Molecular Imaging of the Paracrine Proangiogenic Effects of Progenitor Cell Therapy in Limb Ischemia

Abstract

Background Stem cells are thought to enhance vascular remodeling in ischemic tissue in part through paracrine effects. Using molecular imaging, we tested the hypothesis that treatment of limb ischemia with multipotential adult progenitor cells (MAPC) promotes recovery of blood flow through the recruitment of pro-angiogenic monocytes. Methods and Results Hindlimb ischemia was produced in mice by iliac artery ligation and MAPC were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPC indicated that cells survived for 1 week. Contrast-enhanced ultrasound on day 3, 7 and 21 showed a more complete recovery of blood flow and greater expansion of microvascular blood volume in MAPC-treated mice than in controls. Fluorescent microangiography demonstrated more complete distribution of flow to microvascular units in MAPC-treated mice. On ultrasound molecular imaging, expression of endothelial P-selectin and intravascular recruitment of CX3CR-1-positive monocytes was significantly higher in MAPC-treated than control groups at day 3 and 7 after arterial ligation. Muscle immunohistology showed a >10-fold greater infiltration of monocytes in MAPC-treated than control-treated ischemic limbs at all time points. Intravital microscopy of ischemic or TNF-α-treated cremaster muscle demonstrated that MAPC migrate to peri-microvascular locations and potentiate selectin-dependent leukocyte rolling. In vitro migration of human CD14+ monocytes was 10-fold greater in response to MAPC-conditioned than basal media. Conclusions In limb ischemia, MAPC stimulate the recruitment of pro-angiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond MAPC lifespan suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype.

Ultrasound imaging with bolus delivered contrast agent for the detection of angiogenesis and blood flow irregularities

“…Here, the contrast agent washout was shown to be delayed from these complex vessels, thus indicating possible blood pooling. Evidence of blood pooling has recently also emerged elsewhere (10), in line with our results. What will be important to be determined is whether these kinetic changes in blood circulation are balancing the angiogenic changes so that tissue function is improved ("functional shunting") or whether these changes can be even deleterious to tissue function.…”

Quantification of residual limb skeletal muscle perfusion with contrast-enhanced ultrasound during application of a focal junctional tourniquet

“…Advantages of using CEU for assessment of limb perfusion are that it is rapid and safe, provides spatial assessment of perfusion, and has sufficient sensitivity to detect flow even when it is severely reduced. 15,23 Limb skeletal muscle perfusion imaging with CEU at rest and hyperemic flow during exercise stress has been demonstrated previously to provide information on the severity of PAD and correlates better with claudication symptoms than ABI or exercise ABI does. 8 It has also been used to evaluate the presence of a collateral arterial network in severe PAD.…”

“…Video intensity was measured from regions of interest placed over the thigh rectus femoris and calf soleus and gastrocnemius muscles after subtracting averaged frames obtained during continuous imaging to eliminate signal from tissue and large intramuscular vessels. 14,15 Pulsing interval vs intensity data were fit to the function y = A (1 – e — β t ), where y is intensity at time t , A is the plateau intensity reflecting MBV, and β is the rate constant of microbubble replenishment reflecting microvascular blood flux rate. Skeletal muscle MBV was quantified by the following: A /(1.06 × I B × F ), where 1.06 is tissue density (g/cm 3 ) and F is the scaling factor (3) that corrects for the different infusion rate for measuring I B to avoid dynamic range saturation.…”

Therapies intended for joint regeneration in the horse

“…Other inevitable questions emerge when MSC are considered: do the cells need to be injected, or just the GFs released from the MSC, or even the culture media? (Kurazumi et al 2011, Cargnoni et al 2012, Oshimori and Fuchs 2012, Ryu et al 2013.…”