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Embryonic endothelial progenitor cells expressing a broad range of proangiogenic and remodeling factors enhance vascularization and tissue recovery in acute and chronic ischemia

Christian Kupatt^,1, Jan Horstkotte^,1, Georgios A. Vlastos^*, Achim Pfosser, Corinna Lebherz, Matthias Semisch^*, Mark Thalgott, Kerstin Büttner, Christian Browarzyk, Jörg Mages, Reinhard Hoffmann^||, Alexander Deten^¶, Mathias Lamparter^*,, Fabian Müller^*,, Heike Beck^*, Hildegard Büning^#, Peter Boekstegers and Antonis K. Hatzopoulos^*,,2

^* GSF-Research Center for Environment and Health, Institute for Clinical Molecular Biology and Tumor Genetics, Munich, Germany;
Vanderbilt University Medical Center, Division of Cardiovascular Medicine, Nashville, Tennessee, USA;
Internal Medicine I, Klinikum Großhadern, Munich, Germany;
Technical University, Institute for Medical Microbiology, Munich, Germany;
^|| Max-von-Pettenkoffer Institute, Bacteriology Department, Munich, Germany;
^¶ Leipzig University, Carl-Ludwig Institute of Physiology, Leipzig; and
^# Gene Center, Ludwig-Maximilians-University, Munich, Germany

² Correspondence: Vanderbilt University, Department of Medicine, Division of Cardiovascular Medicine, 2220 Pierce Ave., Nashville, TN 37232-6300, USA. E-mail: antonis.hatzopoulos{at}vanderbilt.edu

SPECIFIC AIMS

The timely and efficient restoration of blood circulation after acute or in chronic ischemia is a key consideration for the treatment of peripheral vascular disease or after myocardial and brain infarction. We recently found that embryonic endothelial progenitor cells (eEPCs), isolated from E7.5 mice at the onset of vasculogenesis, home specifically to hypoxic areas in mouse tumor metastases but spare normal organs and do not form carcinomas. Based on these results, we investigated the potential of eEPCs to enhance vascularization and limit organ dysfunction of ischemic tissues. To further address the molecular basis of the eEPCs’ therapeutic potential, we analyzed their transcriptome to identify secreted factors known to induce angiogenesis, tissue remodeling, and organogenesis. This detailed profile sets the molecular frame to understand how progenitor cells induce blood vessel growth and help tissue recovery.

PRINCIPAL FINDINGS

1. Embryonic EPCs improve hindlimb vascularization
To assess the effect of eEPCs on tissue vascularization in a chronic ischemia model, we applied DiI-labeled eEPCs via retroinfusion into the hindlimbs of rabbits 7 days after excision of the femoral artery (set as day 0). Five days after xenotransplantation (day 12), we detected eEPCs associated with the vascular system of affected muscles in the ischemic area (Fig. 1 A).

View larger version (35K): [in this window] [in a new window]   Figure 1. eEPCs application augments vascularization and improves circulation in chronic ischemic rabbit hindlimbs. A) 5 x 106 DiI-labeled eEPCs were applied via retroinfusion into the hindlimbs of rabbits a week after excision of the femoral artery. The M. gastrognemius (M. gc.) was excised 5 days later, sectioned, and examined for the presence of transplanted cells using fluorescence microscopy. Engrafted eEPCs could be detected within and around the vascular structures of the muscle tissue (left panel). Right panel: phase contrast picture of the same area (bar=100 µm). B) Quantification of eEPCs present within the M.gc. that was excised 2, 5, 7, 10, and 14 days after eEPC retroinfusion (n =3 per time point, #P<0.05 vs. day 2, P<0.05). C) Quantification of capillaries in 3 muscle areas (M. gastrognemius, M. tibialis anterior, M. adductor) reveals an increase in capillary to muscle fiber ratios 28 days after eEPC treatment (day 35). D) Collateral vessel formation increased in eEPC-treated rabbit hind limbs as compared with untreated controls (changes, measured at day 35, are given in percents of day 7 values). E) Angiography of rabbit hind limbs following injection of a contrast agent at day 35 shows a marked improvement in the perfusion score of eEPC-treated animals as compared with untreated controls. C–E) #P< 0.05 vs. control, n = 5 animals/group.

A time course revealed that the mouse cells survived at least 10 days after xenotransplantation in the rabbit, but no significant eEPC numbers could be detected after 14 days (day 21 after excision, Fig. 1B ).

The transient association of eEPCs with the host blood vessels strongly induced vascular growth within the ischemic tissue. Four weeks following eEPC retroinfusion, the ratio of capillaries to muscle fiber improved in all muscles examined (M. gastrognemius, M. tibialis ant., and M. adductor) as compared with control untreated limbs (Fig. 1C ). Retroinfusion of eEPCs also led to an increased number of collateral vessels (201±51% as compared between day 7 and day 35 and between treated and control animals; Fig. 1D ). The increase in capillary and collateral numbers correlated with better perfusion and improved circulation in the ischemic limbs as visualized and measured by fluoroscopy using a contrast agent at day 35 (Fig. 1E ).

2. eEPC-administration improves heart function after myocardial ischemia-reperfusion (I/R) in mice
The efficacy of eEPCs to improve limb perfusion in rabbit hindlimbs prompted us to evaluate their effect after myocardial infarction in mice. To this end, we first tested whether eEPCs can be specifically retained in heart ischemic tissue. DiI-labeled eEPCs were injected into the external jugular vein at the onset of reperfusion after 20 min of coronary artery ligation in vivo. We observed a higher number of adherent cells per microscopic field in ischemic hearts as compared with nonischemic controls (Fig. 2 A). Our previous work on the tumor homing mechanisms of eEPCs showed that selectins expressed in endothelium, and their ligands present on the eEPC surface, mediate this process. In support for a similar homing mechanism in ischemic hearts, we find that pretreatment with the selectin antagonist fucoidin blocks eEPC retention in the ischemic areas (Fig. 2A ).

View larger version (27K): [in this window] [in a new window]   Figure 2. Systemic administration of eEPCs via the tail vein improves heart output in mice after myocardial infarction. A) eEPCs are specifically retained in heart ischemic tissue. After ligation of the left anterior descending coronary artery for 20 min, DiI-labeled eEPCs were injected into the external jugular vein. After 15 min reperfusion, the hearts were isolated, washed and the number of eEPCs retained in the ischemic area was directly counted under a fluorescence microscope (I/R). Sham operated mice, without artery ligation, served as controls. Pretreatment with fucoidin blocked eEPC retention (I/R fucoidin; n=5 per group). B) Systemic infusion of 3 x 105 eEPCs (+EPC post) attenuates I/R injury as compared with saline infusion (control I/R) by improving LVDP as measured 14 days after transplantation. The effect was similar to GM-CSF treatment (0.5µg/d i.p. injection for 10 days, starting at 3 days prior to infarction (+GM-CSF pre), but superior to GM-CSF treatment applied only in the 7 day post-I/R (GM-CSF post). Sham operated mice served as controls. C, D) dP/dtmax and dP/dtmin evaluation 14 days after eEPC administration. Both parameters indicate improvement in heart output after eEPC-treatment or GM-CSF-pretreatment, but not after postischemic GM-CSF application (#P<0.05 vs. sham; n=8 per group).

In a next step, saline solution (300µL) with or without 3x10⁵ eEPCs marked by DiI, or by stable EGFP expression, was infused systemically via the mouse tail vein 24 h after occlusion of the coronary artery for 1 h. The presence of eEPCs, found within or adjacent to vascular structures in the postischemic hearts, was confirmed 7 and 14 days after infusion.

Invasive functional assessment of heart output demonstrated that eEPC treatment improved residual systolic function as measured by an increase of LVDP (Fig. 2B ) and dP/dt_max (Fig. 2C ) when compared with control mice injected with saline solution. In parallel fashion, eEPC treatment attenuated the loss of diastolic function observed in untreated animals (measured by dP/dt_min, Fig. 2D ).

Mice injected subcutaneously for 10 days starting 3 days prior to ischemia/reperfusion with GM-CSF, an agent thought to mobilize adult EPCs, showed a similar improvement and served as a positive control. However, in contrast to the eEPC application one day after infarction, GM-CSF provided no significant enhancement of systolic or diastolic function when it was applied during the postischemic period (days 1–7, Fig. 2B-D ).

CONCLUSIONS AND SIGNIFICANCE

Recent experimental evidence showed that progenitor cells for endothelium and other somatic cell types exist in adults and that they might play an important role in neovascularization and tissue repair processes. Moreover, application of EPCs for therapeutic purposes in animal models and in clinical trials showed that such treatment can improve vascularization and tissue function. The data presented here indicate that embryonic endothelial progenitor cells have a beneficial effect when transplanted in adult ischemic sites. This was true in both settings, acute and chronic ischemia.

Although most studies documented a considerable improvement of tissue vascularization after application of EPCs, the mechanism of this effect remains unclear because the fraction of the newly built vascular beds by the donor cells varies among different experimental approaches and often is low. It has been postulated that besides being a source of building blocks, one reason of improved tissue recovery is the ability of EPCs to induce endogenous angiogenesis through secretion of proangiogenic factors. To begin to understand the molecular basis of this induction, as well as the broader effects on recovery in the tissue surrounding the recruited eEPCs, we have assembled a nearly complete list of expressed genes in eEPCs that encode secreted proteins using Affymetrix Genechips (45,101 gene sequences). The expression profiling data show a rich collection of proteins from a variety of distinct pathways (i.e., cytokines, chemokines, angiogenic factors, and an extensive presence of protease inhibitors that moderate tissue remodeling). The high expression levels of many secreted factors known to induce angiogenesis through distinct pathways—coupled to production of cardioprotective agents like thymosin ß₄—might be a unique advantage of cell-based therapeutic approaches as compared with single agent treatments. Particularly surprising was a prominent contribution of molecules involved in the BMP and Wnt signaling pathways (35% of all proteins classified as growth factors). The importance of such molecules and their receptors for the development of many organs including the heart, raise the possibility that they contribute to the regeneration of heart tissue. This source of developmentally active proteins might be a distinct advantage of the embryonic cells. Our results propose a complex way that embryonic endothelial progenitors influence their microenvironment after recruitment to ischemic sites (Fig. 3 ).

View larger version (54K): [in this window] [in a new window]   Figure 3. A model of eEPC effects on ischemic tissue. Systemically or locally applied eEPCs accumulate in the tissue surrounding the ischemic site. The engrafted eEPCs can ameliorate tissue recovery through: A) direct effects (i.e., contribution in newly forming vascular structures); and B) indirect effects on tissue repair mechanisms. Based on the eEPC-gene expression profile of secreted factors, these effects can be classified as follows: 1) induction of angiogenesis by secretion of angiogenic factors; 2) moderation of local protease activity (of both metalloproteases and serine proteases); and modulation of (3) the wnt signaling and (4) the BMP pathway. We hypothesize that the combinatorial action of these five processes underlines the positive effects on ischemic tissue recovery after eEPC transplantation.

It is of note that we did not observe any adverse effect due to the xenotransplantation of mouse cells in rabbits. Our previous data indicated that murine embryonic EPCs are immunoprivileged in allogeneic settings due to lack of MHC I expression and resistance to nonactivated natural killer cells. The lack of acute inflammation in the xenotransplants could also be an advantage. A close inspection of the expression profile reveals that eEPCs express no measurable levels of proinflammatory cytokines (such as IL-1, IL-2, IL-6, IL-23, etc.) that might account for this effect. A better understanding of the limited immunogenicity of embryonic cells could help design strategies using continuous and readily available sources for cell-based therapies.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.04-3282fje;

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