High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Front. Cardiovasc. Med.

et al. 2022

Self Cite

For patients with chronic limb-threatening ischemia and limited revascularization options, alternate means for therapeutic angiogenesis and limb salvage are needed. E-selectin is a cell adhesion molecule that is critical for inflammation and neovascularization in areas of wound healing and ischemia. Here, we tested the efficacy of modifying ischemic limb tissue by intramuscular administration of E-selectin/AAV2/2 (adeno-associated virus serotype 2/2) to modulate angiogenic and inflammatory responses in a murine hindlimb gangrene model. Limb appearance, reperfusion, and functional recovery were assessed for 3 weeks after induction of ischemia. Mice receiving E-selectin/AAV2/2 gene therapy had reduced gangrene severity, increased limb and footpad perfusion, enhanced recruitment of endothelial progenitor cells, and improved performance on treadmill testing compared to control group. Histologically, E-selectin/AAV2/2 gene therapy was associated with increased vascularity and preserved myofiber integrity. E-selectin/AAV2/2 gene therapy also upregulated a panel of pro-angiogenic genes yet downregulated another group of genes associated with the inflammatory response. This novel gene therapy did not induce adverse effects on coagulability, or hematologic, hepatic, and renal function. Our findings highlight the potential of E-selectin/AAV2/2 gene therapy for improving limb perfusion and function in patients with chronic limb-threatening ischemia.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

E-Selectin/AAV2/2 Gene Therapy Alters Angiogenesis and Inflammatory Gene Profiles in Mouse Gangrene Model

Ribieras

Front. Cardiovasc. Med.

et al. 2022

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“…Briefly, 8- to 10-week-old male and female (50%; 50%) FVB/NJ mice (001800; Jackson Laboratory) underwent femoral artery and vein ligation at proximal and distal sites. Hindlimb ischemia was confirmed by preoperative and immediate postoperative laser Doppler imaging measurements were obtained as described 23–25 . Any mouse that did not demonstrate postoperative perfusion index of 5% or less of preoperative levels were excluded from further analysis in the study.…”

Section: Methodsmentioning

confidence: 99%

“…Hindlimb ischemia was confirmed by preoperative and immediate postoperative laser Doppler imaging measurements were obtained as described. [23][24][25] Any mouse that did not demonstrate postoperative perfusion index of 5% or less of preoperative levels were excluded from further analysis in the study. A cutaneous wound (4 mm punch biopsy) was created over the hindlimb ventral aspect.…”

Section: Murine Model Of Hindlimb Ischemia and Cutaneous Wound Healingmentioning

confidence: 99%

“…Whole-body perfusion with lipophilic carbocyanine dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, #D282; Invitrogen) was performed as described. 25 Imaging with the Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems) was performed using ×5 magnification. Z-stack images were utilized for 3-dimensional image reconstruction and analyzed using Fiji.…”

Section: Perfusion and Confocal Microscopymentioning

confidence: 99%

See 1 more Smart Citation

Novel Gene-Modified Mesenchymal Stem Cell Therapy Reverses Impaired Wound Healing in Ischemic Limbs

Huerta

et al. 2023

Annals of Surgery

Objective: Here, we report a new method to increase the therapeutic potential of mesenchymal stem/stromal cells (MSCs) for ischemic wound healing. We tested biological effects of MSCs modified with E-selectin, a cell adhesion molecule capable of inducing postnatal neovascularization, on a translational murine model. Background: Tissue loss significantly worsens the risk of extremity amputation for patients with chronic limb-threatening ischemia. MSC-based therapeutics hold major promise for wound healing and therapeutic angiogenesis, but unmodified MSCs demonstrate only modest benefits. Methods: Bone marrow cells harvested from FVB/ROSA26SormTmG donor mice were transduced with E-selectin-green fluorescent protein (GFP)/AAV-DJ or GFP/AAV-DJ (control). Ischemic wounds were created via a 4 mm punch biopsy in the ipsilateral limb after femoral artery ligation in recipient FVB mice and subsequently injected with phosphate-buffered saline or 1×106 donor MSCGFP or MSCE-selectin-GFP. Wound closure was monitored daily for 7 postoperative days, and tissues were harvested for molecular and histologic analysis and immunofluorescence. Whole-body DiI perfusion and confocal microscopy were utilized to evaluate wound angiogenesis. Results: Unmodified MSCs do not express E-selectin, and MSCE-selectin-GFP gain stronger MSC phenotype yet maintain trilineage differentiation and colony-forming capability. MSCE-selectin-GFP therapy accelerates wound healing compared with MSCGFP and phosphate-buffered saline treatment. Engrafted MSCE-selectin-GFP manifest stronger survival and viability in wounds at postoperative day 7. Ischemic wounds treated with MSCE-selectin-GFP exhibit more abundant collagen deposition and enhanced angiogenic response. Conclusions: We establish a novel method to potentiate regenerative and proangiogenic capability of MSCs by modification with E-selectin/adeno-associated virus. This innovative therapy carries the potential as a platform worthy of future clinical studies.

E-Selectin/AAV Gene Therapy Promotes Myogenesis and Skeletal Muscle Recovery in a Mouse Hindlimb Ischemia Model

Ribieras

Cardiovascular Therapeutics

et al. 2023

Self Cite

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.