Yeonkyeong Yoo

my ppt slide image is uploaded on my blog
https://blog.naver.com/yk980315/223280155093


 H2O2 induced aged Blood brain barrier in vitro model recapitulate age-related barrier dysfunction and altered Drug and antibody delivery

20225498유연경

Introduction
The human Blood-brain barrier (BBB) serves as a distintive and selective physiological barricade that regulates the exchange of chemical and nutrients between the peripheral and central nervous system (CNS) to maintain homeostasis.[1,2] The BBB is composed of brain microvascular endothelial cell (BMVEC), which stands apart from in peripheral capillaries, due to its strong tight junctions, which restrict paracellular transit and instead, require that transcytosis be used to transport molecules from the bloodstream to CNS. Additionally, BMVEC also express versatile efflux pumps on their luminal surface that obstructing the entry of lipophilic molecules including a multitude of pharmaceutical drugs, in the brain.[3,4]

A promising strategy is to target CNS drugs towards BBB-associated carrier and receptor proteins, among these proteins, the targeting of the transferrin receptor (TfR), expressed by BMVEC.[11] Nonetheless, in the aging brain, a notable shift, ligand-specific receptor-mediated to non-specific caveolar transcytosis occur, and TfR mediated is diminished. Consequently, there is a great need for a human aged-BBB model that could be used to develop targeting therapeutics and delivery technologies as well as advance fundamental and translational research and more effective drug for age related CNS disease, AD, PD, ALS.[12]

In this study, we develop the human aged-BBB model that contain BMVEC, astrocyte, pericyte that have undergone aging induction using Hydrogen peroxide (H2O2)[13] , a well established chemical for simulating the aging process to generate reactive oxygen species (ROS) and is commonly used in this context.
In future, we want to show the aged BBB exhibits distinct physiological changes, with a notable decrease in the expression of key proteins associated with transcytosis, such as TfR and Mfsd2a. Conversely, there is an increase in the expression of caveolin-1, a marker of a non-specific uptake pathway. Consequently, there changes lead to alterations in the transcytosis process of well known BBB-permeable drugs, such as Angio-pep, BBB01. Our aged BBB in vitro model represents a promising and innovate platform for investigating potential therapeutic targets for CNS and neurodegenerative disease.


Results
In order to construct a human blood-brain barrier (BBB) aging model, we investigated proper environmental cues. Various factors associated with endothelial cells, such as iron, H2O2, and calcification, were identified. H2O2, a well-known aging inducer by inhibiting DNA synthesis by reactive oxygen species (ROS) and proved to be the most well accepted reagent.[15,16,17,18,19] Calcification is a well known characteristic feature of aged vessels, including atherosclerosis.[20] Additionally, calcification is the factor that believed to be associated with the increased non-specific caveolar vesicle and transcytosis, which was identified as pericyte loss related disease.[14] However, the experimental procedures are intricate and require a substantial duration.[21] Iron, as a novel factor, was arised from the result about accumulation of iron in aged humans. Indeed, it has been revealed that iron overload is more occured in the brains of aged individuals and Alzheimer’s disease patients.[22] Nevertheless, little is known about the reasons behind iron accumulation, whether it is a consequence of aging, and if iron accumulation accelerates or induces aging. Currently, the prevailing theory suggests that impaired autophagy leads to the accumulation of iron in ferritin due to ineffective degradation.[23] Therefore, if employing iron in studies, it becomes crucial to discern whether the interaction between autophagy and iron is pivotal or if prolonged iron overload alone can lead to the observed effects. In this situation, In vitro studies may play a significant role in addressing these questions.Although Calcification and iron would be contributable topic, However, due to my graduation deadline and the widespread acceptance of H2O2 as a factor for aging,  we use H2O2 as human microvascular endothelial cell aging inducer. Remarkably, there are some existing research findings that indicate the occurrence of calcification[24] and iron-induced ferroptosis[25] as a result of H2O2 exposure.

Our ultimate goal was to create an aging model with factors that are universally accepted by people. Therefore, we aimed to determine the optimal concentration and duration of H2O2 exposure, and subsequently introduce various antibodies, aptamers, and drugs into the model to observe differences compared to a standard model. For selecting time points and concentrations, we intended to utilize qRT-PCR to assess markers such as p21 (an aging marker)[15], tfrc, mfsd2a, and cav1 (markers associated with transcytosis shift).[14]
First, we utilized human brain microvascular endothelial cells obtained from a cell systems provider. We employed the conventional medium, commonly accepted for culturing these cells, which includes 10% Fetal Bovine Serum (FBS).In determining the experimental conditions, we first identified fundamental parameters that needed to be established before optimization H2O2 condition. These included cell density, culture dish selection, and the culture medium. The culture medium remained unchanged, while the cell density was set to a number that was anticipated to achieve 100% confluency in the control group by the final date to account for the potential disruption of the monolayer. For the culture dish, we choose plates in preliminary study than transwells due to the latter being more intricate and expensive. And also we believe that both results would be simillary. Conditions related to H2O2 were established as closely as possible following references.[15,16,17,18] H2O2 treatment is typically acute, administered for a duration of 2 hours rather than chronically. This acute exposure induces issues in DNA synthesis, gradually showing aging features. Additionally, as the observed effects appear until the fourth day, and considering the limitations posed by primary cells, we take a schedule of H2O2 administration every other day to maximize the effects within a one-week.

The results from the initial attempt revealed that the cells exhibited sufficient senescence features. However, Because of cell proliferation significantly decreased, making it impractical to proceed with the experiment at the current density. Moreover, the reduced adhesion strength led more than half of the cells detaching during fixation for staining.In conclusion, the first experiment highlighted that the concentration of H2O2 was too high, and there was a need to increase the cell density. These findings will inform adjustments for subsequent experiments to enhance the overall experimental conditions.

The question arose about the necessity of examining so many markers and whether the desired changes could be observed through qRT-PCR. Consequently, a reevaluation of the relationship between Mfsd2a, Tfrc, and Cav1 was conducted Some researchers reported that this relationship was confirmed through RNA-seq. In summary, Mfsd2a and Cav1 were shown to exhibit an inverse relationship, regulated by lipid composition not related with gene signaling. And general researchers use Mfsd2a as a gene, while Cav1 as a protein, or with electron microscopy revealing a more definitive connection to vesicle formation. Tfrc, on the other hand, appeared unrelated to the former two and seemed to be associated with BBB features, suggesting a potential decrease with aging.[23] As qRT-PCR was the most effective tool for quantifying changes in our lab, we decided to focus on changes in the Mfsd2a gene, narrowing the scope for now, and considering transport-related factors for future investigations.

After several experiments and discussions with professor, we decided to revert to the basics. Instead of administering H2O2 repeatedly, we choose a single treatment. Additionally, we chose to utilize the maturation method commonly employed in our lab for creating blood-brain barrier endothelial cell (BMEC) monolayers to achieve full confluency. Furthermore, we planned to harvest cells at different time points to examine gene expression.The second attempt was successful, revealing a temporal pattern of decreased and then increased expression of mfsd2a. In the subsequent experiments, we plan to verify if this pattern is reproducible and devise experiments to explore trends in transport.

Currently, we are faced with another challenge. Initially, when design the experiment, there was a focus on the features of aging blood-brain barrier (BBB); however, there was a mistake of investigation into whether the same phenomena occur in humans. Papers exclusively studying Human Brain Microvascular Endothelial Cells (BMECs) were extremely scarce, with only two identified, both of which included samples from Alzheimer's disease (AD) patients.

In one study, it was observed that Mfsd2a in normal aged human BMECs increased, contrary to our hypothesis, while in AD patients, it decreased. Thus, in normal aging, the findings contradicted our hypothesis, but in aging with AD, the results aligned with our hypothesis.[27] Another paper suggested a successful decrease in Mfsd2a during the Braak stage V in AD patients, followed by a subsequent increase inBraak VI.[28] This complex situation has led to considerable confusion, raising doubts about considering H2O2 as a factor for AD. Additionally, we grappled with how to address and defend against these unexpected findings.

Three potential alternatives crossed my mind: 1) Slightly shifting the focus of the topic to incorporate AD elements, 2) Generating results using human samples, and 3) Refuting the findings through reference analysis. Considering option 3, One suggested an increase in Mfsd2a in aged humans and a decrease in AD patients, while the other indicated a decrease in Mfsd2a in AD patients at a specific stage. Regarding the first paper, it relied on IHC staining data for normal aging humans, which might yield different results than RNA data. Moreover, the p-value of 0.0505 is not extremely significant, leaving room for alternative interpretations. For the second paper, the observed increase in Mfsd2a in the later stages of Braak suggests a departure from the typical trends, especially considering the unexpected outcomes for amyloid beta and tau.[29] Since this increase represents an advanced and severe stage, potentially accelerating the disease, the decrease in Mfsd2a at Braak V might be more noteworthy While I'm unsure if my considerations are accurate, further discussion with the professor is crucial in determining the next steps.

The power what I gained through omics class is extends beyond RNA expression, emphasizing the presence of a vast expressome in the world. It instills a mindset that caution should be exercised before placing unwavering trust in any given data and fosters an ability to paint a holistic picture. Moreover, through Jong's guidance, I've learned the importance of never losing the romanticism inherent in being a researcher, a quality essential for pursuing ultimate research endeavors.



Conclusion
For the successful discovery of drug delivery agents, particularly those targeting the blood-brain barrier (BBB), it is crucial to Recapitulate it in vitro. Additionally, there is a need for in vitro models to test aging drugs and study the mechanisms of cellular aging in the era of aging.

Our team(only me..?) believes that features such as transcytosis global shift, downregulated Mfsd2a, and upregulated non-specific caveolae transcytosis, which are among the aging features of the BBB, are essential for an in vitro model for drug delivery development. We aimed to observe these changes using hydrogen peroxide (H2O2), commonly considered as a cue for inducing aging. If more studies are conducted successfully to establish this model, it could significantly aid attempts to deliver drugs to the brains of aging and neurodegenerative disease patients. Furthermore, it could serve as a model for testing therapies for aging endothelial cells.

However, the specific changes in aging human samples without disease have not been clearly elucidated, emphasizing the need for further research in humans. Nevertheless, considering the consistency in results from Alzheimer's disease (AD) human RNA sequencing and immunohistochemistry (IHC), along with the many reports related with increase in Cav1 with aging[30,31,32], we consider the model itself to be valid.

SELF
1. Homework : I did not well prepared Presentation work. B+(3.5) .
2. Questions: I think that I did well harsh thinking on this class. A+(4.5)
3. Presenations: I did not make any presentations. So I got B0(3.0)
4. Quiz/Exams: I always tried to make answer and think about Jong’s question. A+(4.5)
5. Evaluation: In my opinion, This class for me to think harsh. And I think harsh, also that’s what Jong wants. A+(4.5)

Average  
4.0 (A0)



 

 

References

[1] Mahringer, A., Ott, M., Reimold, I., Reichel, V. & Fricker, G. The ABC of the blood-brain barrier-regulation of drug efflux pumps. Curr. Pharm. Des. 17, 2762–2770 (2011).
[2] Shawahna, R. et al. Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels. Mol. Pharm. 8, 1332–1341 (2011).
[3] Abbott, N. J. & Friedman, A. Overview and introduction: the blood-brain barrier in health and disease. Epilepsia 53, 1–6 (2012).
[4] Herland, A. et al. Distinct contributions of astrocytes and pericytes to neuroinflammation identified in a 3D human blood-brain barrier on a chip. PLoS One 11, e0150360 (2016).
[5] O’Brien, F. E., Dinan, T. G., Griffin, B. T. & Cryan, J. F. Interactions between antidepressants and P-glycoprotein at the blood-brain barrier: clinical significance of in vitro and in vivo findings. Br. J. Pharmacol. 165, 289–312 (2012).
[6] Jorfi, M., D’Avanzo, C., Kim, D. Y. & Irimia, D. Three-dimensional models of the human brain development and diseases. Adv. Healthc. Mater. 7, 1700723 (2018).
[7] Lauschke, K., Frederiksen, L. & Hall, V. J. Paving the way toward complex blood-brain barrier models using pluripotent stem cells. Stem Cells Dev. 26, 857–874 (2017).
[8] Cecchelli, R. et al. Modelling of the blood–brain barrier in drug discovery and development. Nat. Rev. Drug Discov. 6, 650–661 (2007).
[9] Park, TE., Mustafaoglu, N., Herland, A. et al. Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies. Nat Commun 10, 2621 (2019).
[10] Wolff, A., Antfolk, M., Brodin, B. & Tenje, M. et al. In vitro blood-brain barrier models—an overview of established models and new microfluidic approaches. J. Pharm. Sci. 104, 2727–2746 (2015).
[11]I. Mager, Axel H. Meyer et al. Targeting blood-brain-barrier transcytosis e perspectives for drug delivery . Neuropharmacology 120, 4-7 (2017).
[12]Topiwala, A., Mankia, K., Bell, S. et al. Association of gout with brain reserve and vulnerability to neurodegenerative disease. Nat Commun 14, 2844 (2023). 
[13]Ko, H., Kim, MM. H₂O₂ promotes the aging process of melanogenesis through modulation of MITF and Nrf2. Mol Biol Rep 46, 2461–2471 (2019)
[14]Yang, A.C., Stevens, M.Y., Chen, M.B. et al. Physiological blood–brain transport is impaired with age by a shift in transcytosis. Nature 583, 425–430 (2020).
[15]Koji Itahana et al Methods to detect Biomarkers of cellular Senescence Methods in Mol. Bio. 371 21-31
[16]Ellaine Salvador et al Senescence and associated blood-brain barrier alterations in vitro Histochem. Cell. Bio. 156,283-292 (2021)
[17] Pooi-Fong Wong et al Senescent huvecs-secreted exosomes trigger Endothelial barrier dysfunction in young endothelial cells. EXCLI 18,764-776 (2019)
[18] Q chen et al Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroclast F65 cells PNAS 10 4130-4134 (1994)
[19] Yu Yamazaki et al. Vascular cell senescence contributes to Blood-brain barrier breakdown strokeStroke47 1068–1077 (2016)
[20] Apurba Chakrabarti et alAge-associated arterial calcification: the current pursuit of aggravating and mitigating factorsCurr Opin Lipidol.5: 265–272.(2020)
[21] Elisa Persiani et al Protocol to generate an in vitro model to study vascular calcification using human endothelial and smooth muscle cells , STAR Protocols4, 102328 (2020)
[22]Belaidi, A.A., Masaldan, S., Southon, A. et al. Apolipoprotein E potently inhibits ferroptosis by blocking ferritinophagy. Mol Psychiatry (2022).
[23] Shashank Masaldan et al Iron accumulation in senescent cells is coupled with impaired ferritinophagy and inhibition of ferroptosis 14 110-115 (2018)
[24] Chang Hyun Byon et al,Oxidative Stress Induces Vascular Calcification through Modulation of the Osteogenic Transcription Factor Runx2 by AKT Signaling, THE JOURNAL OF BIOLOGICAL CHEMISTRY 22 15319 –15327 (2008)
[25][Author Yuko Takashi]et al, Mitochondrial dysfunction promotes aquaporin expression that controls hydrogen peroxide permeability and ferroptosis Free Radic Biol Med. 16160–70. (2020)
[26]Yang et al. , Loss of epigenetic information as a cause of mammalian aging ,Cell 186, 305–326
(2023)
[27]Zhao, L., Li, Z., Vong, J.S.L. et al. Pharmacologically reversible zonation-dependent endothelial cell transcriptomic changes with neurodegenerative disease associations in the aged brain. Nat Commun 11, 4413 (2020).
[28]Bryant et al., Endothelial Cell Heterogeneity in Alzheimer’s Disease, The Journal of Neuroscience, 24:4541–4557 (2023)
[29] Miller et al. Neuropathological and transcriptomic characteristics of the aged brain eLife,6:e31126 (2017)
[30] Min Hee park et al. Vascular Neurogenic Rejuvenation in Aging Mice by modulation of ASM Neuron 10 167-182 (2018)
[31] Park Hyun Ju et al. Increased Caveoin-2 Expression in Brain Endothelial cells promotes age-related Neuroinflammation Molecules and cells 12:950-962 (2022)
[32] Huafei Zou et al.Caveolin-1, cellular senescence and age-related diseasesMech Ageing Dev. 11-12 533–542.(2011)