrin and ferritin, the inactivation of glutathione peroxidase 4 (GPX4), and a decrease in glutathione (GSH) levels after stroke favor the ferroptosis (3). Therapies targeting iron chelation with deferoxamine (DFX) and inhibition of ferroptosis by Edaravone, a clinically approved drug for the treatment of ischemic stroke, have been found to be therapeutically promising (3). As diabetes alters several pathways involved in ferroptosis, it is expected that diabetes elevates the risk of ferroptosis. Previous study by Ergul group has shown that iron chelation with DFX protects against ischemic brain injury in male diabetic rats by preventing stroke-induced vasoregression, breach of blood-brain barrier, and neuronal injury (6). Interestingly, epidemiological data have shown that stroke incidence and related mortality are lower in women compared with men until midadulthood but increase with advancing age past the reproductive senescence (7). Thus, ischemic cellular injury in stroke is influenced by not only biological/chromosomal sex but also the availability of hormones (estrogens, progesterone, and androgens; 7). Importantly, in young females, diabetes nullifies the sex-dependent protections against stroke injury that is characterized by increased occurrence of HT following stroke (8, 9). Accordingly, the mechanisms underlying the interaction of diabetes with female sex related to stroke injury are not known.

In this issue of the American Journal of Physiology-Heart and Circulatory Physiology, Li et al. (10) present the findings from their study examining the impact of iron chelation therapy on poststroke outcomes in female rats with and without diabetes. The hypothesis of the study was that diabetes promotes pathological neovascularization, increased HT, and ferroptosis that leads to poststroke NVU remodeling, neurological injury, and functional impairments in female rats. To prevent ferroptosis, authors treated young female rats (12 wk old) with DFX after experimental stroke. Poststroke vascular indices, microglial activation, and neurocognitive function were determined. The findings from diabetic/control female rats untreated and treated with DFX were compared with each other and to the previously published findings from corresponding groups of age-matched male rats (6). Major DFX afforded improvements that are shared by female and male rats include 1) poststroke deficits in sensorimotor function, cognitive function, and working memory in diabetic rats; 2) poststroke vascular indices, NVU remodeling, and BBB disruption in all male and female rats; and 3) ferroptosis cell death in both male and female cultured human brain microvascular endothelial cells (BMVECs) under diabetes and stroke-like conditions in vitro. In contrast, major sex-dependent differences include the following: 1) DFX promoted fine motor skills deficits in male control rats but not in female control rats; 2) working memory deficits at baseline are greater in female rats compared with male cohorts; 3) poststroke vascularization was not observed in female diabetic rats compared with male diabetic rats; 4) poststroke vasoregression was not observed in diabetic female rats compared with diabetic male rats, and further analysis revealed that DFX lowered vascular volume and surface area indices in females but increased it in males; and 5) DFX did not attenuate poststroke microglial activation in diabetic female rats but increased it in control female rats. In contrast, DFX attenuated poststroke microglial activation in diabetic male rats. Interestingly, DFX promoted anti-inflammatory phenotype in diabetic female rats, indicating diverse microglial mechanisms (morphological vs. functional) underlying the actions of DFX in female and male rats.

Experiments in BMVECs from male and female subjects demonstrated that ferroptosis occurs in response to hypoxia in cells exposed to high-glucose levels compared with normal glucose. Notably, male cells displayed significantly higher expression of ferroptosis marker, iron-responsive element-binding protein 2 (IREB2) compared with female cells. Ferroptosis induction with erastin reduced GPX expression in only male cells that were sensitive to ferroptosis inhibitor, Fer-1. Additional experiments also demonstrated the cytoprotective actions of DFX in both male and female BMVECs treated with hemin. However, the cytoprotective actions of DFX displayed subtle sex-dependent differences related to the expression of GPX and key proinflammatory proteins in hemin-treated cells. Finally, the in vitro findings together provocatively suggest that iron chelation under nondiabetic conditions can be detrimental in nature. Overall, the findings from the BMVECs supported the in vivo data showing the therapeutic efficacy of iron chelation in females and males.

Future studies are needed to further advance the mechanistic foundation of the novel findings by Li et al. (10). First, since DFX is detrimental under control conditions in mice and cells, there is an opportunity to investigate the potential role of differential signaling pathways mediating the detrimental or protective actions of DFX. Second, is it possible to replicate the findings from rat studies in other species? Third, what specific sex-dependent signaling mechanisms regulate ferroptosis? What are the impacts of sex chromosomes versus sex hormones on ferroptosis? Fourth, how does the method of experimental stroke used in the studies impacts involving male (filament method) and female (thromboembolic method) rats? Finally, how significantly will the erastin-induced alternate pathways influence the findings from BMVECs?

In summary, the elegant studies by Li et al. (10) made novel observations that diabetes does not promote abnormal cerebral vascularization in female rats. The study also showed that stroke and DFX treatment cause diverse sex-dependent alterations in cerebrovascular density and microglial activation; however, DFX is protective against poststroke injury and neurocognitive functional impairments in diabetic animals of both sexes. Paradoxically, DFX exerts detrimental effects under control conditions in both animals and BMVECs. Authors have acknowledged the limitations of the study, and the one that needs particular mention is that the study could have been stronger with measurement of infarct size or HT and that the study uses only adult animals. However, the justification provided by the authors is reasonable, and these limitations do not diminish the highly significant impact of the novel findings on our understanding of vascular mechanisms linking diabetes to dementia. The study provides convincing evidence to support the potential therapeutic value of iron chelation in preventing PSCI in both sexes, albeit by promoting diverse mechanisms.

GRANTS

This work was supported by National Institutes of Health Grants NS114286 (to P.V.G.K.) and AG074489 (to P.V.G.K.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.S.V.P.S., V.N.S., and P.V.G.K. drafted manuscript; S.S.V.P.S., V.N.S., and P.V.G.K. edited and revised manuscript; S.S.V.P.S., V.N.S., and P.V.G.K. approved final version of manuscript.