Macrophages in collateral arteriogenesis.

Arteriosclerotic vascular disease is the most common cause of death and a major cause of disability in the developed world. Adverse outcomes of arteriosclerotic vascular disease are related to consequences of tissue ischemia and necrosis affecting the heart, brain, limbs, and other organs. Collateral artery growth or arteriogenesis occurs naturally and can help restore perfusion to ischemic tissues. Understanding the mechanisms of collateral artery growth may provide therapeutic options for patients with ischemic vascular disease. In this review, we examine the evidence for a role of monocytes and macrophages in collateral arteriogenesis.

Continuous endothelial cell activation increases angiogenesis: evidence for the direct role of endothelium linking angiogenesis and inflammation.

There is increasing evidence that chronic inflammation is tightly linked to diseases associated with endothelial dysfunction, including the induction of aberrant angiogenesis. While leukocytes have been described as mediators of inflammation-associated angiogenesis, the effects of direct chronic endothelial activation have not been addressed in this context. Using an uncleavable mutant of the transmembrane form of tumor necrosis factor-alpha (TNF-alpha), we have established models of stable TNF-alpha expression in endothelial cells in vitro and in transgenic mice in vivo. In the in vitro model, continuous endothelial activation leads to increased leukocyte cellular adhesion molecule expression and intracellular reactive oxygen species, hallmarks of a proinflammatory and dysfunctional endothelium. In addition, stable expression of TNF-alpha in endothelial cells increased angiogenic sprout formation in the presence but also in the absence of angiogenic growth factors. The partial neutralization of this effect by TNF-alpha antibodies and the inability of conditioned media from stable TNF-alpha-expressing endothelial cells to induce angiogenic activities in control endothelial cells suggest that this effect does not require expression of additional autocrine factors, but is an autonomous effect of the transmembrane TNF on the endothelial cells. Furthermore, using the Matrigel plug assay in vivo, increased angiogenesis was observed in endothelial TNF-alpha-expressing transgenic versus control mice. In conclusion, chronic inflammatory changes mediated by TNF-alpha can induce angiogenesis in vitro and in vivo, suggesting endothelial cell activation as a direct link between inflammation and angiogenesis.

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.

Delayed arteriogenesis in hypercholesterolemic mice.

BACKGROUND: Hypercholesterolemia has been reported to inhibit ischemia-induced angiogenesis. To address its effects on arteriogenesis, we investigated arterial growth in hypercholesterolemic low-density lipoprotein receptor(-/-)/ApoB-48(-/-) (HCE) mice. METHODS AND RESULTS: The extent and the time course of arteriogenesis after femoral artery ligation was evaluated in HCE and strain-matched control mice. Distal limb perfusion was measured by laser Doppler imaging, whereas MRI was used to visualize arterial flow and micro-computed tomography to assess vascular growth. After femoral artery ligation, serial laser Doppler imaging demonstrated significantly delayed restoration of perfusion in untreated HCE compared with control mice (day 3, 0.09 versus 0.19, P<0.05). Treatment with Ad-PR39 in control mice led to a significant restoration of arterial blood flow and tissue perfusion at day 3, whereas in HCE mice, hindlimb perfusion began increasing only by day 7. Micro-CT analysis confirmed increased growth of smaller arterioles (16 to 63 microm in diameter) in the Ad-PR39-treated control compared with HCE mice. The delay in arteriogenesis in HCE mice correlated with delayed tissue appearance of F4/80+ cells. Analysis of gene expression after Ad-PR39 treatment demonstrated that HCE mice had significantly reduced expression of FGF receptor 1, hypoxia-inducible factor-1alpha, vascular cell adhesion molecule-1, macrophage scavenger receptor-1, and cyclophilin A compared with controls 3 days after arterial ligation that equalized by day 7, mimicking relative changes in arteriogenesis and tissue perfusion. CONCLUSIONS: Hypercholesterolemia results in delayed native arteriogenesis because of reduced early monocyte/macrophage influx and delayed and impaired arterial growth response to growth factor therapy.

Synthesis of anticoagulantly active heparan sulfate proteoglycans by glomerular epithelial cells involves multiple 3-O-sulfotransferase isoforms and a limiting precursor pool.

Endothelial and other select cell types synthesize a subpopulation of heparan sulfate (HS) proteoglycans (HSPGs), anticoagulant HSPGs (aHSPGs) that bear aHS-HS chains with the cognate 3-O-sulfated pentasaccharide motif that can bind and activate anti-thrombin (AT). Endothelial cells regulate aHSPG production by limiting levels of HS 3-O-sulfotransferase-1 (3-OST-1), which modifies a non-limiting pool of aHS-precursors. By probing kidney cryosections with (125)I-AT and fluorescently tagged AT we found that the glomerular basement membrane contains aHSPGs, with the staining pattern implicating synthesis by glomerular epithelial cells (GECs). Indeed, cultured GECs synthesized aHS with high AT affinity that was comparable with the endothelial product. Disaccharide analyses of human GEC (hGEC) HS in conjunction with transcript analyses revealed that hGECs express predominantly 3-OST-1 and 3-OST-3(A). aHS production has not been previously examined in cells expressing multiple 3-OST isoforms. This unusual situation appears to involve novel mechanisms to regulate aHS production, as HS structural analyses suggest hGECs exhibit excess levels of 3-OST-1 and an extremely limiting pool of aHS-precursor. A limiting aHS-precursor pool may serve to minimize aHS synthesis by non-3-OST-1 isoforms. Indeed, we show that high in vitro levels of 3-OST-3(A) can efficiently generate aHS. Non-3-OST-1 isoforms can generate aHS in vivo, as the probing of kidney sections from 3-OST-1-deficient mice revealed GEC synthesis of aHSPGs. Surprisingly, Hs3st1(-/-) kidney only expresses 3-OST isoforms having a low specificity for aHS synthesis. Thus, our analyses reveal a cell type that expresses multiple 3-OST isoforms and produces minimal amounts of aHS-precursor. In part, this mechanism should prevent aHS overproduction by non-3-OST-1 isoforms. Such a role may be essential, as 3-OST isoforms that have a low specificity for aHS synthesis can generate substantial levels of aHSPGs in vivo.

Time-of-flight quantitative measurements of blood flow in mouse hindlimbs.

PURPOSE: 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. MATERIALS AND METHODS: Four tubes were imaged simultaneously (diameters = 0.39 mm, 0.59 mm, and two at 1.46 mm) with a 1.0 mM copper sulfate solution for 19 flow velocities. In vivo measurements were performed in the hindlimbs of three mouse strains-C57BL/6 (N = 5), BALB/c (N = 5), and 129S2/Sv (N = 5)-three weeks after femoral artery ligation with a calibration standard. RESULTS: The flow phantom showed that the intensity was linear (r2 = 0.92) over the pertinent blood flow velocities in the mouse hindlimbs. Measurements of the blood flow in the distal hindlimbs in different strains of mice (combination of both the venous and arterial flows) were obtained 21 days after right-sided femoral artery occlusion. The results showed that under similar conditions of anesthesia and temperature, SV129 mice on the nonligated side had the highest flows (0.50 +/- 0.07 mL/minute), followed by C57BL/6 (0.28 +/- 0.04 mL/minute) and BALB/c (0.23 +/- 0.05 mL/minute), P < 0.02. The ligated side measurements (SV129, 0.31 +/- 0.05 mL/minute (P = 0.02); C57BL/6, 0.21 +/- 0.02 mL/minute (P = 0.13); and BALB/c, 0.12 +/- 0.02 mL/minute (P= 0.06)) showed a trend in BALB/c and C57BL/6 and significant differences in SV129 for incomplete recovery three weeks after surgery, compared to the nonligated side. CONCLUSION: Two-dimensional TOF imaging permits quantitative in vivo measurements of hindlimb blood flow in a mouse model of peripheral vascular disease without the need of contrast injection, offering advantages of serial imaging not limited by tissue penetration.

Magnetic resonance angiography of collateral vessels in a murine femoral artery ligation model.

The in vivo detection of growing collateral vessels following arterial occlusion is difficult in small animals. We have addressed the feasibility of performing high resolution time-of-flight angiograms to monitor the growth of collateral vessels after femoral artery occlusion in mice. We will also present a low-pass quadrature birdcage coil construction with a sufficient signal-to-noise ratio to produce high resolution. After a 4-month recovery period a C57BL/6 mouse with a surgical occlusion of the right femoral artery was used to assess the image quality and time requirements to produce magnetic resonance angiograms sufficient to assess collateral artery development using a two-dimensional gradient echo sequence. At a resolution of 100 x 100 x 100 microm and a matrix size of 256 x 128 x 256 for a 2.56 cm isometric volume, three scans were performed with one, two and four repetitions resulting in signal-to-noise ratios for the femoral artery proximal to the ligation site of 58, 126 and 194, respectively. Five C57BL/6 mice were additionally measured 4 weeks after occlusion using two repetitions and the visual collateral vessels were assessed for number and location: 2.0 +/- 1.2 in quadriceps muscle, 0.6 +/- 0.5 in adductor (deep adductor vessel), 0.0 +/- 0.0 in adductor (surface adductor vessels). The results showed a significant difference, two-sided t-test, p < 0.05, in number of vessels in all the locations. We have shown that this method can be utilized to elucidate the contribution of collateral vessels to arterial flow.

Collateral artery growth (arteriogenesis) after experimental arterial occlusion is impaired in mice lacking CC-chemokine receptor-2.

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.

Thromboxane A2 receptor agonists antagonize the proangiogenic effects of fibroblast growth factor-2: role of receptor internalization, thrombospondin-1, and alpha(v)beta3.

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 TPbeta isoform. The deficit in FGFR1 internalization was mediated through activation of TPbeta 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 alpha(v)beta3 integrin on the EC surface. On stimulation, FGFR1 and alpha(v)beta3 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 alpha(v)beta3 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.

Bone marrow-derived cells do not incorporate into the adult growing vasculature.

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.

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.

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: 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.

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.

Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice.

UNLABELLED: The goal of this study was to examine the mechanisms of vascular growth that lead to the restoration of perfusion in a peripheral vascular disease model in mice. We monitored blood flow recovery and measured vascular growth in inbred strains of mice following femoral artery occlusion. Acute collateral blood flow to the hindlimb was lowest in Balb/C mice, causing intense ischemia, and showed a slower recovery (more than 21 days to 50% normal) than C57Bl/6 which had a 7-fold higher acute collateral flow and a fast recovery (3 days). Collateral vessels were enlarged by proliferation of ECs and SMCs. Capillary density increased in the lower limbs of Balb/Cs (1.7-fold) and of sv129s. Tissue oxygen saturation recovered faster than flow in all strains. Morphometry of mature collaterals showed a diameter increase of 2.1-2.4 fold. The increase in total vessel wall area exceeded that of the femoral artery by 1.4-fold and the common lumenal area by 1.6-fold. Infusion of the growth factor peptide FGF-2 by osmotic minipump accelerated arteriogenesis but inhibited the angiogenic response probably because it prevented ischemia. CONCLUSION: the speed of arteriogenesis is inversely related to the intensity of ischemia, and arteriogenesis is by far the most efficient mechanism to increase blood flow after femoral artery occlusion. De novo arteriogenesis was not observed.