Leech Extract Enhances the Pro-Angiogenic Effects of Endothelial Cell-Derived Exosomes in a Mouse Model of Ischemic Stroke
Abstract
1. Introduction
2. Materials and Methods
2.1. Extraction and Identification of EC-Exo
2.1.1. Extraction of EC-Exo
2.1.2. Identification of EC-Exo
2.2. In Vitro Experiments
2.2.1. Cell Culture
2.2.2. Cell Grouping and Drug Administration
2.2.3. Cells Viability Assays
2.2.4. Assessment of Uptake Capacity of MBVP Uptake to EC-Exo
2.2.5. Transwell Experiments
2.2.6. Cell Scratch Assays
2.3. In Vivo Experiments
2.3.1. Animals
2.3.2. Establishment of Mouse MCAO/R Model and Leech Treatment
2.3.3. Animal Euthanasia and Organ Collection
2.3.4. Modified Neurological Severity Score (mNSS)
- (1)
- Motion test
- ①
- Tail suspension test:
- 0 points for balancing;
- 1 point for the flexion of a contralateral forelimb;
- 2 points for the flexion of a contralateral hind limb;
- 3 points for head turns away from the vertical axis by more than 10° within 30 s.
- ②
- Walking test:
- 0 points for going straight normally;
- 1 point for not being able to go straight normally;
- 2 points for turning to the same side;
- 3 points for falling on the same side as the paraplegia.
- (2)
- Sensation test:
- ①
- Placement test—mouse placed on the edge of the table:
- 0 points for forelimbs on the table;
- 1 point for no forelimbs on the table.
- ②
- Proprioception test—mouse pushed to the edge of the table:
- 0 points for resisting with the opposite forelimb;
- 1 point for no resistance.
- (3)
- Balance test—mouse placed on wooden strips with a width of 0.6 cm, height of 1.2 cm, and length of 120 cm:
- 0 points for keeping static balance for greater than 60 s;
- 1 point for grasping the edge of the balance beam tightly;
- 2 points for holding the balance beam tightly and one limb sliding off the balance beam;
- 3 points for holding the balance beam and two limbs sliding down or trying to keep balance for more than 60 s;
- 4 points for holding the balance beam and two limbs sliding down or trying to keep balance for 40 ~ 59 s and then falling down;
- 5 points for holding the balance beam and two limbs sliding down or trying to keep balance for 20 ~ 39 s and then falling down;
- 6 points for falling off the balance beam in less than 20 s or not trying to keep balance.
- (4)
- Reflection test
- ①
- Auricular reflex—touch the external auditory canal of mouse with a cotton swab:
- 0 points for shaking head;
- 1 point for not shaking.
- ②
- Corneal reflex—touch the cornea with cotton:
- 0 points for blinking;
- 1 point for not blinking.
- ③
- Panic reflex—make a noise using fast-elastic cardboard:
- 0 points for jumping;
- 1 point for not jumping.
- (5)
- Abnormal movement: 1 point for dystonia after epilepsy and myoclonia.
2.3.5. Gait Analysis
2.3.6. Observation of Velocity of Erythrocytes in Microvessels
2.3.7. TTC Staining
2.3.8. Hematoxylin–Eosin (H&E) Staining
2.3.9. Observation of Capillary Density
2.3.10. Immunofluorescence Staining
2.3.11. Western Blotting Analysis
2.3.12. Real-Time PCR
2.4. Statistical Analysis
3. Results
3.1. Isolation and Identification of Exosomes
3.2. Leech Enhances the Efficacy of EC-Exo in Facilitating the Proliferation and Migration of Pericytes
3.3. Leech Enhances the Efficacy of EC-Exo in Improving Pathology in MCAO/R Mice
3.4. Leech Enhances the Efficacy of EC-Exo in Improving Motor Function in MCAO/R Mice
3.5. Leech Enhances the Efficacy of EC-Exo in Promoting Collateral Circulation Establishment in MCAO/R Mice
3.6. Leech Modulates the Impact of EC-Exo on the HIFα-VEGF-DLL4-Notch1 Signaling Pathway in MCAO/R Mice
3.7. Leech Regulates the Species and Quantity of Angiogenesis-Related miRNA in EC-Exo
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
Exo | Exosome |
EC | Endothelial cell |
IS | Ischemic stroke |
MBVP | Mouse brain microvascular pericyte |
MCAO/R | Middle cerebral artery occlusion/reperfusion |
tPA | Tissue plasminogen activator |
OGD/R | Oxygen–glucose deprivation/reoxygenation |
mNSS | Modified neurological severity score |
WB | Western blotting |
MWCO | Molecular weight cut-off |
TEM | Transmission electron microscopy |
DLS | Dynamic light-scattering particle |
DMEM | Dulbecco’s Modified Eagle’s Medium |
CCA | Carotid artery |
ICA | Internal carotid artery |
ECA | External carotid artery |
MCA | Middle cerebral artery |
H&E | Hematoxylin–eosin staining |
MMPs | Matrix metalloproteinases |
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Type of miRNA | miRNA | Target | Function | References |
---|---|---|---|---|
Pro-angiogenic | miR-126 | VEGF | Stimulate VEGF-dependent AKT and ERK signal transduction to inhibit P85 | [43] |
PI3K | Inhibit endothelial cell damage and promote angiogenesis | [44] | ||
Pcdh7 | Promote cell proliferation and angiogenesis | |||
miR-210 | Ephrin-A3 | Effects of VEGF on capillary-like formation and endothelial cell chemotaxis | [45] | |
VEGF | ||||
miR-486 | TLR4 | Inhibit TLR4/NF-κB axis, oxidative stress, inflammation, and apoptosis; promote cell proliferation and angiogenesis | [46,47] | |
miR-218 | HMGB1 | Promote endothelial cell proliferation, migration, angiogenesis; reduce inflammatory damage | [48] | |
miR-21 | PTEN | Inhibit PTEN/PI3K/AKT and apoptosis of endothelial cells, promote angiogenesis | [49,50] | |
miR-375 | KLF5 | Endothelial cell migration, proliferation, germination, and vascular network formation; promote angiogenesis and arteriography | [51] | |
miR-214 | ATM | Stimulate angiogenesis by silencing neighboring mutant cells with cerebellar ataxia and vascular expansion through microvascular dilation | [52] | |
PI3K | Improve viability and tube formation of human endothelial cells damaged by high glucose levels | [53] | ||
COX-2 | Downregulate COX-2/PGE2 axis and target COX-2 antagonized indoxyl sulfate (IS)-induced endothelial cell apoptosis | [54,55] | ||
Anti-angiogenic | miR-34 | SirT1 | Inhibit Sirt1 to induce aging and impair EPC-mediated angiogenesis | [56] |
Notch1 | Inhibit angiogenesis induced by VEGF | [57,58] | ||
miR-15 | CCNB1 | Inhibit angiogenesis | [59] |
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Cao, Y.; Sun, J.; Guo, L.; Wang, M.; Su, L.; Zhang, T.; Wang, S.; Chai, L.; Yuan, Q.; Hu, L. Leech Extract Enhances the Pro-Angiogenic Effects of Endothelial Cell-Derived Exosomes in a Mouse Model of Ischemic Stroke. Curr. Issues Mol. Biol. 2025, 47, 499. https://doi.org/10.3390/cimb47070499
Cao Y, Sun J, Guo L, Wang M, Su L, Zhang T, Wang S, Chai L, Yuan Q, Hu L. Leech Extract Enhances the Pro-Angiogenic Effects of Endothelial Cell-Derived Exosomes in a Mouse Model of Ischemic Stroke. Current Issues in Molecular Biology. 2025; 47(7):499. https://doi.org/10.3390/cimb47070499
Chicago/Turabian StyleCao, Yushuang, Jin Sun, Lichen Guo, Meng Wang, Linlin Su, Tong Zhang, Shaoxia Wang, Lijuan Chai, Qing Yuan, and Limin Hu. 2025. "Leech Extract Enhances the Pro-Angiogenic Effects of Endothelial Cell-Derived Exosomes in a Mouse Model of Ischemic Stroke" Current Issues in Molecular Biology 47, no. 7: 499. https://doi.org/10.3390/cimb47070499
APA StyleCao, Y., Sun, J., Guo, L., Wang, M., Su, L., Zhang, T., Wang, S., Chai, L., Yuan, Q., & Hu, L. (2025). Leech Extract Enhances the Pro-Angiogenic Effects of Endothelial Cell-Derived Exosomes in a Mouse Model of Ischemic Stroke. Current Issues in Molecular Biology, 47(7), 499. https://doi.org/10.3390/cimb47070499