The Range of Adaptation by Collateral Vessels After Femoral Artery Occlusion
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Inka Eitenmüller, Oscar Volger, Alexande
参见附件。
the Max-Planck-Institute for Heart and Lung Research (I.E., K.T., M.H., F.P., W.S.), Bad Nauheim, Germany
Department of Medical Biochemistry, Academic Medical Centre (O.V., A.J.G.H.), University of Amsterdam, The Netherlands
Kerckhoff Clinic (A.K.), Bad Nauheim, Germany
Division of Vascular and Endovascular Surgery (T.S.), Goethe-University of Frankfurt/Main, Germany
Slovak Academy of Sciences (M.B.), Bratislava, Slovakia
Department of Anatomy, Xiangsha School of Medicine (W.C.), Central South University, Xiangsha, Hunan, P.R. China
Department of Medical Biochemistry (S.F.), Liebig-University Giessen, Germany.
Abstract
Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only 35% of adenosine-recruitable maximal conductance (Cmax) probably because initially elevated fluid shear stress (FSS) quickly normalizes. We tested the hypothesis whether this deficit can be mended by artificially increasing FSS or whether anatomical restraints prevent complete restitution. We chronically increased FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein. After reclosure of the shunt collateral flow was measured at maximal vasodilatation. Cmax reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the Cmax of the normal vasculature before occlusion. Expression profiling showed upregulation of members of the Rho-pathway (RhoA, cofilin, focal adhesion kinase, vimentin) and the Rho-antagonist Fasudil markedly inhibited arteriogenesis. The activities of Ras and ERK-1,-2 were markedly increased in collateral vessels of the shunt experiment, and infusions of L-NAME and L-NNA strongly inhibited MAPK activity as well as shunt-induced arteriogenesis. Infusions of the peroxinitrite donor Sin-1 inhibited arteriogenesis. The radical scavengers urate, ebselen, SOD, and catalase had no effect. We conclude that increased FSS can overcome the anatomical restrictions of collateral arteries and is potentially able to completely restore maximal collateral conductance. Increased FSS activates the Ras-ERK-, the Rho-, and the NO- (but not the Akt-) pathway enabling collateral artery growth.
Key Words: arteriogenesis fluid shear stress shunt growth factors microarrays
Introduction
The restoration of maximal conductance (Cmax) in animals after arterial occlusion remains defective (35% in the canine coronary circulation1 and 40% in the rabbit hind limb2) in spite of the fact that normal resting blood flow is reached early. As a consequence exercise testing in experimental animals reveals defects similar to those in human patients.3 It was not known until now whether collateral vessels are potentially able to restore the full dilatory reserve of a normal vascular bed. Many observations would predict that this is not the case: the multitude of small vessels that replace an occluded artery is inefficient according to Poissieulle’s Law, and the tortuosity of collateral vessels offers finite resistance because of curvature flow and increased collateral length.4 One reason for the defective adaptation may lie in the fact that fluid shear stress normalizes prematurely: FSS falls by the third power of the growing radius. We tested the hypothesis whether a sustained increase of FSS is able to prolong the growth process and to restore normal maximal conductance. The method to achieve this was the creation of a shunt between the distal stump of the occluded femoral artery and the accompanying vein.5
The novelty of the present findings is the demonstration that Cmax can be reached and even surpassed by a drastic increase of fluid shear stress. Signaling pathways involved in the adaptation to fluid shear stress were interrogated, and it could be shown that three pathways have to converge to induce arterial growth, ie, the Ras-ERK pathway, the Rho-, and the NO-pathway.
Materials and Methods
An expanded Materials and Methods section can be found in the supplement available at http://circres.ahajournals.org.
We subjected rabbits to femoral artery occlusion and studied the effects of an arteriovenous shunt on the development of the collateral circulation (Figure 1). We measured collateral blood flow by ultrasonic flow probes and by MRI. The morphology of the vessels was studied in vivo by CT-scanning and post mortem by arteriography following pressure-controlled contrast medium injection. Collateral artery tissue was prepared for RNA extraction, which was fashioned for microarray studies and for Western blot analysis. Animals were treated with a variety of tool drugs to interrogate the NO, Rho-, and Ras pathways. Smooth muscle cells in culture were treated with several agents used in the pharmacological in vivo studies.
Results
No animals were lost because of the surgical procedure or postoperatively. Clinical signs of peripheral ischemia were transiently present in a few animals but did not lead to ulceration or amputation. In two thirds of the treated animals, the A-V shunt was still open at termination of the experiment. Only animals with a functional shunt were included in the study.
Hemodynamics
Immediately after occlusion of the femoral artery blood pressure in the peripheral stump of the occluded artery fell to low values of 31±4.2% of the aortic pressure. Peripheral pressure rose to 50±8% after 7 days and remained at that level for several weeks. In contrast, in animals with femoral artery ligature and an additional A-V shunt the peripheral arterial pressure dropped to 6±1% of the arterial pressure (immediately after the operation), increased within 7 days to 17±3%, and reached 29±3% after 4 weeks. When the shunt was occluded—routinely done during the terminal experiment—7 days after femoral artery occlusion, peripheral pressure increased to 60±3% of the aortic pressure and to 73±1% when measured 4 weeks after femoral artery occlusion.
Collateral blood flow ratio (shunt versus control side) measured by MRI was 5.8±0.8 at day 7 and 13.7±3.7 at day 28. One week after shunt treatment calculated Cmax values under optimal vasodilatation were 88±4% of Cmax of nonoccluded legs, representing a 2-fold increase in comparison to Cmax of control ligated legs (44±7%). After 4 weeks, shunt treated legs showed a Cmax of 199±19% of nonligated legs. In contrast, legs with control ligatures did not show any further improvement after 4 weeks when compared with the results after 1 week (Figure 2A).
Effects of Pharmacological Inhibitors
Fasudil, a Rho-pathway inhibitor, and L-NAME and L-NNA, both inhibitors of endothelial NO synthetase (eNOS), almost completely blocked the positive effects of high shear stress on collateral artery growth (shunt 306±15 versus Fasudil 176±4 versus L-NAME 163±8 versus control 155±27 mL/min/100 mm Hg) (Figure 2B).
Moreover, the hypothesis that shear stress leads to an uncoupling of eNOS resulting in increased production of oxygen- and nitrogen-based radicals was investigated. To demonstrate such an effect, 3-Morpholinosydnonimine (Sin-1), which acts as a NO- and superoxide anion radical donor (both react to produce peroxinitrite), was infused but arteriogenesis was inhibited in a dose-dependent manner. In contrast, infusion of the scavengers Ebselen, urate, catalase, and superoxide dismutase showed no effect (data not shown).
Angiography and Computerized Tomography
Postmortem angiograms (Figure 3A and 3B) showed marked increases in the number of enlarged collaterals on the shunt side (day 7: 42±2.5 versus 18±0.95 [control]; day 28: 48±2.3 versus 12±2.0 [control]), which corresponded with the number and size of collaterals detected in vivo by CT (Figure 4A and 4B). Treatment with Fasudil or L-NAME resulted in a significant decrease in collateral count (fasudil 31±0.9 versus L-NAME 23.8±1.2 versus shunt 42±2.5). In contrast to the natural course of collateral growth, which leads to the elimination of most vessels that had initially participated in the growth transformation in favor of a few large ones ("pruning"), no pruning had occurred on the shunt side without further treatment: all preexistent arteriolar connections had enlarged and stayed that way.
High Shear Stress Activates the Ras/ERK Pathway and Increases H-Ras Protein Levels
Using antibodies that react specifically with dual (Thr202/Tyr204) phosphorylated extracellular signal-regulated kinases (ERK), an activation of ERK-1 and ERK-2 in fluid shear stress induced collaterals was detected in Western blot analysis. Quantitative analysis showed that levels of phosphorylated (activated) ERK-1 and ERK-2 were increased in both cytosolic (2.5-fold) and particulate fractions (1.5-fold) obtained from collateral arteries of the shunted side when compared with collaterals of the control side. This shear stress–induced ERK activation was abrogated by L-NAME. Total ERK-1 and ERK-2 protein levels showed no difference between control and shunted side collaterals with or without L-NAME treatment (Figure 5).
We next sought to analyze expression levels of H-Ras in collaterals from the shunted and control side. H-ras is a member of GTP-binding proteins of small molecular weight, known to act as biological switches for various cellular processes. As shown in Figure 6A, Western blot analyses showed a 2.3-fold increase in expression of H-Ras on the shunted side. This effect was reduced by 50% if shunt collaterals were additionally treated with L-NAME (Figure 6C).
Fluid Shear Stress and L-NAME Treatment Did Not Influence the Activation of SEK/JNK and Akt Kinase Pathways
Western blot analysis revealed that neither shear stress nor inhibition of NO synthesis by L-NAME influence the activation of SEK/MKK4, an upstream activator of JNK, and Akt kinase (Figure 6D). No differences in P-SEK1/MKK4 levels were observed when comparing collaterals of control and shunted side. Furthermore, no differences in P-SEK1/MKK4 levels were detectable when collaterals of the shunted side (without L-NAME) were compared with collaterals of shunted side with L-NAME. Moreover, FSS and L-NAME treatment did not change the levels and specific phosphorylation (activation) of Akt kinase (Figure 6F).
Microarray Expression Profiling and Pathway Analysis
For details see supplemental methods section.
SNAP and L-NAME (But Not L-NNA) Inhibited Cell Proliferation in MVSMC
For details see supplemental methods section.
Discussion
That flow determines arterial size, ie, that form follows function, was known for a long time. Thoma,6 Schretzenmayr,7 Rodbard,8 Langille,9 Holtz,10 Tronc,11 and Ben Driss12 made the observation that arterial size depends on flow during development, that adult arteries respond with structural changes to changes in blood flow, and that the lumen is controlled by "an immediate physiological adjustment in vascular tone induced by the change in flow, and a delayed anatomical change that occurs when the change in flow persists". Thereafter many studies identified a host of molecules (vasoactive, growth regulatory, inflammatory, adhesive) whose endothelial production is also mediated by fluid shear stress.13–15 Recent studies by the laboratories of Tedgui,16–18 Busse,19 Dejana,20 and from our own group21 have shed light on the mechanisms of transduction of the mechanical stimulus into a growth response.
The most important present new findings are that markedly increased fluid shear stress, created by high shunt flows, overcomes the anatomical restrictions of collateral vessels and leads to complete normalization and overcompensation of maximal conductance. The pathways involved are the Ras-Raf-MEK-ERK- and the Rho-pathway. NO plays an important but complex role.
An often-repeated observation is that the spontaneous growth of collateral arteries remains defective in that only 35% to 40% of the maximal physiological resistance is restored after arterial occlusion.1,22 This defect in the natural restoration of maximal conductance was often tried to ameliorate with the application of angiogenic growth factors, but even in the rigorous studies by Unger23,24 high doses of FGF-2 increased Cmax from only 40% to 50% of Cmax. One explanation for the limited usefulness of growth factors could be that because of anatomical restrictions collateral vessels had reached their limit and even if further stimulated do not respond with a functional improvement. The tortuosity of collateral vessels, leading to energy-wasting curvature flow, their increase in length, and the relatively large number of small vessels would make them inefficient from the viewpoint of Poissieulle’s Law.4 However, if this defective natural restoration is caused by premature normalization of FSS by the initial increases in collateral diameter, (which reduces FSS by the third power of the radius), an increase in FSS should lead to further growth. In our distal-stump arterio-venous shunt experiments we show for the first time that collateral vessels are not growth-limited and that their mentioned shortcomings do not restrict the restoration of maximal collateral blood flow. In fact, their growth is finally only limited by the size of the surgical anastomosis and by the increased flow-load on the heart. Four weeks after installment of the shunt, the collateral vessels are able to conduct twice as much maximal blood flow compared with the normal femoral artery bed (see Figure 2a). It could be argued that even under the conditions of the shunt, FSS should fall with the radius in 3rd power caused by the enlargement of the collateral vessels. However because the venous system acts like a flow sink, Poissieulle’s law with the radius in the 4th power applies in the absence of a flow limiting peripheral resistance, creating a positive feedback loop.
Arteriogenic Pathways
Our present studies provide evidence for 3 signaling pathways in addition to the already described cell–cell interaction largely based on bone marrow–derived cells adhering to activated endothelium.25,26 The 3 signaling pathways are the Ras-ERK pathway, the Rho-pathway, and the NO-pathway. For the RAS-ERK pathway we could show a marked increase of phosphorylation in Western blots of collateral vessel tissue. Whereas the protein amount of ERK did not change, that of Ras had increased. The RAS-ERK pathway plays a prominent role in the transmission of growth factor–initiated signals leading to cell proliferation.27 It is therefore not surprising to find members of that signaling chain in high activity in growing collateral vessels. However, within the constraints of the present experiment, we cannot identify with any certainty the growth factor(s) that had caused this. Our observation that classical growth factors did not show changes of expression in the microarray screen makes their role uncertain.
A new finding is the prominent role of the Rho-pathway in arteriogenesis, which is based on the observed upregulation of RhoA itself, and members of the Rho signaling chain like cofilin (CFL1), vimentin and the increased protein expression, and increased degree of phosphorylation of focal adhesion kinase (FAK). Furthermore, the Rho inhibitor Fasudil inhibited arteriogenesis. Although the Rho-pathway is essential for cell motility, necessary for collateral remodeling, a role in smooth muscle cell proliferation was also discussed in a recent review.28 Its main role lies in the modification of the actin cytoskeleton via cofilin and destrin as we could recently show29 and confirm with our present microarray experiments. The dissolution of the contractile phenotype of the SMCs is a prerequisite for their mobility during remodeling30 and is paralleled by the transcriptional downregulation of alpha smooth muscle actin. Cofilin exists in 2 forms: cofilin1, which is expressed in endothelial cells, and cofilin 2, which is expressed in smooth muscle cells. The cofilins are activated by effectors of Rho GTPases, involving LIM kinases, and the phosphatase slingshot. The fact that focal adhesion kinase is also increased in SMCs indicates that this flow sensing system is probably also a general stress sensor in SMCs that are under increased circumferential wall stress because of the vasodilating influence of NO. The high pressure– and stretch-related forces must now be borne by the SMCs alone because the elastin skeleton had dissolved under the influence of the monocyte elastase.31 High fluid shear stress is one of the activators of eNOS.32,33 It is therefore not surprising to discover that NO plays a role in arteriogenesis which is highlighted by our previous finding of its strong expression in growing collateral vessels31 and by the almost complete inhibition of the shunt effect by treatment with L-NAME which also caused a shutdown of the RAS-ERK pathway. The role of eNOS may be more complex than the clear-cut L-NAME effect at first suggests, because NO is a known antimitogen for SMCs32 and it prevents leukocyte adhesion which we showed is crucial for collateral vessel remodeling.26 Our studies with cultured smooth muscle cells treated with the NO donor SNAP in nontoxic concentrations showed a marked 50% reduction of 3HTHymidine uptake. L-NAME, apart from its inhibitory action on eNOS,45 is a known antiproliferative agent for vascular smooth muscle and an inhibitor of the cell cycle34 which we also confirmed in SM cell culture studies. Reports on the negative effects of L-NAME on positive remodeling of large conductance arteries under high fluid shear stress35 may therefore allow for a different interpretation. However, L-NAME is not the most specific antagonist of eNOS, and we tried therefore the more specific antagonist L-NNA in our shunt studies and found that it exerted also a marked antiarteriogenic effect without exhibiting an antiproliferative effect in cultured smooth muscles cells. This strengthened the NO-hypothesis beyond reasonable doubt but did not eliminate the complexity of the action of NO because of the strong antiproliferative action of NO itself, because NO donor infusion did not increase Cmax, and because eNOS overexpression in transgenic mice does not stimulate arteriogenesis.36,37 We hypothesize therefore that NO, produced by high shear stress, most probably induces a Rho-activating factor. Both pathways, the NO as well as the Rho-pathway, assume equal rank because the inhibition of either one inhibits arteriogenesis. The final common pathway for both Rho- and NO is most probably the Ras/ERK signaling chain.
We explored yet another hypothesis: an uncoupling of eNOS may have happened under the influence of the intense and long lasting shear stress38 and under the influence of oxygen based radical production.39 NO plus superoxide might have given rise to the formation of peroxinitrite, a radical recently described as being involved in vascular remodeling.16,40 Radical scavengers like Ebselen and urate had no effect on Cmax and the peroxiradical donor SIN-1 markedly and significantly decreased collateral conductance thereby refuting this hypothesis.
Our own present and previous findings would favor the view that stress-induced deformation of the endothelial cell and the change in cytoskeletal tension and arrangement may initiate signals that lead to a variety of responses that act in concert41,42: chemokine expression,2 adhesion molecule production,43 marked upregulation of integrins,44 Rho-activation (present report), and NO-production.31
Conclusion
Sustained high fluid shear stress caused by a surgically created shunt between the distal end of an occluded femoral artery and its accompanying vein leads to a much prolonged growth process of collateral arteries which completely restores full maximal conductance, even surpassing normal maximal conductance of a normal arterial bed by a factor of 2. At least 3 signaling pathways converge, ie, the RAS-ERK-, the Rho-, and the NO-pathway, which enable the substantial and sustained growth of collateral arteries and allow for the great range of adaptation potential.
Acknowledgments
Sources of Funding
This study was funded by grants from the German Cardiac Society, from the Kuehl-Foundation, and from the Kerckhoff-Foundation.
Disclosures
None.
Footnotes
Original received December 16, 2005; resubmission received June 8, 2006; revised resubmission received July 21, 2006; accepted August 10, 2006.
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the Max-Planck-Institute for Heart and Lung Research (I.E., K.T., M.H., F.P., W.S.), Bad Nauheim, Germany
Department of Medical Biochemistry, Academic Medical Centre (O.V., A.J.G.H.), University of Amsterdam, The Netherlands
Kerckhoff Clinic (A.K.), Bad Nauheim, Germany
Division of Vascular and Endovascular Surgery (T.S.), Goethe-University of Frankfurt/Main, Germany
Slovak Academy of Sciences (M.B.), Bratislava, Slovakia
Department of Anatomy, Xiangsha School of Medicine (W.C.), Central South University, Xiangsha, Hunan, P.R. China
Department of Medical Biochemistry (S.F.), Liebig-University Giessen, Germany.
Abstract
Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only 35% of adenosine-recruitable maximal conductance (Cmax) probably because initially elevated fluid shear stress (FSS) quickly normalizes. We tested the hypothesis whether this deficit can be mended by artificially increasing FSS or whether anatomical restraints prevent complete restitution. We chronically increased FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein. After reclosure of the shunt collateral flow was measured at maximal vasodilatation. Cmax reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the Cmax of the normal vasculature before occlusion. Expression profiling showed upregulation of members of the Rho-pathway (RhoA, cofilin, focal adhesion kinase, vimentin) and the Rho-antagonist Fasudil markedly inhibited arteriogenesis. The activities of Ras and ERK-1,-2 were markedly increased in collateral vessels of the shunt experiment, and infusions of L-NAME and L-NNA strongly inhibited MAPK activity as well as shunt-induced arteriogenesis. Infusions of the peroxinitrite donor Sin-1 inhibited arteriogenesis. The radical scavengers urate, ebselen, SOD, and catalase had no effect. We conclude that increased FSS can overcome the anatomical restrictions of collateral arteries and is potentially able to completely restore maximal collateral conductance. Increased FSS activates the Ras-ERK-, the Rho-, and the NO- (but not the Akt-) pathway enabling collateral artery growth.
Key Words: arteriogenesis fluid shear stress shunt growth factors microarrays
Introduction
The restoration of maximal conductance (Cmax) in animals after arterial occlusion remains defective (35% in the canine coronary circulation1 and 40% in the rabbit hind limb2) in spite of the fact that normal resting blood flow is reached early. As a consequence exercise testing in experimental animals reveals defects similar to those in human patients.3 It was not known until now whether collateral vessels are potentially able to restore the full dilatory reserve of a normal vascular bed. Many observations would predict that this is not the case: the multitude of small vessels that replace an occluded artery is inefficient according to Poissieulle’s Law, and the tortuosity of collateral vessels offers finite resistance because of curvature flow and increased collateral length.4 One reason for the defective adaptation may lie in the fact that fluid shear stress normalizes prematurely: FSS falls by the third power of the growing radius. We tested the hypothesis whether a sustained increase of FSS is able to prolong the growth process and to restore normal maximal conductance. The method to achieve this was the creation of a shunt between the distal stump of the occluded femoral artery and the accompanying vein.5
The novelty of the present findings is the demonstration that Cmax can be reached and even surpassed by a drastic increase of fluid shear stress. Signaling pathways involved in the adaptation to fluid shear stress were interrogated, and it could be shown that three pathways have to converge to induce arterial growth, ie, the Ras-ERK pathway, the Rho-, and the NO-pathway.
Materials and Methods
An expanded Materials and Methods section can be found in the supplement available at http://circres.ahajournals.org.
We subjected rabbits to femoral artery occlusion and studied the effects of an arteriovenous shunt on the development of the collateral circulation (Figure 1). We measured collateral blood flow by ultrasonic flow probes and by MRI. The morphology of the vessels was studied in vivo by CT-scanning and post mortem by arteriography following pressure-controlled contrast medium injection. Collateral artery tissue was prepared for RNA extraction, which was fashioned for microarray studies and for Western blot analysis. Animals were treated with a variety of tool drugs to interrogate the NO, Rho-, and Ras pathways. Smooth muscle cells in culture were treated with several agents used in the pharmacological in vivo studies.
Results
No animals were lost because of the surgical procedure or postoperatively. Clinical signs of peripheral ischemia were transiently present in a few animals but did not lead to ulceration or amputation. In two thirds of the treated animals, the A-V shunt was still open at termination of the experiment. Only animals with a functional shunt were included in the study.
Hemodynamics
Immediately after occlusion of the femoral artery blood pressure in the peripheral stump of the occluded artery fell to low values of 31±4.2% of the aortic pressure. Peripheral pressure rose to 50±8% after 7 days and remained at that level for several weeks. In contrast, in animals with femoral artery ligature and an additional A-V shunt the peripheral arterial pressure dropped to 6±1% of the arterial pressure (immediately after the operation), increased within 7 days to 17±3%, and reached 29±3% after 4 weeks. When the shunt was occluded—routinely done during the terminal experiment—7 days after femoral artery occlusion, peripheral pressure increased to 60±3% of the aortic pressure and to 73±1% when measured 4 weeks after femoral artery occlusion.
Collateral blood flow ratio (shunt versus control side) measured by MRI was 5.8±0.8 at day 7 and 13.7±3.7 at day 28. One week after shunt treatment calculated Cmax values under optimal vasodilatation were 88±4% of Cmax of nonoccluded legs, representing a 2-fold increase in comparison to Cmax of control ligated legs (44±7%). After 4 weeks, shunt treated legs showed a Cmax of 199±19% of nonligated legs. In contrast, legs with control ligatures did not show any further improvement after 4 weeks when compared with the results after 1 week (Figure 2A).
Effects of Pharmacological Inhibitors
Fasudil, a Rho-pathway inhibitor, and L-NAME and L-NNA, both inhibitors of endothelial NO synthetase (eNOS), almost completely blocked the positive effects of high shear stress on collateral artery growth (shunt 306±15 versus Fasudil 176±4 versus L-NAME 163±8 versus control 155±27 mL/min/100 mm Hg) (Figure 2B).
Moreover, the hypothesis that shear stress leads to an uncoupling of eNOS resulting in increased production of oxygen- and nitrogen-based radicals was investigated. To demonstrate such an effect, 3-Morpholinosydnonimine (Sin-1), which acts as a NO- and superoxide anion radical donor (both react to produce peroxinitrite), was infused but arteriogenesis was inhibited in a dose-dependent manner. In contrast, infusion of the scavengers Ebselen, urate, catalase, and superoxide dismutase showed no effect (data not shown).
Angiography and Computerized Tomography
Postmortem angiograms (Figure 3A and 3B) showed marked increases in the number of enlarged collaterals on the shunt side (day 7: 42±2.5 versus 18±0.95 [control]; day 28: 48±2.3 versus 12±2.0 [control]), which corresponded with the number and size of collaterals detected in vivo by CT (Figure 4A and 4B). Treatment with Fasudil or L-NAME resulted in a significant decrease in collateral count (fasudil 31±0.9 versus L-NAME 23.8±1.2 versus shunt 42±2.5). In contrast to the natural course of collateral growth, which leads to the elimination of most vessels that had initially participated in the growth transformation in favor of a few large ones ("pruning"), no pruning had occurred on the shunt side without further treatment: all preexistent arteriolar connections had enlarged and stayed that way.
High Shear Stress Activates the Ras/ERK Pathway and Increases H-Ras Protein Levels
Using antibodies that react specifically with dual (Thr202/Tyr204) phosphorylated extracellular signal-regulated kinases (ERK), an activation of ERK-1 and ERK-2 in fluid shear stress induced collaterals was detected in Western blot analysis. Quantitative analysis showed that levels of phosphorylated (activated) ERK-1 and ERK-2 were increased in both cytosolic (2.5-fold) and particulate fractions (1.5-fold) obtained from collateral arteries of the shunted side when compared with collaterals of the control side. This shear stress–induced ERK activation was abrogated by L-NAME. Total ERK-1 and ERK-2 protein levels showed no difference between control and shunted side collaterals with or without L-NAME treatment (Figure 5).
We next sought to analyze expression levels of H-Ras in collaterals from the shunted and control side. H-ras is a member of GTP-binding proteins of small molecular weight, known to act as biological switches for various cellular processes. As shown in Figure 6A, Western blot analyses showed a 2.3-fold increase in expression of H-Ras on the shunted side. This effect was reduced by 50% if shunt collaterals were additionally treated with L-NAME (Figure 6C).
Fluid Shear Stress and L-NAME Treatment Did Not Influence the Activation of SEK/JNK and Akt Kinase Pathways
Western blot analysis revealed that neither shear stress nor inhibition of NO synthesis by L-NAME influence the activation of SEK/MKK4, an upstream activator of JNK, and Akt kinase (Figure 6D). No differences in P-SEK1/MKK4 levels were observed when comparing collaterals of control and shunted side. Furthermore, no differences in P-SEK1/MKK4 levels were detectable when collaterals of the shunted side (without L-NAME) were compared with collaterals of shunted side with L-NAME. Moreover, FSS and L-NAME treatment did not change the levels and specific phosphorylation (activation) of Akt kinase (Figure 6F).
Microarray Expression Profiling and Pathway Analysis
For details see supplemental methods section.
SNAP and L-NAME (But Not L-NNA) Inhibited Cell Proliferation in MVSMC
For details see supplemental methods section.
Discussion
That flow determines arterial size, ie, that form follows function, was known for a long time. Thoma,6 Schretzenmayr,7 Rodbard,8 Langille,9 Holtz,10 Tronc,11 and Ben Driss12 made the observation that arterial size depends on flow during development, that adult arteries respond with structural changes to changes in blood flow, and that the lumen is controlled by "an immediate physiological adjustment in vascular tone induced by the change in flow, and a delayed anatomical change that occurs when the change in flow persists". Thereafter many studies identified a host of molecules (vasoactive, growth regulatory, inflammatory, adhesive) whose endothelial production is also mediated by fluid shear stress.13–15 Recent studies by the laboratories of Tedgui,16–18 Busse,19 Dejana,20 and from our own group21 have shed light on the mechanisms of transduction of the mechanical stimulus into a growth response.
The most important present new findings are that markedly increased fluid shear stress, created by high shunt flows, overcomes the anatomical restrictions of collateral vessels and leads to complete normalization and overcompensation of maximal conductance. The pathways involved are the Ras-Raf-MEK-ERK- and the Rho-pathway. NO plays an important but complex role.
An often-repeated observation is that the spontaneous growth of collateral arteries remains defective in that only 35% to 40% of the maximal physiological resistance is restored after arterial occlusion.1,22 This defect in the natural restoration of maximal conductance was often tried to ameliorate with the application of angiogenic growth factors, but even in the rigorous studies by Unger23,24 high doses of FGF-2 increased Cmax from only 40% to 50% of Cmax. One explanation for the limited usefulness of growth factors could be that because of anatomical restrictions collateral vessels had reached their limit and even if further stimulated do not respond with a functional improvement. The tortuosity of collateral vessels, leading to energy-wasting curvature flow, their increase in length, and the relatively large number of small vessels would make them inefficient from the viewpoint of Poissieulle’s Law.4 However, if this defective natural restoration is caused by premature normalization of FSS by the initial increases in collateral diameter, (which reduces FSS by the third power of the radius), an increase in FSS should lead to further growth. In our distal-stump arterio-venous shunt experiments we show for the first time that collateral vessels are not growth-limited and that their mentioned shortcomings do not restrict the restoration of maximal collateral blood flow. In fact, their growth is finally only limited by the size of the surgical anastomosis and by the increased flow-load on the heart. Four weeks after installment of the shunt, the collateral vessels are able to conduct twice as much maximal blood flow compared with the normal femoral artery bed (see Figure 2a). It could be argued that even under the conditions of the shunt, FSS should fall with the radius in 3rd power caused by the enlargement of the collateral vessels. However because the venous system acts like a flow sink, Poissieulle’s law with the radius in the 4th power applies in the absence of a flow limiting peripheral resistance, creating a positive feedback loop.
Arteriogenic Pathways
Our present studies provide evidence for 3 signaling pathways in addition to the already described cell–cell interaction largely based on bone marrow–derived cells adhering to activated endothelium.25,26 The 3 signaling pathways are the Ras-ERK pathway, the Rho-pathway, and the NO-pathway. For the RAS-ERK pathway we could show a marked increase of phosphorylation in Western blots of collateral vessel tissue. Whereas the protein amount of ERK did not change, that of Ras had increased. The RAS-ERK pathway plays a prominent role in the transmission of growth factor–initiated signals leading to cell proliferation.27 It is therefore not surprising to find members of that signaling chain in high activity in growing collateral vessels. However, within the constraints of the present experiment, we cannot identify with any certainty the growth factor(s) that had caused this. Our observation that classical growth factors did not show changes of expression in the microarray screen makes their role uncertain.
A new finding is the prominent role of the Rho-pathway in arteriogenesis, which is based on the observed upregulation of RhoA itself, and members of the Rho signaling chain like cofilin (CFL1), vimentin and the increased protein expression, and increased degree of phosphorylation of focal adhesion kinase (FAK). Furthermore, the Rho inhibitor Fasudil inhibited arteriogenesis. Although the Rho-pathway is essential for cell motility, necessary for collateral remodeling, a role in smooth muscle cell proliferation was also discussed in a recent review.28 Its main role lies in the modification of the actin cytoskeleton via cofilin and destrin as we could recently show29 and confirm with our present microarray experiments. The dissolution of the contractile phenotype of the SMCs is a prerequisite for their mobility during remodeling30 and is paralleled by the transcriptional downregulation of alpha smooth muscle actin. Cofilin exists in 2 forms: cofilin1, which is expressed in endothelial cells, and cofilin 2, which is expressed in smooth muscle cells. The cofilins are activated by effectors of Rho GTPases, involving LIM kinases, and the phosphatase slingshot. The fact that focal adhesion kinase is also increased in SMCs indicates that this flow sensing system is probably also a general stress sensor in SMCs that are under increased circumferential wall stress because of the vasodilating influence of NO. The high pressure– and stretch-related forces must now be borne by the SMCs alone because the elastin skeleton had dissolved under the influence of the monocyte elastase.31 High fluid shear stress is one of the activators of eNOS.32,33 It is therefore not surprising to discover that NO plays a role in arteriogenesis which is highlighted by our previous finding of its strong expression in growing collateral vessels31 and by the almost complete inhibition of the shunt effect by treatment with L-NAME which also caused a shutdown of the RAS-ERK pathway. The role of eNOS may be more complex than the clear-cut L-NAME effect at first suggests, because NO is a known antimitogen for SMCs32 and it prevents leukocyte adhesion which we showed is crucial for collateral vessel remodeling.26 Our studies with cultured smooth muscle cells treated with the NO donor SNAP in nontoxic concentrations showed a marked 50% reduction of 3HTHymidine uptake. L-NAME, apart from its inhibitory action on eNOS,45 is a known antiproliferative agent for vascular smooth muscle and an inhibitor of the cell cycle34 which we also confirmed in SM cell culture studies. Reports on the negative effects of L-NAME on positive remodeling of large conductance arteries under high fluid shear stress35 may therefore allow for a different interpretation. However, L-NAME is not the most specific antagonist of eNOS, and we tried therefore the more specific antagonist L-NNA in our shunt studies and found that it exerted also a marked antiarteriogenic effect without exhibiting an antiproliferative effect in cultured smooth muscles cells. This strengthened the NO-hypothesis beyond reasonable doubt but did not eliminate the complexity of the action of NO because of the strong antiproliferative action of NO itself, because NO donor infusion did not increase Cmax, and because eNOS overexpression in transgenic mice does not stimulate arteriogenesis.36,37 We hypothesize therefore that NO, produced by high shear stress, most probably induces a Rho-activating factor. Both pathways, the NO as well as the Rho-pathway, assume equal rank because the inhibition of either one inhibits arteriogenesis. The final common pathway for both Rho- and NO is most probably the Ras/ERK signaling chain.
We explored yet another hypothesis: an uncoupling of eNOS may have happened under the influence of the intense and long lasting shear stress38 and under the influence of oxygen based radical production.39 NO plus superoxide might have given rise to the formation of peroxinitrite, a radical recently described as being involved in vascular remodeling.16,40 Radical scavengers like Ebselen and urate had no effect on Cmax and the peroxiradical donor SIN-1 markedly and significantly decreased collateral conductance thereby refuting this hypothesis.
Our own present and previous findings would favor the view that stress-induced deformation of the endothelial cell and the change in cytoskeletal tension and arrangement may initiate signals that lead to a variety of responses that act in concert41,42: chemokine expression,2 adhesion molecule production,43 marked upregulation of integrins,44 Rho-activation (present report), and NO-production.31
Conclusion
Sustained high fluid shear stress caused by a surgically created shunt between the distal end of an occluded femoral artery and its accompanying vein leads to a much prolonged growth process of collateral arteries which completely restores full maximal conductance, even surpassing normal maximal conductance of a normal arterial bed by a factor of 2. At least 3 signaling pathways converge, ie, the RAS-ERK-, the Rho-, and the NO-pathway, which enable the substantial and sustained growth of collateral arteries and allow for the great range of adaptation potential.
Acknowledgments
Sources of Funding
This study was funded by grants from the German Cardiac Society, from the Kuehl-Foundation, and from the Kerckhoff-Foundation.
Disclosures
None.
Footnotes
Original received December 16, 2005; resubmission received June 8, 2006; revised resubmission received July 21, 2006; accepted August 10, 2006.
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