Tibor Ziegelhoeffer

Bone Marrow-Derived Cells Do Not Incorporate Into the Adult Growing Vasculature

Abstract— Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into growing vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial growth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increase versus nonoccluded side, P <0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular growth by incorporating into vessel walls but may function as supporting cells.

VEGFR-1–Selective VEGF Homologue PlGF Is Arteriogenic: Evidence for a Monocyte-Mediated Mechanism

Abstract— Two signaling receptors for vascular endothelial growth factor (VEGF) in the vasculature are known with not yet well-understood roles in collateral vessel growth (arteriogenesis). In this study, we examined the involvement of the two VEGF receptors in arteriogenesis. Therefore, we used the VEGF homologue placenta growth factor (PlGF), which only binds to VEGFR-1 and VEGF-E, which only recognizes VEGFR-2. These peptides were locally infused over 7 days after ligation of the femoral artery in the rabbit. Evaluation of collateral growth by determining collateral conductance and angiographic scores demonstrated that the VEGFR-1–specific PlGF contributed significantly more to arteriogenesis than the VEGFR-2 specific VEGF-E. The combination of VEGF-E and PlGF did not exceed the effect of PlGF alone, indicating that cooperation of the two VEGF receptors in endothelial cell signaling is not required for arteriogenesis. In an in vitro model of angiogenesis, VEGF and VEGF-E were comparably active, whereas PlGF displayed no activity when given alone and did not further increase the effects of VEGF or VEGF-E. However, PlGF was as potent as VEGF when monocyte activation was assessed by monitoring integrin surface expression. In addition, accumulation of activated monocytes/macrophages in the periphery of collateral vessels in PlGF-treated animals was observed. Furthermore, in monocyte-depleted animals, the ability of PlGF to enhance collateral growth in the rabbit model and to rescue impaired arteriogenesis in PlGF gene–deficient mice was abrogated. Together, these data indicate that the arteriogenic activity observed with the VEGFR-1–specific PlGF is caused by its monocyte-activating properties.

Collateral Artery Growth (Arteriogenesis) After Experimental Arterial Occlusion Is Impaired in Mice Lacking CC-Chemokine Receptor-2

Abstract— Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. Induced macrophage migration in vivo is driven by the binding of monocyte chemoattractant protein-1 (MCP-1, or CCL2 in the new nomenclature) to the CCR2-chemokine receptor on macrophages. To determine whether the CCL2-CCR2 signaling pathway is involved in the accumulation of macrophages in growing collateral vessels, we used mice that are deficient in CCR2 in a model of experimental arterial occlusion and collateral vessel growth. In an in vitro CCL2-driven chemotaxis assay, mononuclear cells isolated from wild-type BALB/c mice exhibited CCL2 concentration–dependent migration, whereas this migration was abolished in cells from CCR2−/− mice on a BALB/c genetic background. In vivo, blood flow recovery as measured by laser Doppler (LDI) and MRI (MRI) was impaired in CCR2−/− mice on either the BALB/c or C57BL/6 genetic backgrounds. Three weeks after femoral artery ligation, LDI perfusion ratio of operated versus nonoperated distal hindlimb in BALB/c wild-type mice increased to 0.45±0.06 and in CCR2−/− animals only to 0.21±0.03 (P <0.01). In C57BL/6 mice, ratio increased to 0.96±0.09 and 0.85±0.08 (P <0.05), respectively. MRI at 3 weeks (0.76±0.06 versus 0.62±0.01; P <0.05) and hemoglobin oxygen saturation measurements confirmed these findings. Active foot movement score significantly decreased and gastrocnemius muscle atrophy was significantly greater in CCR2−/− mice. Morphometric analysis showed a lesser increase in collateral vessel diameters in CCR2−/− mice. Importantly, the number of invaded monocytes/macrophages in the perivascular space of collateral arteries of CCR2−/− animals was dramatically reduced in comparison to wild-type mice. In conclusion, our results demonstrate that the CCR2 signaling pathway is essential for efficient collateral artery growth.

A Different Outlook on the Role of Bone Marrow Stem Cells in Vascular Growth: Bone Marrow Delivers Software not Hardware

Blood vessel growth in adult organisms is a process occurring under various physiological and pathological conditions. Two forms of blood vessel growth have been described after birth: angiogenesis, ie, capillary sprouting, and arteriogenesis, ie, growth of large conductance arteries from preexisting arterial anastomoses (collaterals).1,2 It is increasingly recognized that blood flow deficits caused by occlusion or stenosis of a major artery can only be efficiently compensated by arteriogenesis. Fluid shear stress (FSS) is suggested to be the molding force for arteriogenesis. The activation of the collateral endothelium caused by increased FSS is reflected by an upregulation of adhesion molecules and release of cytokines that attract circulating blood cells, mainly monocytes, to adhere to and invade the collateral vessel wall.3 Besides this monocyte/macrophage accumulation around growing collaterals, also an increase in numbers of T cells and granulocytes has been reported, underlining the paradigm that circulating cells are of a great importance in this type of vascular growth.4,5 A variety of different cytokines and proteases (ie, MCP-1, FGF-2, TGF-β, uPA, and MMPs) produced by these invading cells have been identified to stimulate endothelial and smooth muscle cell proliferation and migration as well as tissue degradation.6 This has led to a number of animal studies and clinical phase I trials demonstrating the short-term safety of administering several cytokines. However, recent clinical phase II trials have failed to extrapolate the promising results from animal studies into the therapeutic realm in human patients.7,8 New therapeutic approaches to promote arteriogenesis have evolved when …

Impact of Mouse Strain Differences in Innate Hindlimb Collateral Vasculature

Objective—To assess the importance of genetic background for collateral artery development. Methods and Results—C57BL/6, BALB/c and 129S2/Sv mice were studied after femoral artery ligation by laser Doppler imaging, visible light oximetry, time-of-flight–magnetic resonance imaging, and treadmill testing; C57BL/6 and BALB/c also underwent electron paramagnetic resonance (EPR) oximetry, x-ray angiography, and histology. C57BL/6 had the least initial distal ischemia and most complete recovery. BALB/c had the most severe initial ischemia and poorest recovery. BALB/c had some vasodilatory reserve in their ligated limbs not seen in the other strains at 3 weeks. By in vivo TOF-magnetic resonance angiography, C57BL/6 had larger preexistent and developed collaterals. By x-ray angiography, C57BL/6 had a higher collateral-dependent filling score and number of visible collaterals immediately after femoral ligation and a higher number of visible collaterals at 1 week but not at 4 weeks. EPR oximetry and histology revealed hypoxia and tissue damage in regions of collateral growth of BALB/c but not C57BL/6 mice. In C57BL/6 BrdUrd uptake in the thigh was limited to larger vessels and isolated perivascular cells. Proliferative activity in collateral arterioles was similar in both strains. Conclusions—Genetic differences in preexistent collateral vasculature can profoundly affect outcome and milieu for compensatory collateral artery growth after femoral artery occlusion.

Notch Ligand Delta-Like 1 Is Essential for Postnatal Arteriogenesis

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.

Circulating Vascular Progenitor Cells Do Not Contribute to Compensatory Lung Growth

Abstract— The biological principles that underlie the induction and process of alveolization in the lung as well as the maintenance of the complex lung tissue structure are one of the major obstacles in pulmonary medicine today. Bone marrow–derived cells have been shown to participate in angiogenesis, vascular repair, and remodeling of various organs. We addressed this phenomenon in the lung vasculature of mice in a model of regenerative lung growth. C57BL/6 mice were transplanted with bone marrow from one of three different reporter gene–transgenic strains. flk-1+/lacZ mice, tie-2/lacZ transgenic mice (both exhibiting endothelial cell–specific reporter gene expression), and ubiquitously enhanced green fluorescent protein (eGFP)-expressing mice served as marrow donors. After hematopoietic recovery, compensatory lung growth was induced by unilateral pneumonectomy and led to complete restoration of initial lung volume and surface area. The lungs were consecutively investigated for bone marrow–derived vascular cells by lacZ staining and immunohistochemistry for phenotype identification of vascular cells. lacZ- or eGFP-expressing bone marrow–derived endothelial cells could not be found in microvascular regions of alveolar septa. Single eGFP-positive endothelial cells were detected in pulmonary arteries at very low frequencies, whereas no eGFP-positive vascular smooth muscle cells were observed. In conclusion, we demonstrate in a model of lung growth and alveolization in adult mice the absence of significant bone marrow–derived progenitor cell contribution to the concomitant vascular growth and remodeling processes.

Participation of Bone Marrow-Derived Cells in Long-Term Repair Processes after Experimental Stroke

Bone marrow-derived cells participate in remodeling processes of many ischemia-associated diseases, which has raised hopes for the use of bone marrow as a source for cell-based therapeutic approaches. To study the participation of bone marrow-derived cells in a stroke model, bone marrow from C57BL/6-TgN(ACTbEGFP)1Osb mice that express green fluorescent protein (GFP) in all cells was transplanted into C57BL/6J mice. The recipient mice underwent permanent occlusion of the middle cerebral artery, and bone marrow-derived cells were tracked by fluorescence. The authors investigated the involvement of bone marrow-derived cells in repair processes 6 weeks and 6 months after infarction. Six weeks after occlusion of the artery, more than 90% of the GFP-positive cells in the infarct border zone were microglial cells. Very few GFP-positive cells expressed endothelial markers in the infarct/infarct border zone, and no bone marrow-derived cells transdifferentiated into astrocytes, neurons, or oligodendroglial cells at all time points investigated. The results indicate the need for additional experimental studies to determine whether therapeutic application of nonselected bone marrow will replenish brain cells beyond an increase in microglial engraftment.

Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp)

OBJECTIVE Collateral artery growth (arteriogenesis) can be induced in rabbit and mice by occlusion of the femoral artery. We aimed to identify genes that are differentially expressed during arteriogenesis. METHODS 24 h after femoral ligation or sham operation collateral arteries were isolated from New Zealand white rabbits, mRNAs were extracted and amplified using the SMART technique. cDNAs were subjected to suppression subtractive hybridization. The differential expression was confirmed by Northern blot, Real time PCR and Western blot. Additionally, the gene expression was modulated in vivo by application of cytokines via osmotic minipumps. RESULTS We found the cardiac ankyrin repeat protein (carp) mRNA to be upregulated at 24 h and already at 6 h and 12 h after surgery as shown by Northern blot hybridization and real time PCR. The carp mRNA was also increased in our mouse model of arteriogenesis. Western blot results on nuclear extracts of rabbit collaterals 24 h after surgery indicated that carp, which we showed to be expressed in endothelial cells and smooth muscle cells of collateral arteries by immunohistochemistry, was also upregulated on the protein level. We infused MCP-1, TGF-beta1 or doxorubicin for 24 h in rabbits and found that only TGF-beta1 led to an additional increase of carp mRNA. Overexpression of carp in cos-1 cells resulted in a 3.7-fold increase of the immediate early gene egr-1. CONCLUSIONS Our results implicate that carp is associated with the initiation and regulation of arteriogenesis.

Improved Arteriogenesis with Simultaneous Skeletal Muscle Repair in Ischemic Tissue by SCL+ Multipotent Adult Progenitor Cell Clones from Peripheral Blood

Background: The CD34– murine stem cell line RM26 cloned from peripheral blood mononuclear cells has been shown to generate hematopoietic progeny in lethally irradiated animals. The peripheral blood-derived cell clones expresses a variety of mesodermal and erythroid/myeloid transcription factors suggesting a multipotent differentiation potential like the bone marrow-derived ‘multipotent adult progenitor cells’ (MAP-C). Methods: SCL+ CD34– RM26 cells were transfused intravenously into mice suffering from chronic hind-limb ischemia, evaluating the effect of stem cells on collateral artery growth and simultaneous skeletal muscle repair. Results: RM26 cells are capable of differentiating in vitro into endothelial cells when cultured on the appropriate collagen matrix. Activation of the SCL stem cell enhancer (SCL+) is mediated through the binding to two Ets and one GATA site and cells start to express milieu- and growth condition-dependent levels of the endothelial markers CD31 (PECAM) and Flt-1 (VEGF-R1). Intravenously infused RM26 cells significantly improved the collateral blood flow (arteriogenesis) and neo-angiogenesis formation in a murine hind-limb ischemia transplant model. Although transplanted RM26 cells did not integrate into the growing collateral arteries, cells were found adjacent to local arteriogenesis, but instead integrated into the ischemic skeletal muscle exclusively in the affected limb for simultaneous tissue repair. Conclusion: These data suggest that molecularly primed hem-/mesangioblast-type adult progenitor cells can circulate in the peripheral blood improving perfusion of tissues with chronic ischemia and extending beyond the vascular compartment.