GM-CSF: a strong arteriogenic factor acting by amplification of monocyte function.

We investigated the role of the colony stimulating factor for monocytes (GM-CSF) to test the hypothesis whether prolongation of the monocyte's life cycle will support arteriogenesis (rapid growth of preexisting collateral arteries). This appeared logical in view of our discovery that circulating monocytes play an important part in the positive remodeling of small preexisting arterioles into arteries to compensate for arterial occlusions (arteriogenesis) and especially following our findings that MCP-1 markedly increases the speed of arteriogenesis. The continuous infusion of GM-CSF for 7 days into the proximal stump of the acutely occluded femoral artery of rabbits by osmotic minipump produced indeed a marked arteriogenic response as demonstrated by an increase (2-fold) in number and size of collateral arteries on postmortem angiograms and by the increase of maximal blood flow during vasodilation measured in vivo by blood pump perfusion of the hindquarter (5-fold). When GM-CSF and MCP-1 were simultaneously infused the effects on arteriogenesis were additive on angiograms as well as on conductance. GM-CSF was also able to widen the time window of MCP-1 activity: MCP-1 treatment alone was ineffective when given after the third week following occlusion. When administered together with GM-CSF about 80% of normal maximal conductance of the artery that was replaced by collaterals were achieved, a result that was not reached before by any other experimental treatment. Experiments with cells isolated from treated animals showed that monocyte apoptosis was markedly reduced. In addition we hypothesize that GM-CSF may aid in releasing pluripotent monocyte (stem-) cells from the bone marrow into the circulation. In contrast to MCP-1, GM-CSF showed no activity on monocyte transmigration through- and also no influence on monocyte adhesion to cultured endothelial cells. In conclusion we have discovered a new function of the hemopoietic stem cell factor GM-CSF, which is also a powerful arteriogenic peptide that acts via prolongation of the life cycle of monocytes/macrophages.     

Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease

After birth two forms of vessel growth can be observed; angiogenesis and arteriogenesis. Angiogenesis refers to the formation of capillary networks. Arteriogenesis refers to the growth of preexistent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. The process of arteriogenesis is initiated when shear stresses increase in the preexistent collateral pathways upon narrowing of a main artery. The increased shear stress leads to an upregulation of cell adhesion molecules for circulating monocytes, which accumulate subsequently around the proliferating arteries and provide the several required cytokines and growth factors. Several strategies are currently tested for their potential to stimulate the process of arteriogenesis. These strategies focus either at shear stress, at direct stimulation of endothelial and smooth muscle cell growth or at the monocytic pathway and promising results were obtained from experimental studies. However, some important questions remain to be answered before arteriogenesis can be brought from bench to bedside.     

Factors regulating arteriogenesis

Growth of collateral vessels is potentially able to preserve structure and a variable degree of function in subtended tissues in the presence of arterial occlusions. The process of transformation of a small arteriole into much larger conductance artery is called arteriogenesis. Small arterioles that interconnect side branches proximal from the arterial occlusion with distal ones experience increased fluid shear stress because of the increased blood flow velocity attributable to the pressure gradient along the bridging collaterals. This activates the endothelium and leads to monocyte adhesion and infiltration with the subsequent production of growth factors and proteases. Preexistent arterioles are essential. Their presence is genetically determined. Arteriogenesis is not organ- or species-specific; coronary or peripheral collateral vessels develop following the same design principles in mice, rats, rabbits, or dogs. In contrast to angiogenesis, arteriogenesis is not dependent on the presence of hypoxia/ischemia.     

Arteriogenesis - is this terminology necessary?

The role of the collateral circulation has been controversially discussed over many decades. With the availability of purified growth factors such as the fibroblast growth factors several studies provided data, that the growth of collateral arteries, termed arteriogenesis, is not limited to its natural timecourse. When applied in experimental models FGF in particular led to a significant increase in collateral conductance upon arterial occlusion. Thus the proliferation of preexisting bypassing arterioles could be enhanced therapeutically. The purpose of this review is to discuss the physiological importance of different kinds of vascular growth (e.g. vasculogenesis, angiogenesis, arteriogenesis) to enhance blood flow to ischemic limbs or the heart. It is outlined that large conductance arteries rather than capillary networks are needed to compensate for perfusion deficits due to atherosclerotic stenosis or occlusion.     

Involvement of the fibroblast growth factor system in adaptive and chemokine-induced arteriogenesis

Fibroblast growth factors (FGFs) have been applied in a variety of therapeutic and experimental studies to improve collateral blood flow. However, the pathophysiological role and the temporospatial expression of the FGFs and their receptors during arteriogenesis have never been elucidated in vivo. Here, we report that collateral artery growth in its early phase is associated with an increased expression of FGF receptor-1 (FGFR-1) and syndecan-4 on mRNA and protein levels as well as with an increased kinase activity of FGFR-1 in a rabbit model of arteriogenesis. However, the mRNA levels of FGF-1 and -2 remained constant. Our data suggest that these growth factors are supplied by endothelial attracted monocytes that, in turn, produce and deliver the FGFs to growing collateral arteries. Monocyte chemoattractant protein-1-stimulated arteriogenesis was strongly reduced in rabbits by application of the FGF inhibitor polyanetholesulfonic acid, indicating that the monocyte-related arteriogenesis (as well as the unstimulated adaptation proper) is promoted by FGFs. In summary, this study shows that arteriogenesis is associated with an increased expression of the FGFRs at the site of the vessel, whereas the growth-promoting ligands are supplied by monocytes in a paracrine way.     

Influence of inflammatory cytokines on arteriogenesis

Blood vessel growth after birth is limited to two major processes. Angiogenesis is the growth of new capillaries by sprouting or intussusception. The major stimulus for angiogenesis is ischemia. In contrast, arteriogenesis describes the remodeling and growth of collateral arteries from a preexisting arteriolar network. Arteriogenesis is induced after the occlusion of a major artery which induces hemodynamic and mechanical effects on the collateral vessel wall which occur with increasing blood flow velocity due to the low pressure at the reentrant site of the collateral vessel. A variety of different cytokines that act by stimulating endothelial and smooth muscle cell proliferation and migration or recruitment and activation of monocytes have been identified to stimulate angiogenesis and/or arteriogenesis (i.e., MCP-1, FGF-2, TGF-beta, VEGF, and GM-CSF). Several clinical trials have been published in that field to suggest the feasibility and safety of treatment with such cytokines or their genes. However, the results indicate that further studies are needed before proangiogenic and proarteriogenic therapies are ready for clinical application.     

Connexin37, not Cx40 and Cx43, is induced in vascular smooth muscle cells during coronary arteriogenesis

W.-J. Cai, S. Koltai, E. Kocsis, D. Scholz, W. Schaper and J. Schaper. Connexin37, not Cx40 and Cx43, is Induced in Vascular Smooth Muscle Cells During Coronary Arteriogenesis. Journal of Molecular and Cellular Cardiology (2001) 33, 957-967. The hypothesis that an altered expression of gap junction (GJ) proteins, connexin37 (Cx37), Cx40 and Cx43 will contribute to adaptive arteriogenesis was tested in growing coronary collateral vessels (CV) of the dog heart by immunoconfocal microscopy and transmission electron microscopy (TEM). We found that: (1) in the normal coronary system Cx37 and Cx40 were only expressed in endothelial cells (EC) from artery to capillary; (2) during collateral growth Cx37 was significantly induced in smooth muscle cells (SMC) from small-large arteries to precapillary arterioles (?=15 microm), while Cx40 was still only present in EC; (3) both homogeneous and heterogeneous distribution of Cx37 was observed in normal vessels (NV) and growing vessels (GV); (4) in mature vessels (MV), Cx37 was downregulated, similar to NV; (5) dual immunostaining revealed an inverse correlation between expression of Cx37 and desmin in GV occurring prior to downregulation of alpha-smooth actin and calponin; (6) Cx43 was undetectable in any vascular cells, both in NV and GV; (7) GJ were not found in SMC by TEM. Our data for the first time show the profile of connexin expression in the coronary system and provide evidence for existence of GJ proteins in capillaries. It is a novel finding that an altered expression of Cx37 is characteristic of adaptive arteriogenesis in the dog heart and may be used as a marker of vascular growth. Induced Cx37 may be an early signal indicating that SMC are responding to haemodynamic changes, i.e. increased shear stress.     

Time course of arteriogenesis following femoral artery occlusion in the rabbit

OBJECTIVE: We examined the time course of arteriogenesis (collateral artery growth) after femoral artery ligation and the effect of monocyte chemoattractant protein-1 (MCP-1). METHODS: New Zealand White rabbits received MCP-1 or phosphate buffered saline (PBS) for a 1-week period, either directly or 3 weeks after femoral artery ligation (non-ischemic model). A control group was studied with intact femoral arteries and another 1 min after acute femoral artery ligation. RESULTS: Collateral conductance index significantly increased when MCP-1 treatment started directly after femoral artery ligation (acute occlusion: 0.94+/-0.19; without occlusion: 168.56+/-15.99; PBS: 4.10+/-0.48; MCP-1: 33.96+/-1.76 ml/min/100 mmHg). However, delayed onset of treatment 3 weeks after ligation and final study of conductance at 4 weeks showed no significant difference against a 4-week control (PBS: 79.08+/-7.24; MCP-1: 90.03+/-8.73 ml/min/100 mmHg). In these groups increased conductance indices were accompanied by a decrease in the number of visible collateral vessels (from 18 to 36 identifiable vessels at day 7 to about four at 21 days). CONCLUSION: We conclude that the chemokine MCP-1 markedly accelerated collateral artery growth but did not alter its final extent above that reached spontaneously as a function of time. We show thus for the first time that a narrow time window exists for the responsiveness to the arteriogenic actions of MCP-1, a feature that MCP-1 may share with other growth factors. We show furthermore that the spontaneous adaptation by arteriogenesis stops when only about 50% of the vasodilatory reserve of the arterial bed before occlusion are reached. The superiority of few large arterial collaterals in their ability to conduct large amounts of blood flow per unit of pressure as compared to the angiogenic response where large numbers of small vessels are produced with minimal ability to allow mass transport of bulk flow is stressed.     

冠状动脉侧支循环及其临床意义

目的研究冠状动脉侧支循环开放的条件、影响因素及其对心肌缺血、心功能的保护作用。方法回顾性分析１６３例经冠状动脉造影证实管腔直径狭窄≥７５％的２１４支血管的侧支循环开放情况。结果７９支见侧支循环开放,６３例７２支完全闭塞血管中,３１例３８支侧支循环发展良好（Ａ组）,３２例３４支侧支循环发展不良（Ｂ组）。心肌缺血病程长、无高血压病或糖尿病史及右冠状动脉闭塞者,侧支循环发展良好率高。心功能指标Ａ组明显优于Ｂ组,运动负荷试验阴性率两组间无差异。结论侧支循环开放依赖冠状血管完全闭塞或次全闭塞,发展受多种因素影响,良好的侧支循环对缺血心肌和心功能有保护作用,但多数不能消除负荷所致心肌缺血。     

Biological bypass in cardiovascular surgery

Protein and gene therapy offer a tremendous opportunity to improve the care of critically ill patients with ischemic heart and peripheral artery occlusion disease. With the availability of purified growth factors such as vascular endothelial and fibroblast growth factors (FGF), several experimental and clinical studies provided data, that the growth of capillaries (angiogenesis) and of collateral arteries (arteriogenesis) is not limited to its natural time course. When applied in experimental models and in conjunction with coronary artery bypass operations, FGF in particular, led to a significant increase in endogenous rerouting of blood flow by collateral vessels inside the tissue itself. Thus, the proliferation of preexisting bypassing arterioles could be enhanced therapeutically (biological bypass). The purpose of this review is to discuss the physiological importance of different kinds of cytokines which are able to induce angio- and arteriogenesis in ischemic limbs or the heart. It is outlined that a combination of a sufficient amount of large arterioles and a capillary network are needed to compensate perfusion deficits. Each patient, who has an ischemic area and cannot be conventionally revascularized, is a potential candidate for the biological bypass.     

Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice

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.     

Coronary wedge pressure in relation to spontaneously visible and recruitable collaterals

Coronary angiography demonstrates only collateral arteries that are already in use (spontaneously visible collaterals). Percutaneous transluminal coronary angioplasty (PTCA) provides an opportunity to uncover collaterals ready to become functional in case of occlusion of the recipient artery (recruitable collaterals). The incidence of recruitable collaterals and their relation to the distal pressure in the occluded artery (coronary wedge pressure) during a 30 sec or longer balloon occlusion was assessed in 57 coronary arteries of 49 patients undergoing PTCA for a proximal coronary stenosis or occlusion. Collateral to 75% of the arteries were present. Spontaneously visible collaterals were four times as frequent as recruitable collaterals. Coronary wedge pressure was significantly higher in arteries with spontaneously visible and recruitable collaterals (41 +/- 12 and 36 +/- 12 mm Hg, respectively) than in arteries without collaterals (18 +/- 4 mm Hg). A coronary wedge pressure of 30 mm Hg or higher was found exclusively in the presence of collaterals. Electrocardiographic changes during balloon occlusion were found more frequently with arteries without collaterals than with arteries with spontaneously visible or recruitable collaterals. Chest pain was more frequent in patients without collaterals or with recruitable collaterals than in those with spontaneously visible collaterals. Major in-hospital events occurred in three patients with collaterals, with a salutary influence of the collaterals in two. The coronary wedge pressure allows prediction of recruitable collaterals. Their clinical impact remains to be investigated in long-term studies on large patient populations.     

Pathophysiology of collateral development

The formation of collateral arteries in patients suffering from occlusive atherosclerotic vascular diseases has been frequently reported. The growth of these collateral arteries has been termed 'arteriogenesis'. Clinical observations and investigations using various animal models support the hypothesis that the mechanism of arteriogenesis is based on the remodelling of pre-existing collateral anastomoses. This process seems to be mainly triggered by fluid shear stress which is induced by the altered blood flow conditions after an arterial occlusion. Early arteriogenesis involves the activation of collateral endothelial cells, the attraction of leukocytes to the collateral vascular wall and subsequently their invasion into the perivascular space of the collateral vessel. In a second phase, proliferation of vascular cells is initiated by growth factors mainly released from accumulated leukocytes. Furthermore, tissue degradation and changes in the extracellular matrix are observed. Unravelling the mechanisms of arteriogenesis is crucial to the development of successful therapeutic approaches for the treatment of patients with ischemic vascular diseases.     

Angiogenesis and arteriogenesis--not yet for prescription

Angiogenesis is the growth of new vessels from preexisting vessels by sprouting and intussusception with ischemia being the major stimulus. Circulating endothelial precursor cells have recently been found to participate in this process. The remodeling of preexisting bridging collateral arterioles, i.e., arteriogenesis, should be a much more efficient mechanism to compensate for the gradual or intermittent occlusion of a major epicardial or peripheral artery. Arteriogenesis is associated with an active growth process. It is probably not dependent on ischemia but initiated by local hemodynamic and mechanical effects on the vessel that occur with increasing blood flow. A variety of growth factors that act not only by stimulating endothelial and smooth muscle cell proliferation and migration, as well as substances that increase recruitment and activation of monocytes have been demonstrated to stimulate angiogenesis and arteriogenesis. Several clinical phase I trials suggest the feasibility and short-term safety of treatment with growth factors or their genes. The VIVA trial, the only phase II trial that has been published in this field, employed VEGF165 by intracoronary infusion followed by several intravenous infusions and did not demonstrate any increase of exercise time or angina by VEGF over placebo. The strong sustained placebo effect was surprising. Concerns about the long-term exacerbation of angiogenesis-dependent pathologic processes, like malignant tumors, atherosclerotic plaque formation and proliferative retinopathies, will require careful follow-up. Pro-angiogenic and pro-arteriogenic therapies may need further sophistication before they enter clinical practice.     

Transgenic myocardial overexpression of fibroblast growth factor-1 increases coronary artery density and branching

Fibroblast growth factor (FGF)-1 plays important roles during myocardial and coronary morphogenesis. FGF-1 is also involved in the physiological response of the adult heart against ischemia, which includes cardiomyocyte protection and vascular growth. In the present study, we have generated transgenic mice with specific myocardial overexpression of the gene. Transgene expression was verified by Northern blot, and increased FGF-1 protein content was assessed by Western blot and immunoconfocal microscopy. Anatomic, histomorphological, and ultrastructural analyses revealed no major morphological or developmental abnormalities of transgenic hearts. Capillary density was unaltered, whereas the density of coronary arteries, especially arterioles, was significantly increased, as was the number of branches of the main coronary arteries. In addition, the coronary flow was significantly enhanced in transgenic mice ex vivo. These differences in the anatomic pattern of the coronary vasculature are established during the second month of postnatal life. The present findings demonstrate an important role of FGF-1 in the differentiation and growth of the coronary system and suggest that it is a key regulatory molecule of the differentiation of the arterial system.     

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.     

Quantitative assessment of coronary arterial diameter before and after coronary artery bypass grafting

Because coronary artery bypass graft patency is related to the size of the artery, a preoperative assessment of arterial diameter is important. However, the relation between coronary arterial diameter (assessed by angiography) and true luminal size of arteries that are occluded and filled by collaterals has not been completely characterized. This study was done to measure the luminal diameter of coronary arteries before and after bypass grafting. Twenty-six patients (20 men and 6 women, aged 34 to 72 years) had coronary angiography before and from 1 to 35 months after bypass surgery. Coronary arterial luminal diameter was similar before and after operation for the 18 insignificantly narrowed arteries and the 24 arteries with narrowings that filled by anterograde flow before operation and were bypassed. In contrast, in the 21 arteries that were occluded and filled by collateral flow, coronary arterial diameter was significantly larger (p less than 0.001) after operation. Thus, when a coronary artery is occluded and filled by collaterals, its true luminal size is consistently underestimated by the angiogram.