Bone marrow derived cells in collateral formation : studies toward therapeutic arteriogenesis
Hellingman, A.A.
Citation
Hellingman, A. A. (2011, September 15). Bone marrow derived cells in collateral formation : studies toward therapeutic arteriogenesis. Retrieved from https://hdl.handle.net/1887/17838
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Chapter 9
Summary and General Discussion
General discussion and summary 151
SUMMARY
Peripheral artery disease (PAD) remains a major cause of morbidity and mortality in the Western world1. Therapeutic strategies to obtain revascularization of affected limbs are frequently needed in these patients. Unfortunately, the initial success of surgical and/
or endovascular revascularization can be threatened by progressive disease ultimately leading to an amputation. A better understanding of the complex cellular and molecular processes involved in post-ischemic neovascularization may reveal novel options for therapeutic interventions for PAD patients. This thesis describes reports that contrib- ute to an increase in the knowledge and insights into different cell types involved in collateral artery growth in limb ischemia with the aim of developing new therapeutic strategies for PAD.
The immune system, its cellular components and factors play a critical role in arterio- genesis2, 3. Last years, these are extensively studied in the hind limb ischemia mouse model, mimicking PAD. For studying potentially pro-arteriogenic cells in vivo, the development of a hind limb ischemia mouse model with a therapeutic window that is large enough to study improved blood flow recovery is required. This is pointed out in Chapter 3, which describes a model of double electro-coagulation of both the femoral artery and iliac artery. Next to enlargement of the therapeutic window for studying ef- fects of pro-arteriogenic cells, this model more closely resembles clinical situation of multi-level occluding PAD. Furthermore, in this research field, awareness of the fact that the extent of the arterial defect to induce hind limb ischemia in mice does have physi- ological consequences and is associated with different patterns of perfusion recovery is very important. The impact of technical variations in hind limb ischemia mouse models on the outcome could be illustrated with conflicting outcomes of several pre-clinical experiments on vascular endothelial growth factor (VEGF)-mediated gene therapy for stimulation of collateral artery formation4-6.
Insights into which cell types and factors are involved in the post-ischemic arteriogenic response will enlarge the toolbox for new strategies to stimulate arteriogenesis. Al- though experimental studies in the stimulation of arteriogenesis are promising, none of these inflammatory cells have been demonstrated to be applicable in clinical practice.
This emphasizes that there are currently gaps in this research field and some of them are tackled by the studies of this thesis. In chapter 2, various inflammatory key play- ers in the arteriogenic response are reviewed. It illustrates the complexity and subtle balance of the inflammatory cascade during arteriogenesis. However, it is necessary to further refine our knowledge on which exact (subtype of) leukocytes are involved in col- lateral artery formation to design more effective strategies to stimulate arteriogenesis
152 Chapter 9
in PAD-patients. Besides, communication and relationships of different cells involved in the complex web of arteriogenesis have to be further unravelled, since it seems that leukocytes do not act as a soloist but are dependent of cross talks with each other for their pro-arteriogenic phenotype. Recent reports show that monocytes require a certain pre-stimulation by other inflammatory cells or their soluble factors in order to improve neovascularisation in a hind limb ischemia mouse model7, 8. In chapter 5 we showed that pre-stimulation of human monocytes with CD4+ T cell derived soluble factors significantly improves neovascularization as compared to unstimulated monocytes in a hind limb ischemia mouse model. This emphasizes the capacity of pre-stimulated monocytes to improve vascular regeneration. Although effective, delineation of those T cell derived factors that drive pro-arteriogenic monocyte differentiation is needed. Fu- ture challenge is to find one or a combination of molecular factors that is responsible for pro-arteriogenic pre-stimulation of these cells. The importance of the activation status of monocytes in arteriogenesis is once more underscored in chapter 6. Pro-angiogenic reparative monocytes (Ly6Clo monocytes) in protease-activated receptor-2 (PAR-2) knock out mice showed hampered capacity to become activated and this was associ- ated with severely impaired blood flow recovery in PAR-2 knock out mice. The present study indicates a pro-arteriogenic role of PAR-2, but not PAR-1, and renders PAR-2 as a new therapeutic target for therapeutic arteriogenesis. Researchers are challenged to develop new therapeutic strategies for selectively stimulating arteriogenesis and not simultaneously influencing atherosclerosis formation. Unfortunately, there is overlap in inflammatory processes involved in atherogenesis and arteriogenesis9, 10 and monocytes do play a role in atherosclerosis as well as in arteriogenesis11-14. This trade-off of shared mechanisms contributing to collaterogenesis and atherogenesis is called the Janus phenomenon15, impeding the implementation of pre-stimulated monocyte treatment into the clinical situation to date.
Next to pre-stimulation of inflammatory cells, new insights unravelled subpopula- tions of inflammatory cells that are also in twined in the arteriogenic response, but their exact role and working mechanisms must be further elucidated. Regulatory T cells (CD4+CD25+FoxP3+ T cells) are specialized in the suppression of CD4+ and CD8+ T cell reactions and they are indispensable for balancing immune responses16-19. This prompted us to study regulatory T cells more into detail in arteriogenesis. Chapter 7 reports that regulatory T cells do contribute to the arteriogenic response, but some results astonished us. Only moderate effects on post-ischemic neovascularization were observed after major modulations of regulatory T cell number in the mouse model. Even full depletion of regulatory T cells in a hind limb ischemia mouse model showed only moderate effects on arteriogenesis. To investigate potential mechanisms involved in the pro-arteriogenic effects of regulatory T cells, we showed that regulatory T cells were not present in the post-ischemic skeletal muscle, but only in the lymph nodes in this
General discussion and summary 153
area. These results suggest that regulatory T cells act not directly by local induction of collateral artery formation, but rather in a paracrine manner by affecting other inflam- matory cells in the ischemic muscle tissue from a distance. However, further research is warranted to study the actual upregulation of T cell and monocyte recruitment into the ischemic area by this paracrine function of regulatory T cells.
Recently, bone marrow derived precursor cell therapy has emerged from bench to bedside as a treatment for severe PAD. Since the pioneering work of Tateishi-Yuyama et al.20, numerous clinical trials have been performed evaluating either intramuscular or systemic injections of bone marrow derived cells into the ischemic leg. Although results from first studies were promising, these results have not been confirmed by large ran- domized clinical trials so far. Therefore, we studied bone marrow derived mononuclear cell (BM-MNC)-behavior in the ischemic mouse hind limb in chapter 4. The presence of BM-MNCs after transplantation is monitored by molecular imaging and showed short cell survival of a maximum of 20 days after transplantation into the ischemic adduc- tor muscle. Nevertheless, increasing evidence21-23 indicates that bone marrow derived progenitor cells do not promote vascular growth by incorporation into the vessel walls but may function as supporting cells to secrete growth factors and cytokines and contribute to neovascularization through paracrine effects. In this respect, 20-days cell survival of BM-MNCs may be sufficient to recruit inflammatory cells to the ischemic region to contribute to collateral artery formation. However, this activity may be limited by the absence of pre-stimulation of these BM-MNCs, which may explain the lack of effects in our hind limb ischemia mouse model and some clinical trials. Therefore, one major goal might be to develop strategies that improve pro-arteriogenic cell function by pre-stimulation next to improvement of cell survival. To complete research toward an optimal cell therapy, the interaction of autologous bone marrow derived cells with the environment leading to modulation of cell function must be acknowledged. Patient’
risk factors and the vascular disease itself can impair the function of autologous cells24. Analyses of risk factors indicate that smoking, hypertension, serum LDL-cholesterol levels, age and positive family history contribute to a reduced number and migratory capacity of bone marrow derived endothelial progenitor cells25 and may negatively influence outcome of a cell therapy using these cells. Based on these findings, it seems very important to study bone marrow derived cells in a proper local microenvironment.
For that purpose, diseased animal models were developed like the diet-dependent hyperlipidemic apolipoprotein (APO)E*3Leiden transgenic mouse, atherosclerotic APOE knock out mouse (APOE-/-) and the low-density lipoprotein (LDL) receptor knock out mouse (LDR-/-).
154 Chapter 9
Non-viral gene therapy through shRNA is a new area of investigation for therapeutic approaches for PAD26. Chapter 8 studies the upregulation of an important regulatory factor involved in angiogenesis, HIF-1_, through PHD2 knockdown by an shRNA. We studied the use of a new minicircle vector27 since minicircles have advantages over viral systems such as less immunogenicity and a prolonged gene expression. Importantly, HIF-1_ is an upstream transcriptional factor, involved in the upregulation of several pro- angiogenic factors like VEGF, fibroblast growth factor (FGF), stromal cell derived growth factor-1 (SDF-1) and endothelial nitric oxide synthase (eNOS)28. This new approach based on up regulation of the master regulator HIF-1_, may be a more effective choice since evidence suggest that the expression of a single angiogenic factor like VEGF may not be sufficient for functional revascularization in the ischemic limb. We reported a sig- nificantly enhanced post-ischemic neovascularization after treatment with minicircles carrying a shRNA targeting PHD2 in a hind limb ischemia mouse model. So, this might be a potential new target in the field of vascular gene therapy, but further research to- ward transfection efficiency and the absence of immunostimulation is warranted before clinical translation.
Concerns and recommendations for future clinical research
Considering clinical trials testing new therapeutic strategies for PAD, several con- cerns earn attention. To date, participants of these clinical trials are largely end-stage PAD patients. It is difficult to obtain any beneficial effect of new treatments in these no-option patients. However, for patients with claudication intermittent, known benefit with non-invasive treatment like exercise programmes, restrain the inclusion of these patients into randomized clinical trials studying therapeutic arteriogenesis. Next to the stage of the disease, other factors contribute to patient diversity participating into clinical trials such as diabetes mellitus, medication use and factors involving the inflammatory and regenerative response capacity. This is underscored by the fact that patients can respond completely different to the same cell therapy and justify stratifi- cation of patients by pre-treatment parameters. (Inter)national experts in the field of vascular regeneration should unite and develop shared clinical trial protocols to include large numbers of patients with PAD for testing new therapeutic strategies. Only after national or international collecting patients with PAD, patient number is proper for patient stratification within the trial. Also clues in the precise composition of the often- heterogeneous cellular product should be linked to patient factors and respondership.
A thorough characterization of the cell product and its functionality in vitro and in vivo is needed. Data banking of patients-materials participating in the clinical trials is important in this respect. Given the heterogeneity of the patient and its cellular product, one could argue that there is a need for tailor-made cell therapy for patients with PAD. Finally, more insight into whether genetic predisposition involves the capacity for vascular repair in
General discussion and summary 155
patients with PAD is desirable. Differences in pre-existent collateral networks may be genetically determined. This could also explain why one patient form good collateral arteries and others do not. Fundamental research studying different mice strains sug- gests that genetic determinants convey the capacity for arteriogenesis. C57Bl6 mice are good responders to hind limb ischemia and are exuberant collateral formers. Conversely, BalbC mice have an inferior collateral artery forming capacity and are bad responders to hind limb ischemia induction. Therefore, regenerative response capacity appears to apply to genetic predisposition and needs to be studied in the clinical setting.
In conclusion, an increased understanding of the complex mechanism of post-ischemic neovascularization will finally lead to potential and efficient therapeutic strategies to improve outcomes for patients with PAD. In this thesis, a mouse model, mimicking PAD was used to study cellular mechanisms of arteriogenesis. This thesis reports growing possibilities of pre-stimulation of monocytic cells, subpopulations of T cells (regulatory T cells) and non-viral gene therapy carrying an shRNA, but further research is required be- fore translation into the clinical situation. For this purpose, close collaboration between basic scientists and clinicians is of great significance.
156 Chapter 9
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