Armin Helisch

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.

Arteriogenesis The Development and Growth of Collateral Arteries

In patients with atherosclerotic vascular diseases, collateral vessels bypassing major arterial obstructions have frequently been observed. This may explain why some patients remain without symptoms or signs of ischemia. The term “arteriogenesis” was introduced to differentiate the formation of collateral arteries from angiogenesis, which mainly occurs in the ischemic, collateral flow‐dependent tissue. Many observations in various animal models and humans support that the remodeling of preexisting collateral vessels is the mechanism of collateral artery formation. This remodeling process seems to be mainly flow‐mediated. It involves endothelial cell activation, basal membrane degradation, leukocyte invasion, proliferation of vascular cells, neointima formation (in most species studied), and changes of the extracellular matrix. The contribution of ischemia to arteriogenesis is still unclear, but arteriogenesis clearly can occur in the absence of any significant ischemia. It is questionable, whether collateral arteries also form de novo in ischemic vascular diseases. A better understanding of the mechanisms of arteriogenesis will be important for the design of more effective strategies for the treatment of patients with ischemic vascular diseases.

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.

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.

Mouse gridlock: No Aortic Coarctation or Deficiency, but Fatal Cardiac Defects in Hey2 −/− Mice

Gridlock (grl) is one of the first mutations characterized from the large zebrafish mutagenesis screens, and it results in an arterial (aortic) maturation defect, which was proposed to resemble aortic coarctation, a clinically important human malformation. While the grl mutation appears to be a hypomorph, grl knockdown experiments have shown even stronger effects on arterial development. We have generated a knockout of the murine Hey2 (gridlock) gene to analyze the mammalian phenotype. Surprisingly, Hey2 loss does not affect aortic development, but it instead leads to a massive postnatal cardiac hypertrophy with high lethality during the first 10 days of life. This cardiomyopathy is ameliorated with time in surviving animals that do not appear to be manifestly impaired during adult life. These differences in phenotypes suggest that changes in expression or function of genes during evolution may lead to quite different pathological phenotypes, if impaired.

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.

Thromboxane A2 Receptor Agonists Antagonize the Proangiogenic Effects of Fibroblast Growth Factor-2. Role of Receptor Internalization, Thrombospondin-1, and αvβ3

Abstract— Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of the pathogenesis of many vascular events, including angiogenesis. Endothelial cells express TXA2 receptors (TP) but the effects of TP stimulation on angiogenesis remain controversial. In this study, we show that stimulation of endothelial cell TP impairs ligand-induced FGF receptor internalization and consequently abrogates FGF-2-induced endothelial cell migration in vitro and angiogenesis in vivo. Prevention of FGF-2-induced angiogenesis was associated with expression of the TP&bgr; isoform. The deficit in FGFR1 internalization was mediated through activation of TP&bgr; preventing the FGF-2-mediated decrease in p53 expression, thus enhancing thrombospondin-1 (TSP-1) release from EC and reducing FGFR1 internalization. Once released TSP-1 interacted with the &agr;v&bgr;3 integrin on the EC surface. On stimulation, FGFR1 and &agr;v&bgr;3 were found to associate in a complex. We determined that complex formation was important for receptor internalization as conditions that inhibit FGFR1 internalization, such as inappropriate ligation of &agr;v&bgr;3 by either TSP-1 or a neutralizing antibody, disrupted the complex. These results establish a novel role for isoform specific regulation of angiogenesis by TP, provide the first functional significance for the existence of two TP isoforms in humans, and clarify the mechanism by which TP signaling regulates FGFR1 kinetics and signaling.

Time‐of‐flight quantitative measurements of blood flow in mouse hindlimbs

To evaluate the feasibility of using time‐of‐flight (TOF) imaging to directly measure hindlimb blood flow in a mouse model of peripheral vascular disease.

Inhibition of Collateral Artery Growth by Mibefradil: Possible Role of Volume-Regulated Chloride Channels

Endothelial cell swelling is one of the earliest hallmarks of arteriogenesis, the growth and maturation of collaterals. Mibefradil was found to block endothelial Cl(-) channels that control the volume of endothelial cells. Thus the authors investigated whether the blockade of volume-controlling endothelial cell channels would translate into an inhibition of arteriogenesis. In BALB/c mice, the right femoral artery was ligated and the animals received either mibefradil or solvent (phosphate-buffered saline [PBS]) via osmotic minipumps. Laser Doppler perfusion ratio (R/L) of ligated versus nonligated distal hindlimb increased from 0.06 +/- 0.01 (immediately after ligation) to 0.25 +/- 0.02 (day 7) in the PBS group and only from 0.07 +/- 0.02 to 0.13 +/- 0.02 in the mibefradil group (p <.01). Collateral artery diameters were significantly smaller in the mibefradil group (61 +/- 4.7 microm) versus controls (77.3 +/- 0.9 microm) (p <.05). Relative hemoglobin oxygen saturation measurements confirmed these findings (p <.02). The inhibition of arteriogenesis in the mibefradil group suggests that endothelial Cl(-) channels are involved in the initiation of arteriogenesis.

Therapeutic Angiogenesis for Ischemic Heart Disease

The de-novo formation of vessels (angiogenesis) and the remodelling of preexisting collateral vessels (arteriogenesis) are processes that occur naturally in ischemic heart disease. Promoting these processes by administration of various substances or other physical stimuli (therapeutic angiogenesis) may provide a future strategy for the treatment of ischemic vascular diseases. Mechanisms of angiogenesis and arteriogenesis, as well as trials of therapeutic angiogenesis in animal models and humans are reviewed.