In the run up to the World Congress of Microcirculation, Beijing in September this year (20th-24th), we are looking at one of our more common applications in various perspectives: The pre-clinical use of the hind limb ischemia assessment techniques to develop novel therapies for peripheral arterial diseases and related applications.
Here, in the second episode, we briefly consider recent use of the hind limb ischemia model for the assessment of cell treatments of peripheral arterial disease in studied on vascularisation following femoral artery occlusion.
We hope this provides useful background information on current uses of the measurement, practicalities. Ultimately we aim to help you make a well-informed choice on the equipment options available to you.
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Apoptosis and oxidative stress are factors that can limit the effectiveness of injected cells. Strategies to overcome these obstacles are investigated: Ishizaki et al administered the synthetic triterpenoid RTA dh404 prior to injection of adipose-derived regenerative cells (ADRCs) into mice with ischemic lower limbs. They measured a 25% decrease in apoptotic ADRCs, a 10% increase in neovascularization and a 63% increase in salvage ratio for RTA dh404 treated compared with control mice.
This unfavourable microenvironment of ischemic tissue was addressed by Tang et al, with use of platelet extracellular vesicles (PEV) to protect and enhance the proangiogenic potential of adipose-derived mesenchymal stem cells (ADSC). Hind limb reperfusion, over the 21-day recovery period, assessed by moorFLPI2 Laser Speckle Contrast Imager, was consistently higher for ADSC-PEV than for ADSC alone and both were higher than for saline control.
Infusion of endothelial progenitor cells but not of mature endothelial cells promotes neovascularization after ischemia. Presence of the protease cathepsin L was found by Ubrich et al to be essential for the promotion of neovascularization after ischemia, assessed by moorLDI2 Laser Doppler Imaging. Infused cathepsin L-deficient endothelial progenitor cells failed to home to sites of ischemia and to augment neovascularization whereas over-expression of cathepsin L in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo.
The therapeutic efficacy of stem cell-derived extracellular vesicles (EVs) is limited due to their degradation and rapid diffusion after injection. Hao et al developed a new generation of collagen binding EVs by conjugation with the collagen binding peptide SILY (RRANAALKAGELYKSILY, a high-affinity collagen binding ligand derived from platelet membrane receptors that bind to α1 chains in collagen). Efficacy was assessed, with murine hind limb ischemia and a range of tests including moorLDI which found blood perfusion in the SILY-EV treatment group was significantly improved compared to the EV treatment group on days 14 (p<0.05) and 21 (p<0.01) after surgery.
The hind limb ischemia study of van Rhijn-Brouwer et al on human bone marrow mononuclear cells, by intramuscular administration into murine hind limbs, 24 hours post hind limb ischemia, found a reperfusion pattern, assessed by moorLDI on days 0, 1, 4, 7, 10, and 14 post hind limb ischemia, that was practically the same as vehicle control. However, bone marrow-derived mesenchymal stromal cells significantly increased perfusion compared with both (p<0.001 at day14).
The wide range of cell therapy studies are reviewed by Beltrán-Camacho et al, 2021.
Devices used for these studies include:
The moorLDI2 Laser Dopper Imaging series (red, IR and HIR) standard and high resolution imagers; these provide relatively deep tissue penetration with a scan resolution down to 50 microns with the HIR version, 100 microns standard.
The moorFLPI-2 Laser Speckle Contrast Imager has a resolution of 5microns in temporal mode and is capable of up to 100 frames per second in spatial mode.
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These studies also contain a wealth of references to earlier important studies.
Beltrán-Camacho L , Rojas-Torres M and Durán-Ruiz MC.
Current Status of Angiogenic Cell Therapy and Related Strategies Applied in Critical Limb Ischemia.
Int J Mol Sci. 2021 Feb 26;22(5):2335.
Hao D, Lu L, Song H, Duan Y, Chen J, Carney R, Li JJ, Zhou P, Nolta J, Lam KS, Leach JK, Farmer DL, Panitch A, Wang A.
Engineered extracellular vesicles with high collagen-binding affinity present superior in situ retention and therapeutic efficacy in tissue repair.
Theranostics. 2022 Aug 8;12(13):6021-6037.
Ishizaki Y, Sasaki KI, Yoshikawa T, Nakayoshi T, Sasaki M, Ohtsuka M, Hatada-Katakabe S, Takata Y, Fukumoto Y.
RTA-dh404 decreased oxidative stress in mice ischemic limbs and augmented efficacy of therapeutic angiogenesis by intramuscular injection of adipose-derived regenerative cells in the limbs.
Eur J Pharmacol. 2023 Jan 5;938:175422.
van Rhijn-Brouwer FCCC, Gremmels H, Ouden KD, Teraa M, Fledderus JO, Verhaar MC.
Human Bone Marrow Mononuclear Cells Do Not Improve Limb Perfusion in the Hindlimb Ischemia Model
Stem Cells Dev. April 2022; 31(7-8): 176–180.
Tang Y, Li J, Wang W, Chen B, Chen J, Shen Z, Hou J, Mei Y, Liu S, Zhang L, Li Z and Lu S.
Platelet extracellular vesicles enhance the proangiogenic potential of adipose‑derived stem cells in vivo and in vitro.
Stem Cell Research & Therapy volume 12, Article number: 497 (2021).
Urbich C, Heeschen C, Aicher A, Sasaki K-I, Bruhl T, Farhadi MR, Vajkoczy P, Hofmann WK, Peters C, Pennacchio LA, Abolmaali ND, Chavakis E, Reinheckel T, Zeiher AM, Dimmeler S.
Cathepsin L is required for endothelial progenitor cell-induced neovascularization.
Nat Med. 2005 Feb;11(2):206-13.