These results present the first step toward development of an x-ray-visible cellular therapy for the treatment of PAD. While most cellular clinical PAD trials have used autologous cells and a recent meta-analysis of these intramuscular PAD cell therapy trials has shown potential efficacy, [26
] an increasing body of evidence suggests that patients with cardiovascular disease often have native cells that are impaired [27
]. Thus, allogeneic cellular therapy from healthy donors may be preferred. In this study, an x-ray-visible cellular therapy comprises allogeneic bone marrow-derived MSCs encapsulated in alginate impregnated with barium sulfate demonstrated high MSC viability with a high sensitivity for detection.
MSC-Xcaps improved hind limb perfusion in an endovascular rabbit model of PAD with easy visualization of the therapeutic at the time of implantation and up to day 14 using standard x-ray clinical imaging suites. MSC-Xcaps improved hind limb perfusion through both arteriogenic and angiogenic mechanisms. Whereas arteriogenesis is the development of conductance vessels through the remodeling of pre-existing arterioles resulting in an increase in luminal area, this is a different process than angiogenesis. Angiogenesis is the sprouting of new capillaries from pre-existing capillaries. Angiogenesis is important for local tissue oxygen delivery but cannot substitute for a proximal obstruction in a conductance vessel. In this endovascular model of hind limb ischemia, the SFA, the main conductance vessel to the calf, was occluded. The lower modified TIMI frame count in the MSC-Xcap group, that is, the more rapid contrast opacification of the conductance calf vessels in the MSC-Xcap group compared to the control groups, demonstrates the arteriogenic effect of this therapy. The latter was confirmed with almost a twofold increase in the number of small arterioles in the MSC-Xcap group relative to sham or naked cell injections. Interestingly, we were unable to detect a difference in either the size of the DDFA or the number of collateral vessels between groups by angiography to explain the increased conductance blood flow. We hypothesize that the remodeled arterioles in the MSC-Xcap group are larger than in the control group. However, accurate measurement of these small diameter vessels is difficult on digital subtraction angiography as the vessels approach the spatial resolution of the images or on post-mortem sections due to artifacts due to freezing. Furthermore, while Xcaps have the desirable effect of visibility on x-ray imaging, they do obscure the detection of small neovessels. Nonetheless, in addition to the MSC-Xcaps’ proarteriogenic effect, this group also demonstrated a proangiogenic effect of increased capillary density.
In ischemic hind limb experiments, MSCs have been studied extensively. The precise role that MSCs have in promoting arteriogenesis is unclear. There is debate about whether the administered cells have the “leading role” marked by incorporation and/or fusion within the enlarging vessel wall or whether they function in a “supporting role” to produce multiple cytokines in the perivascular tissue in order to enlarge the blood vessel [29
]. MSCs are known to act in a paracrine fashion through expression of a variety of arteriogenic cytokines: vascular endothelial growth factor; fibroblastic growth factor; placental growth factor; monocyte chemoattractant protein; insulin-like growth factor; interleukins, and so forth [30
]. Additionally, MSCs have the potential to differentiate into components of the vascular wall [11
]. Since there was no microscopic evidence of capsule rupture and capillary density adjacent to the MSC-Xcaps was increased, the MSCs within the XCaps were unable to directly incorporate into new vessels. As a result, the mechanism of enhanced hind limb perfusion in MSC-XCap-treated animals appears to be through paracrine mechanisms, exclusively. Unfortunately, determining the exact profile of minute quantities of cytokines by exogenously delivered allogeneic MSCs from native proteins would be extremely difficult to perform in vivo.
The “cellular protection” afforded by the Xcaps could explain the improved hind limb perfusion in the MSC-Xcaps group compared to the naked MSCs. Cellular encapsulation isolates the MSCs immunologically; the pores within the capsules are large enough to permit the diffusion of nutrients into the capsule, but small enough to prevent antibody or immune cell entry [17
]. An alternate or complementary explanation may be that MSC microencapsulation enhances cellular survival in vivo via an ischemic preconditioning mechanism [37
]. Ischemic preconditioning refers to short bouts of hypoxia that upregulate endogenous mechanisms to protect against future cell death when exposed to lethal low oxygenation conditions. In this study, cell survival after microencapsulation in culture, where ideal oxygenation and nutrient supply are available, was neither enhanced nor impaired. However, other investigators have shown that the MSC administration in an ischemic environment leads to significant death of naked MSCs shortly after administration in part due to the host immune response and/or the poor oxygenation and nutrient supply [14
]. Thus, if encapsulation could prevent early destruction, the therapeutic effect of MSCs, via a paracrine mechanism, could potentially be prolonged or enhanced. Practically, prolonging cell survival will have commercial impact as MSCs are a rare component of the bone marrow, that is, 0.01%–0.001%. Therefore, ex vivo expansion, a time and resource intensive process, is required to produce sufficient quantities for a therapeutic dose. By protecting the cells from the immune system, the administered dose could potentially be decreased and, if combined with the utilization of banked allogeneic stem cells, could result in a more cost-effective acute therapy. In this particular study, a positive angiogenic effect was achieved using an administered dose of MSCs in the MSC-Xcap group that was more than 40-fold reduced compared to naked stem cells. In this study, the MSC-Xcap dose was chosen based on a comparable volume to naked MSC injections. Future studies will be needed to determine whether there will be an additional dose response to larger numbers of MSC-Xcaps or whether microencapsulation could provide an off-the-shelf therapy that can be tracked with minimal cell manipulation, unlike gene therapy [39
] or preincubation with proangiogenic cocktails [40
Having an x-ray-visible stem cell therapeutic could have direct bearing on patient PAD trials. Because of the large volume of tissue in the human leg, the locations where the cells are administered need to be more precisely targeted than in rodent models with minimal leg muscle mass. Also, unlike rodent models, there are significant differences between the muscle mass/subcutaneous fat ratios in patients, which can affect delivery location. Prior clinical trials have delivered the therapeutic agent via blind intramuscular injections 1.5–2.5 cm deep into the thigh and calf [41
]. Because of the differences in body habitus, some patients received the agent deep within the muscle, while others received the agent in the subcutaneous fat. Potential reasons for the disappointing clinical results [43
] are poor injection localization and poor cell survival; two obstacles the Xcap approach could help to overcome. In our study, the MSC-Xcaps were clearly visible on clinical imaging systems, both at the time of implantation and on day 14, which would permit the treating physician to confirm accurate delivery and persistence of the therapeutic payload.
The initial MSC-Xcaps biocompatibility profile appears promising. Once the cells were encapsulated, there was neither significant cell death nor proliferation in the MSC-Xcaps over a 30-day period. Furthermore, naked MSCs and MSCXcaps neither demonstrated a significant difference in the inflammatory scores nor adversely affected hematologic parameters. Indeed, MSC-Xcaps in our study had a lower inflammatory score (mean score, <3) than was seen previously with normal wound healing after surgical ligation of the femoral artery in rabbits (mean score, 5.5) [18
]. Whereas encapsulated allogeneic islet cells have often resulted in an inflammatory response that leads to suffocation and cell death [45
], the native MSC immunotolerance properties [47
] may contribute to further enhanced survival and therapeutic effect in Xcaps. In fact, while the total administered dose of MSCs in Xcaps was several fold smaller than naked MSCs, survival of encapsulated MSCs did not elicit a foreign body reaction. Nonetheless, only a short-term follow-up of 2 weeks was performed in this study. Whereas previous work by our group has shown that barium microcapsules remain intact for at least 1 month [17
] and given the positive results of this study, long-term biocompatibility and stability of barium sulfate-impregnated alginate microcapsules beyond 2–4 weeks would be warranted.
The surgical and endovascular animal PAD models, like most animal models, have limitations. Both involve a relatively acute occlusive event in a single vessel segment in an otherwise healthy animal. Our endovascular PAD model in the rabbit hind limb, however, has several features that make it superior to surgical models for the study of therapeutic arteriogenesis. The endovascular model benefits are: (a) like atherosclerosis, vessel occlusion is achieved from within the lumen; (b) there is no hind limb surgical wound-healing response to induce local stem cell recruitment [6
]; (c) pre-existing collateral vessels in the thigh are not disrupted as occurs with surgical dissection; and (d) there is no postoperative pain in the treated limb to impair mobility. As such, endovascular occlusion causes significantly less disturbance to the surrounding tissue and a minimal wound-healing response thereby better resembling the pathophysiology of PAD in patients [18