Stem Cells Combined With Gene Transfer for Therapeutic Vasculogenesis
Discoveries in one scientific field sometimes have the unexpected result of igniting a major step forward or new direction in another field. We are in the early phases of one of these sea changes. Advances in stem cell therapies are progressing at a rapid clip and are providing the field of gene therapy with an expanded platform of technologies for the introduction of recombinant genes into human tissues. Likewise, a decade worth of experience in gene therapy provides stem cell researchers with a jump-start. Indeed, it is likely and probable that these 2 fields will eventually merge due to significant scientific and technical overlap.
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The major contribution of the study by Iwaguro et al1 in this issue of Circulation is the demonstration that gene transfer of vectors encoding VEGF into endothelial progenitor cells (EPCs) ex vivo and injection of gene modified EPCs into ischemic muscle in vivo increases neovascularization in an animal model of hindlimb ischemia. Peripheral blood mononuclear cells from human volunteers were cultured in a special cocktail that facilitates growth of EPCs—plating on fibronectin and grown in the presence of multiple growth factors, such as endothelial cell basal medium, fetal bovine serum, human VEGF-A, human FGF-2, human EGF, IGF-1, and ascorbic acid—as previously characterized by this group.2 After 7 days in culture, EPCs were transduced with adenoviral vectors encoding a murine VEGF164 gene or a control reporter gene. Proliferative indices and adhesion properties were measured ex vivo in order to characterize the phenotype of the cells. The authors then conducted pilot studies in vivo to determine the dose of gene-modified cells required to produce a similar magnitude of neovascularization achieved with nontransduced EPCs. Interestingly, they found that 30 times fewer VEGF-transduced EPCs (1.5×104 cells) produced equivalent neovascularization compared with 4.5×105 nontransduced EPCs. VEGF-expressing EPCs were injected into the tail vein of athymic nude mice. The fate of transplanted EPCs was tracked by DiI fluorescent tagging, plasma VEGF levels were measured, and a histological and physiological evaluation of the transplanted animals was performed out to 28 days.
Several important observations were made. First, ex vivo the proliferation rate of the VEGF expressing EPCs was two-fold higher than the nontransduced or reporter gene–transduced EPCs. Adhesion of VEGF-EPCs to activated and nonactivated HUVEC monolayers was also significantly increased in the gene-modified cells. These findings suggested that the phenotype of the EPCs had been shifted toward a proangiogenesis state. Second, in vivo detectable levels of circulating human VEGF were quantified in the athymic mice at 2 log–fold levels higher at day 1 compared with the 2 groups of control mice. The levels of human VEGF declined rapidly after 7 days, yet were still discernable at 28 days, consistent with the time course of gene expression after adenoviral gene transfer. As predicted, the human VEGF protein was tolerated in the absence of an intact immune system in the athymic nude mouse. Hence, endogenous levels of growth factors or chemokines can be augmented by the expression of exogenous proteins in gene-transduced cells.
An important question is what is the fate of the gene-modified cells in vivo? Pilot experiments were performed to identify the DiI-labeled human EPCs in tissue sections. The cells were injected into the tail vein of mice, and one could predict that most of the cells would be retained in the terminal vasculature of the lung, taken up by macrophages in the liver and spleen, with a minority of cells reaching ischemic target tissue. The authors report an ≈60% increase in the number of DiI-labeled, VEGF-transduced EPCs and the density of vascular cells in the distribution of the ischemic limb compared with the 2 control groups of mice. The percent of the total number of cells injected was not reported, nor was the distribution of the cells throughout the mice. These data are important from a regulatory perspective to understand the potential safety and toxicity of this approach.
Nonetheless, the fact that more EPCs are found in the region of the ischemic tissue raises 2 additional important points. First, the association with an increase in the density of native vascular endothelial cells in the same regions suggests, but does not prove, that the VEGF-transduced EPCs may have induced native angiogenesis; that is, sprouting of new capillaries from preexisting mature endothelial cells (ECs) by activation of native ECs by secreted VEGF, leading to endothelial cell proliferation, disruption of the basement membrane, and migration of ECs into the interstitial space, possibly in response to an ischemic stimulus and subsequent capillary formation. Second, in addition to native angiogenesis, does vasculogenesis from the newly injected EPCs occur? The authors attempt to address these more difficult questions through a series of physiological assessments of blood flow and clinical outcome. Three parameters were measured. Serial examinations of hindlimb perfusion by laser Doppler perfusion imaging were performed at days 0 and 28. The ratio of ischemic to normal blood flow was calculated, and the authors found that there was an approximate 2-fold greater increase in blood flow in the VEGF-transduced EPC group compared with the 2 control groups. Histological examination of skeletal muscle retrieved from the ischemic hindlimbs of mice euthanized at 28 days revealed an increase in capillary density in the VEGF-transduced EPC mice compared with the reporter gene–transduced or nontransduced EPCs. Finally, clinical outcome was determined by the extent of toe necrosis and autoamputation of the ischemic limb. Mice in the VEGF-transduced EPC group had fewer autoamputations and limited toe necrosis, consistent with the clinical concept of limb salvage. Thus, it appears from histological, perfusion, and clinical data that the VEGF-transduced cells had a superior outcome on limb salvage.
This article by Iwaguro et al1 adds a new twist to both the therapeutic angiogenesis gene therapy and stem cell EPC stories. VEGF-transduced EPCs promote neovascularization in ischemic tissue in greater quantity and with improved clinical outcomes compared to nontransduced or reporter gene–transduced cells. That is, EPCs secreting VEGF are better “therapeutic bullets” than EPCs alone. However, are the VEGF-transduced EPCs better than VEGF gene transfer into ischemic tissue? This question was not addressed in the present study, but a side-by-side comparison study would be straightforward to do and should be done. So now, here are potentially 2 powerful methods in our therapeutic armentarium for promoting therapeutic angiogenesis that, when combined, may be synergistically more powerful than either alone.
Still, several tantalizing questions remain to be answered. What is the mechanism for the improved neovascularization—vasculogenesis, angiogenesis, or both? Vasculogenesis refers to the in situ formation of blood vessels from EPCs or angioblasts, beginning with the formation of cell clusters or blood islands.3 Growth and fusion of multiple blood islands in the embryo ultimately give rise to the capillary network structure, and after the onset of blood circulation, this network differentiates into an arteriovenous vascular system.4 Although vasculogenesis was initially considered to be restricted to embryos,5 recent evidence suggests that postnatal vasculogenesis is a critical paradigm for repair of adult ischemic tissue. Pioneering work from this group lead to the discovery that endothelial progenitor cells circulate in adult peripheral blood.6 This discovery was rapidly confirmed by others; transplantation of either culture-expanded EPCs or adult stem cells isolated from bone marrow have been recently shown to effectively enhance angiogenesis in ischemic tissues.2,7⇓ In other words, the sprouting of new blood vessels from differentiated EPCs is not be limited to the developing embryo, but extends to adult tissue. With regard to the present study, it is possible, although not proven, that VEGF-transduced EPCs induce vasculogenesis within the ischemic hindlimb. This process appears to be enhanced by the autocrine effects of transduced human VEGF164 and possibly by paracrine effects of native murine VEGF. Support for this concept comes from other recent studies demonstrating that EPCs derived from the bone marrow circulate in peripheral blood in response to ischemic stimuli in adult animals,8,9⇓ and that EPCs are also derived from human umbilical cord blood.10
A second mechanism by which VEGF-transduced EPCs promote neovascularization is through enhancement of native angiogenesis. In contrast to vasculogenesis, angiogenesis is the sprouting of new capillaries from preexisting mature endothelial cells. Angiogenesis begins with the activation of endothelial cells within a parent vessel and endothelial cell proliferation, followed by disruption of the basement membrane, migration of endothelial cells into the interstitial space, further proliferation, pericyte recruitment, and formation of an intact capillary vessel.11 It is possible, although again not proven in this study, that VEGF-transduced EPCs may activate native endothelial cells and promote the process of angiogenesis in the native vasculature; secreted human VEGF164 may enhance this process. A likely hypothesis is that both mechanisms—vasculogenesis and angiogenesis—are at play simultaneously. This hypothesis requires investigation in order to completely understand the biological activity of gene-transduced EPCs in vivo.
It is intriguing that biologically modified EPCs may be more potent therapeutic bullets than nonmodified or nontransduced cells alone. Data to support this concept comes from 2 sources. Recent studies suggest that the statins (HMG-CoA reductase inhibitors), like VEGF, also promote vasculogenesis.12,13⇓ Although Dimmeler et al13 show that statins increase the number of differentiated EPCs via the PI3-kinase/Akt pathway, Llevadot et al12 demonstrate that the statins mobilize bone marrow–derived EPCs and that these cells promote angiogenesis—the cells proliferate, migrate, and form new blood vessels. Second, the data from the present study consistently shows that the VEGF-transduced EPCs are better biological actors than the reporter gene–transduced EPCs, implying that autocrine and paracrine effects of the secreted transgene modifies the biology of the cells.
Several caveats should be raised. These studies were performed in small animals lacking an intact immune system. The experiments need to be repeated in a larger animal model, more similar to human physiology. However, if the data proceeds in an encouraging manner, then it would be logical to consider proceeding to early Phase I clinical testing of VEGF-transduced EPCs in human to promote therapeutic vasculogenesis and angiogenesis in ischemic peripheral muscle. As noted above, the mechanisms for the assumed vasculogenesis and angiogenesis must be worked out. Finally, the fate of the gene-transduced EPCs that do not “home” to the ischemic tissue must be determined. Where do they go, and do they produce toxic effects on other tissues? These experiments would fall under the purview of regulatory studies that would be required before Phase I human protocols.
In closing, I am humbled to write an editorial posthumously on an exciting study performed by a colleague and friend, Jeffrey M. Isner. Jeff was a pioneer in cardiovascular research and gene therapy whose tragic death was a terrible personal and professional loss to medicine and cardiovascular biology. Jeff’s vision was to revascularize ischemic limbs and heart using gene therapy to promote therapeutic angiogenesis; in 1997, he reported the surprising discovery (at the time) that endothelial progenitor cells enter the circulation from the bone marrow and that the process was augmented by VEGF. Now, he has brought the 2 concepts together in this preclinical animal model study. Jeff was also a caring and compassionate physician who worked arduously to help and to provide hope to his patients. His legacy will be this field; stem cell biology and gene transfer have now come together to team up to become potentially powerful angiogenic therapies.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Iwaguro H, Yamaguchi J, Kalka C, et al. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation. 2001; 105: 732–738.
- ↵Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000; 97: 3422–3427.
- ↵Riseau W. Differentiation of endothelium. FASEB J. 1995; 9: 926–933.
- ↵Riseau W, Sariola H, Zerwes HG, et al. Vasculogenesis and angiogenesis in embryonic stem cell-derived embryoid bodies. Development. 1988; 102: 471–478.
- ↵Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 965–967.
- ↵Shintani S, Murohara T, Ikeda H, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation. 2001; 103: 897–903.
- ↵Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999; 85: 221–228.