Rejuvenation of brain endothelial cells: a novel paradigm for treating BBB disorders

Paromita Paul PinkyVivek BasudkarBalaji GovindaswamyPurva KhareDevika S Manickam^*

• School of Pharmacy, Duquesne University, Pittsburgh, United States

The blood-brain barrier (BBB) is a complex structure consisting of brain endothelial cells (BECs), astrocytes and pericytes forming a selectively-permeable membrane barrier between systemic circulation and the central nervous system (CNS) (1)(2)(3). Tight junctions of the BBB formed by BECs consist of transmembrane proteins and membrane associated cytoplasmic proteins such as zona occludens, atypical protein kinase C and caveolin. Tight junction complexes allows the movement of selective molecules into the brain to facilitate brain homeostasis (4). BECs lining the BBB contain nearly a 5-fold greater concentration of mitochondria compared to peripheral endothelial cells (5)-suggesting the increased reliance of BECs on their mitochondrial load to maintain their metabolic function and structural integrity (6). BBB mitochondrial functions can be impaired due to various stress conditions such as oxidative stress, hypoxia (see graphical abstract), genetic mutations, inflammatory cytokines and metabolic disorders (7,8). ATP-binding cassette (ABC) transporters are efflux transporters present in the BBB that require ATP for its function. These transporters play a key role in facilitating the movement of toxic compounds and drugs across the BBB. Mitochondrial dysfunction leads to dysfunction of ABC transporters causing the accumulation of toxic compounds eventually damaging the BBB (9). Such a disruption of the BBB may lead to the progression of various CNS disorders like Parkinson's disease (PD), Alzheimer disease (AD), ischemic stroke, amyotrophic lateral sclerosis (ALS) and epilepsy. Oxidative stress and mitochondrial dysfunction caused by environmental toxins result in the progression of PD by disrupting the BBB. Downregulation of peroxisome proliferator-activated receptor α-coactivator 1-alpha, a central modulator of mitochondrial biogenesis, has been reported in animal as well as cellular models of Parkinson's disease, suggesting the key role of mitochondria in PD pathogenesis (10,11). Accumulation of amyloid beta plaques is reported to be one of the primary causes of AD (12). Mitochondrial dysfunction also leads to upregulation of amyloid precursor protein that eventually leads to increased accumulation of amyloid beta plaques in brain (13,14). Loss of mitochondrial function at the BBB leads to both BEC death as well as loss of tight junction integrity (see graphical abstract), leading to long term neurological dysfunction in stroke (6). Mitochondrial dysfunction causes damaged calcium buffering and oxidative stress in motor neurons that accelerates the progression of ALS (15). Similarly, ATP loss and oxidative stress due to mitochondrial dysfunction is one of the potential causes of epilepsy (16). To sum up, mitochondrial dysfunction leads to a severe imbalance in brain bioenergetics that contributes to the progression of numerous CNS disorders.As discussed in the above section, mounting evidence reveal that oxidative stress-induced mitochondrial dysfunction of BECs leads to BBB disruption and neurodegeneration in several pathophysiological conditions including ischemic stroke, PD, AD, ALS, epilepsy and septic encephalopathy (9,(17)(18)(19)(20). Therefore, increasing mitochondrial function in the damaged BECs can restore BBB bioenergetics and alleviate the risk of BBB disruption, thus serving as a novel approach to treat diseases associated with BBB dysfunction. Considering the evidence linking mitochondrial function and BBB health and homeostasis, delivery of functional mitochondria, mitochondrial components or even adenosine triphosphate (ATP) as such to dysfunctional BECs can be a promising therapeutic approach to restore BBB bioenergetics and tight junction integrity (see graphical abstract) (21).Large EVs with mitochondria. Several studies have reported that cell-derived extracellular vesicles (EVs) contain functional mitochondria and mitochondrial components (mitochondrial proteins and mitochondrial DNA) that have shown significant beneficial effects under various pathological conditions (22). EVs are cell-derived heterogeneous entities with particle diameters ranging from 100 -1000 nm that carry an array of bioactive cargo such as lipids, proteins, short nucleic acids such as miRNA and mitochondria enclosed within their lipid membranes (22,23). Among the several subtypes of EVs, two major subtypes of EVs have gained interest because of their promising therapeutic potential in various pathological conditions. These two EV subtypes has been categorized based on their particle diameters and are referred to as large EVs (lEVs >200 nm particle diameter) and small EVs (sEVs <200 nm particle diameter) and are formed via distinct biogenesis pathways (22,(24)(25)(26). lEVs and sEVs carry distinct cargo likely reflective of their cellular biogenesis pathways and interactions of EVs with the recipient cells result in a variety of responses as a function of EV cargo. For instance, lEVs isolated from bonemarrow derived stromal cells transferred their mitochondrial load into recipient alveolar epithelial cells that increased cellular ATP levels and exhibited protective effects against acute lung injury in mice (27). Improvement of cellular ATP levels and mitochondrial biogenesis was observed in doxorubicin-injured cardiomyocytes treated with lEVs. These outcomes were attributed to the presence of lEV-mitochondria (28). In contrast, sEVs have been reported to carry mitochondrial proteins, mitochondrial DNA and heat shock proteins that have demonstrated substantial therapeutic efficacy in several pathological conditions (29)(30)(31)(32). sEVs derived from astrocytes and glioblastoma cells carrying mitochondrial DNA has shown potential pathogenic implications for several neurodegenerative diseases (29). C2C12 myoblast-secreted sEVs carrying mitochondrial DNA and mitochondrial proteins influenced several transduction mechanisms in the recipient cells (33). sEVs derived from Tsuchiya Human Peripheral-1: THP-1 macrophages contained HSP27 that stimulated NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) and release of interleukin-10 contributing to anti-inflammatory effects in recipient THP-1 and human embryonic kidney cells (32).EVs are biocompatible, less immunogenic and stable in biological environments, making them a promising system to deliver therapeutics to the BBB because of their intrinsic tissuehoming capabilities (34)(35)(36)(37)(38). Here, we briefly describe our findings on the treatment outcomes of lEV mitochondria, and we encourage interested readers to refer to our published works (39)(40)(41)(42)(43)(44). In our recent work, we have discussed mechanisms of mitochondria incorporation into lEVs secreted from healthy BECs (45). We have previously demonstrated that BEC-derived lEVs but not sEVs carry mitochondria in their lumen (31,46).We compared the functional effects of lEVs and sEVs in oxygen-glucose deprived (OGD) BECs (31,46). We demonstrated that lEVs transferred their mitochondrial load to both normoxic and OGD BECs (43), see graphical abstract. Delivery of lEVmitochondria significantly improved relative intracellular ATP levels and rescued mitochondrial respiration of recipient OGD BECs (31,46,47), see graphical abstract. Moreover, intravenously injected lEVs caused a 50% reduction in infarct volume and showed a 33% improvement in neurological functions in a mouse model of transient ischemic stroke (47). Collectively, our findings have demonstrated that the delivery of lEV-mitochondria to hypoxic BECs can be a promising therapeutic approach to treat ischemic stroke.As an alternative approach to using large EVs containing mitochondria, delivery of ATP to BECs is also an effective strategy for reviving the cell viability of BECs post-duress (see graphical abstract). Restoring ATP levels in BECs can help preserve their metabolic functions and structural integrity, thereby mitigating long-term damage to brain tissue (46,(48)(49)(50). Mitochondrial dysfunction leads to decreased ATP production in BECs, resulting in an energy deficit that contributes to BBB breakdown in ischemic stroke (48,51). However, delivering ATP, a negatively charged molecule poses significant challenges due to its short half-life and limited ability to permeate cellular membranes. The extracellular half-life of free ATP is approximately 10 minutes ( 52), and its negative charge at the physiological pH of 7.4 necessitates the use of carriers for effective intracellular delivery. Liposomes have been extensively studied for ATP loading and have shown promise in enhancing energy levels in ischemic liver, cardiac, and brain tissues (53)(54)(55)(56)(57)(58). Despite being a well-established delivery platform, the thin film hydration method used for loading ATP into liposomes typically achieves only a modest ATP loading of about 5 mol% (56). Advances in processing techniques, such as reverse phase evaporation and freezing-thawing methods, have improved ATP entrapment up to a maximum of 36 to 38 mol% (56).Lipid nanoparticles (LNPs) have transformed non-viral delivery systems (59,60) and serve as mRNA carriers in the Pfizer-BioNTech (Comirnaty) and Moderna (Spikevax) COVID-19 vaccines (61). Onpattro was the first FDA-approved siRNA-LNP drug for treating polyneuropathies caused by hereditary transthyretin amyloidosis (62). LNPs can effectively transport ATP to BECs that are experiencing hypoxic conditions due to stroke, potentially increasing intracellular ATP levels and restoring energy balance in these cells. LNPs are composed of an ionizable cationic lipid and various helper lipids, including cholesterol, polyethylene glycoldimyristoyl glycerol (PEG-DMG), and distearoylphosphatidylcholine (63,64). LNPs offer several advantages, such as high cargo encapsulation efficiency up to 80-90%, protection from enzymatic degradation, improved pharmacokinetics, and reduced toxicity and immunogenicity (62,(65)(66)(67)(68)(69). The use of ionizable cationic lipids enhances the encapsulation of anionic cargo and facilitates efficient intracellular release through endosomal escape (70). We have previously demonstrated the feasibility of preparing ATP-loaded LNPs as a method for delivering ATP to injured BECs (71). Our formulation approach utilized a C12-200 ionizable cationic lipid along with other helper lipids for preparing ATP-loaded LNPs (71). Notably, PEG-DMG played a critical role in maintaining the colloidal stability of LNPs over time, and the inclusion of both ATP and PEG-DMG preserved LNP stability in the presence of serum proteins. The ATP-LNPs formulated with PEG-DMG exhibited 7.7-fold and 6.6-fold increases in ATP uptake in normoxic and hypoxic BECs, respectively (71) (see graphical abstract). In summary, our findings highlighted the potential of LNPs as innovative carriers for delivering ATP to BECs.Translating EVs and LNPs from in vitro to in vivo applications brings significant challenges in stability, targeting efficiency, and host responses to EVs (72). The biocompatibility and innate targeting ability of EVs make them attractive for drug delivery applications (73).However, developing EVs for BBB delivery will require addressing issues related to EV heterogeneity and non-specific biodistribution (74). Once administered in vivo, the EVs often accumulate in the liver and spleen due to rapid clearance by the mononuclear phagocyte system (75). Modifying the EV surfaces with peptides or proteins for receptors at the BBB can potentially increase EV accumulation at the BBB. Furthermore, the isolation and purification of EVs is associated with technical challenges: current methods such as ultracentrifugation, polymer-based precipitation, and size exclusion chromatography produce EVs with varied characteristics influencing their therapeutic efficiency (76). Development of optimized protocols for isolating mitochondria-containing large EVs is key to harnessing the promising potential of EV-mediated mitochondria delivery.A significant challenge with ATP-LNPs is related to achieving increased uptake into BECs and effective endosomal escape to release encapsulated drug cargo into the cytosol. Though LNPs have demonstrated efficacy in the delivery of nucleic acids such as mRNA vaccines and siRNA (77,78), alternative engineering techniques are required to further increase endosomal escape and cytosolic bioavailability of small molecules like ATP. Inadequate release or premature leakage of ATP from the administered LNPs can cause ATP accumulation, which may stimulate purinergic receptors and pro-inflammatory responses (79). Both EVs and ATP-LNPs are promising drug delivery platforms but their translation to in vivo therapeutic applications demands a concerted focus on improving production methods, batch to batch reproducibility, stability, BBB targeting, safety and immune compatibility. Addressing these translational hurdles is critical to unlocking their therapeutic potential.In this commentary, we have highlighted the critical role of mitochondrial function in maintaining BBB integrity and the implications of loss of BBB mitochondrial function in various CNS diseases. We propose that increasing BBB energetics through the delivery of functional mitochondria or ATP may restore BBB integrity and mitigate disease progression. We have discussed and presented evidence based on two promising therapeutic strategies to mitigate BBBrelated disorders: the use of lEVs carrying mitochondria and engineered LNPs for ATP delivery.While lEVs have demonstrated potential in improving mitochondrial function in BECs under ischemic conditions and improved outcomes in an in vivo mouse model of stroke, LNPs offer a novel method for delivering ATP to increase BEC cellular viability post-duress.