Cerebral arteriogenesis is enhanced by pharmacological as well as fluid-shear-stress activation of the Trpv4 calcium channel.

OBJECTIVES: This study aimed to determine the importance of the shear-stress-sensitive calcium channels Trpc1, Trpm7, Trpp2, Trpv2 (transient receptor potential cation channel, subfamily V, member 2) and Trpv4 for cerebral arteriogenesis. The expression profiles were analysed, comparing the stimulation of collateral growth by target-specific drugs to that achieved by maximum increased fluid shear stress (FSS). DESIGN: A prospective, controlled study wherein rats were subjected to bilateral carotid artery ligature (BCL), or BCL + arteriovenous fistula, or BCL + drug application. METHODS: Messenger RNA (mRNA) abundance and protein expression were determined in FSS-stimulated cerebral collaterals by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Drugs were applied via osmotic mini pumps and arteriogenesis was evaluated by post-mortem angiograms and Ki67 immunostaining. RESULTS: Trpv4 was the only mechanosensitive Trp channel showing significantly increased mRNA abundance and protein expression after FSS stimulation. Activation of Trpv4 by 4α-phorbol-12,13-didecanoate caused significantly enhanced collateral growth (length: 4.43 ± 0.20 mm and diameter: 282.6 ± 8.1 µm) compared with control (length: 3.80 ± 0.06 mm and diameter: 237.3 ± 5.3 µm). Drug application stimulated arteriogenesis to almost the same extent as did maximum FSS stimulation (length: 4.61 ± 0.07 mm and diameter: 327.4 ± 12.6 µm). CONCLUSIONS: Trpv4 showed significantly increased expression in FSS-stimulated cerebral collaterals. Pharmacological Trpv4 activation enhanced cerebral arteriogenesis, pinpointing Trpv4 as a possible candidate for the development of new therapeutic concepts.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Involvement of neuronal NO synthase in collateral artery growth.

To evaluate the role of neuronal nitric oxides synthase (nNOS) in collateral artery growth (arteriogenesis), we analyzed the expression pattern of nNOS at distinct time points on RNA and protein levels in a rabbit and a murine model of peripheral arteriogenesis. In the rabbit model, Northern blot analyses revealed a significant upregulation of nNOS at 6 h (1.6-fold), 12 h (2.2-fold) and 24 h (2.0-fold) after induction of arteriogenesis via femoral artery ligation, when compared to the sham operated side. In mice, an upregulation of nNOS was also detected using Northern blot (at 6 h, 12 h) and qRT-PCR (12 h: 2.4-fold). On the protein level, nNOS was found to be upregulated 24 h after femoral artery ligation. Immunohistochemical staining showed that nNOS was localized in endothelial and smooth muscle cells of collateral arteries, as well as in skeletal muscle and nerves. In summary, our data provide evidence that nNOS is not constitutively expressed, but is induced during arteriogenesis, playing a role in supplying reactive oxygen species such as H2O2 and low levels of NO.

Calcium-dependent signalling is essential during collateral growth in the pig hind limb-ischemia model.

We investigated the effect of pharmacological activation of the Ca(2+)-channel transient receptor potential cation channel, subfamily V, member 4 (TRPV4) on collateral growth in a pig hind limb-ischemia model thereby identifying subcellular mechanisms. Domestic pigs received femoral artery ligature and were randomly assigned to one of the following groups (each n=6): (1) 4alpha-phorbol 12,13-didecanoate (4alphaPDD) treatment; (2) treatment with an arterio-venous shunt (AV-shunt) distal to the occlusion; or (3) implantation of NaCl-filled minipump. Six sham-operated pigs acted as controls. Aortic and peripheral mean arterial pressure (MAP) measurements were performed to assess the collateral flow index (CFI). Tissue was isolated from M. quadriceps for immunohistochemistry and from isolated collateral arteries for quantitative real time PCR (qRT-PCR). Shortly after ligature the CFI dropped from 0.96+/-0.02 to 0.21+/-0.02 in all ligature-treated groups. In ligature-only-treated pigs CFI increased to 0.56+/-0.03 after 7days. Treatment with 4alphaPDD led to an enhancement of CFI compared with ligature alone (0.73+/-0.03). CD31-staining showed improved arteriolar density. Increased Ki67 staining in collaterals indicated proliferation. qRT-PCR and Western blot analysis showed upregulation or modulation of Ca(2+)-dependent transcription factors nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), Kv channel interacting protein 3, calsenilin (KCNIP3/CSEN/DREAM), and myocyte enhancer factor 2C (MEF2C) in 4alphaPDD- and AV-shunt-treated pigs compared with controls. Improved CFI after 4alphaPDD treatment identifies TRPV4 as an initial fluid shear-stress sensor and collateral remodelling and growth trigger. Subcellularly, modulation of Ca(2+)-dependent transcription factors indicates a pivotal role for Ca(2+)-signalling during arteriogenesis.

Exercise linked to transient increase in expression and activity of cation channels in newly formed hind-limb collaterals.

OBJECTIVE: This study aimed to compare arteriogenesis after femoral artery occlusion as influenced by exercise or arteriovenous shunt and follow changes in collateral transient receptor potential cation channel, subfamily V, member 4 (Trpv4). DESIGN: A prospective, controlled study wherein rats were subjected to femoral artery ligation (FAL), or FAL+arteriovenous shunt. Collateral Trpv4 was determined 0.5 and 6h post exercise. METHODS: Rats were subjected to exercise for 15 min, twice daily. The number and diameter of collaterals were assessed after 7 days. Collateral Trpv4 expression was quantified by reverse transcription-polymerase chain reaction. RESULTS: Collateral number and diameter per limb were significantly higher in the shunt group (number: 16.0+/-2.4 and diameter: 216.0+/-34 microm) compared to the ligature (number: 9.4+/-2 and diameter: 144+/-21 microm) and exercise groups (number: 9.9+/-2.5 and diameter: 151+/-15 microm). Trpv4 expression in collaterals harvested 0.5h post exercise was not significantly different from expression in shunted rats. It was significantly lower in collaterals harvested 6h post exercise (comparable to that in ligated rats). CONCLUSION: Collateral formation was greater in the shunt group than in the exercise group. Exercise-induced Trpv4 up-regulation, not significantly different from that achieved with shunt, returned to control values when evaluated 6h post exercise. More frequent exercise to chronically increase fluid shear stress, as with a shunt model, may be required for sufficient arteriogenesis to compensate for peripheral occlusion.

Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.

We have previously shown that monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis). In this study we investigated the association of monocyte accumulation with both the production of the cytokines-basic fibroblast growth factor (bFGF) and TNF-alpha-and vessel proliferation in the rabbit after femoral artery occlusion. In particular, we studied the effects of an increase in monocyte recruitment by LPS on capillary density as well as collateral and peripheral conductance after 7 d of occlusion. Monocytes accumulated around day 3 in collateral arteries when maximal proliferation was observed, and stained strongly for bFGF and TNF-alpha. In the lower limb where angiogenesis was shown to be predominant, macrophage accumulation was also closely associated with maximal proliferation (around day 7). LPS treatment significantly increased capillary density (424+/-26.1 n/mm2 vs. 312+/-20.7 n/mm2; P < 0.05) and peripheral conductance (109+/-33.8 ml/min/100 mmHg vs. 45+/-6.8 ml/min/100 mmHg; P < 0.05) as compared with untreated animals after 7 d of occlusion. These results indicate that monocyte activation plays a major role in angiogenesis and collateral artery growth.

The delivery of angiogenic factors to the heart by microsphere therapy.

Microspheres offer the possibility of local noninvasive delivery of drugs over an extended period of time. We adsorbed fibroblast growth factor (FGF) to microspheres of precapillary size that were injected via a coronary catheter. We showed that FGF was released from these microspheres and taken up by endothelial cells, which proliferated following translocation of FGF to the nucleus. This method for application of growth factors allows the precise delivery of angiogenic substances to any selected part of the heart or other organs without causing inflammation or ischemia.

Local monocyte chemoattractant protein-1 therapy increases collateral artery formation in apolipoprotein E-deficient mice but induces systemic monocytic CD11b expression, neointimal formation, and plaque progression.

Monocyte chemoattractant protein-1 (MCP-1) stimulates the formation of a collateral circulation on arterial occlusion. The present study served to determine whether these proarteriogenic properties of MCP-1 are preserved in hyperlipidemic apolipoprotein E-deficient (apoE-/-) mice and whether it affects the systemic development of atherosclerosis. A total of 78 apoE-/- mice were treated with local infusion of low-dose MCP-1 (1 microg/kg per week), high-dose MCP-1 (10 microg/kg per week), or PBS as a control after unilateral ligation of the femoral artery. Collateral hindlimb flow, measured with fluorescent microspheres, significantly increased on a 1-week high-dose MCP-1 treatment (PBS 22.6+/-7.2%, MCP-1 31.3+/-10.3%; P<0.05). These effects were still present 2 months after the treatment (PBS 44.3+/-4.6%, MCP-1 56.5+/-10.4%; P<0.001). The increase in collateral flow was accompanied by an increase in the number of perivascular monocytes/macrophages on MCP-1 treatment. However, systemic CD11b expression by monocytes also increased, as did monocyte adhesion at the aortic endothelium and neointimal formation (intima/media ratio, 0.097+/-0.011 [PBS] versus 0.257+/-0.022 [MCP-1]; P<0.0001). Moreover, Sudan IV staining revealed an increase in aortic atherosclerotic plaque surface (24.3+/-5.2% [PBS] versus 38.2+/-9.5% [MCP-1]; P<0.01). Finally, a significant decrease in the percentage of smooth muscle cells was found in plaques (15.0+/-5.2% [PBS] versus 5.8+/-2.3% [MCP-1]; P<0.001). In conclusion, local infusion of MCP-1 significantly increases collateral flow on femoral artery ligation in apoE-/- mice up to 2 months after the treatment. However, the local treatment did not preclude systemic effects on atherogenesis, leading to increased atherosclerotic plaque formation and changes in cellular content of plaques.

Role of ischemia and of hypoxia-inducible genes in arteriogenesis after femoral artery occlusion in the rabbit.

Vascular endothelial growth factor (VEGF) is known to play an important role in angiogenesis. Its place in collateral artery growth (arteriogenesis), however, is still debated. In the present study, we analyzed the expression of VEGF and its receptors (Flk-1 and Flt-1) in a rabbit model of collateral artery growth after femoral artery occlusion. Hypoxia presents the most important stimulus for VEGF expression. We therefore also investigated the expression level of distinct hypoxia-inducible genes (HIF-1alpha, LDH A) and determined metabolic intermediates indicative for ischemia (ATP, creatine phosphate, and their catabolites). We found that arteriogenesis was not associated with an increased expression of VEGF or the mentioned hypoxia-inducible genes. Furthermore, the high-energy phosphates and their catabolites were entirely within normal limits. Despite the absence of an increased expression of VEGF and its receptors, collateral vessels increased their diameter by a factor of 10. The speed of collateral development could be increased by infusion of the chemoattractant monocyte chemotactic protein-1 but not by infusion of a 30 times higher concentration of VEGF. From these data, we conclude that under nonischemic conditions, arteriogenesis is neither associated with nor inducible by increased levels of VEGF and that VEGF is not a natural agent to induce arteriogenesis in vivo.

CD44 regulates arteriogenesis in mice and is differentially expressed in patients with poor and good collateralization.

BACKGROUND: Arteriogenesis refers to the development of collateral conductance arteries and is orchestrated by circulating monocytes, which invade growing collateral arteries and act as suppliers of cytokines and growth factors. CD44 glycoproteins are involved in leukocyte extravasation but also in the regulation of growth factor activation, stability, and signaling. Here, we explored the role of CD44 during arteriogenesis. METHODS AND RESULTS: CD44 expression increases strongly during collateral artery growth in a murine hind-limb model of arteriogenesis. This CD44 expression is of great functional importance, because arteriogenesis is severely impaired in CD44-/- mice (wild-type, 54.5+/-14.9% versus CD44-/-, 24.1+/-9.2%, P<0.001). The defective arteriogenesis is accompanied by reduced leukocyte trafficking to sites of collateral artery growth (wild-type, 29+/-12% versus CD44-/-, 18+/-7% CD11b-positive cells/square, P<0.01) and reduced expression of fibroblast growth factor-2 and platelet-derived growth factor-B protein. Finally, in patients with single-vessel coronary artery disease, the maximal expression of CD44 on activated monocytes is reduced in case of impaired collateral artery formation (poor collateralization, 1764+/-572 versus good collateralization, 2817+/-1029 AU, P<0.05). CONCLUSIONS: For the first time, the pivotal role of CD44 during arteriogenesis is shown. The expression of CD44 increases during arteriogenesis, and the deficiency of CD44 severely impedes arteriogenesis. Maximal CD44 expression on isolated monocytes is decreased in patients with a poor collateralization compared with patients with a good collateralization.

Reperfusion of ischemic myocardium: ultrastructural and histochemical aspects.

The effects of reperfusion on ischemic myocardium generally depend on the severity of the preceding ischemic injury. Reperfusion of myocardium, irreversibly injured by ischemia, produces further progression of myocardial necrosis that is accompanied by simultaneously occurring stimulation of interstitial cell proliferation resulting in scar formation. Reperfusion of reversibly injured myocardium leads to structural improvement and reorganization. Thus, it may be stated from the ultrastructural part of this study that reperfusion of ischemic myocardium induces 1) slow structural recuperation after reversible injury, and 2) accelerated cellular destruction and symptoms of scar formation after irreversible ischemic injury. We observed that the reduced tissue content of nicotinamide adenine dinucleotide (NAD), rather than reduced dehydrogenase activity, is the basis of histochemical reactions employing tetrazolium salts. Directly measured NAD tissue content in ischemic tissue correlated well with the degree of ultrastructural injury and with macroscopic differential staining. Occlusion of two small coronary arteries in the same heart followed by reperfusion of only one artery (identical occlusion times for both arteries) showed identical infarct sizes for reperfused and nonreperfused myocardium for occlusion times of 3 and 6 hours. When the effects of occlusion times of less than 3 hours are studied with tetrazolium salts, a difficult technical problem arises: during that time, tissue-NAD concentrations have not decreased enough to enable differential staining. Reperfusion leads to washout of NAD, thus producing differential staining; this may be a harmful effect of reperfusion. However, because early reperfusion leads to significant structural and functional recovery and to small infarcts, reperfusion injury is unlikely to occur. Both ultrastructural and histochemical evidence suggest that reperfusion is beneficial for reversibly injured tissue but accelerates the decay of irreversibly injured tissue.

Thrombolysis in acute experimental myocardial infarction.

Lysis of thrombi by intracoronary application of streptokinase has become a new therapeutic approach in patients with acute myocardial infarction. To simulate the clinical situation of myocardial infarction a new experimental model was developed, which was based on a thrombotic coronary occlusion at the site of a high degree stenosis created by a constrictor. In 20 dogs, two ligations 15 mm apart were prepared at the left anterior descending or circumflex coronary artery. After closure of the distal ligation, 2 IU of thrombin was injected through a catheter directly in front of the proximal ligation. The catheter was withdrawn and the proximal ligation was closed. Occlusion time ranged from 1 to 6 hours. At 1, 2, 4 and 6 hours after occlusion, streptokinase was infused for 1 hour (100,000 IU in 200 ml of saline solution) into the left main coronary artery. Hemodynamic variables and coronary blood flow to the ischemic and normal myocardial areas were recorded continuously. Myocardial perfusion was measured six times with tracer microspheres. Reinstatement of blood flow, as well as normalization of myocardial perfusion in the ischemic area, was achieved by streptokinase at 5 minutes after 1 hour of occlusion, 8 minutes after 2 hours, 15 minutes after 4 hours, and 30 minutes after 6 hours; no hyperemic flow occurred. Postmortem staining of infarct size revealed more than 50% of viable myocardium in the perfusion area of the thrombotic vessel even after 6 hours of occlusion. Hemorrhage occurred only after 6 hours of occlusion and was limited to the central area of necrosis in the subendocardial layer. Serious reperfusion arrhythmias occurred only after 1 and 2 hours of occlusion and seemed to be independent of the mode of reperfusion; however, the total number of episodes of ventricular fibrillation after reperfusion was probably decreased compared with that after sudden and hyperemic reflow.

Molecular biologic concepts of coronary anastomoses.

The discovery that collateral development after progressive coronary stenosis proceeds by means of DNA synthesis, mitosis and proliferation of endothelial and smooth muscle cells in preformed small interconnecting arterioles (canine heart) and capillaries (porcine heart) has stimulated research into the molecular mechanisms of vascular growth. Growth is tightly controlled under physiologic conditions, and several factors must act in concert to overcome control. Because the result of growth is a much larger orderly structure of complex design, we expect the existence of a genetic blueprint for its construction. Peptide growth factors have recently been isolated from a variety of organs, including the heart. We have provided experimental evidence that the heparin-binding growth factor beta-ECGF shows an increased transcription in growing pig collateral vessels. Because the chain of events probably originates in the ischemic cardiac myocyte, it appears logical to search there for the initiating factor. In addition to local production, growth factors can also be transported into ischemic myocardium by blood-borne cells. Monocytes adhere to altered endothelium in a potentially ischemic region and start to produce growth factors in situ. Platelets are rich sources of transforming growth factor-beta (TGF-beta), platelet-derived endothelial cell growth factor (PDECGF) and platelet-derived growth factor (PDGF), all of which are known angiogenic factors or mitogens.

Myocyte degeneration and cell death in hibernating human myocardium.

OBJECTIVES: The aim of this study was to analyze the morphologic characteristics of myocyte degeneration leading to replacement fibrosis in hibernating myocardium by use of electron microscopy and immunohistochemical techniques. BACKGROUND: Data on the ultrastructure and the cytoskeleton of cardiomyocytes in myocardial hibernation are scarce. Incomplete or delayed functional recovery might be due to variable degree of cardiomyocyte degeneration in hibernating myocardium. METHODS: In 24 patients, regional wall motion abnormalities were analyzed by use of the centerline method before and 6 +/- 1 months after coronary artery bypass surgery. Preoperative technetium-99m sestamibi uptake was measured by single-photon emission computed tomography for assessing regional perfusion. Fluorine-18 fluorodeoxyglucose uptake was measured by positron emission tomography to assess glucose metabolism. Transmural biopsy specimens were taken during coronary artery bypass surgery from the center of the hypocontractile area of the anterior wall. RESULTS: The myocytes showed varying signs of mild-to-severe degenerative changes and an increased degree of fibrosis. Immunohistochemical analysis demonstrated disruption of the cytoskeletal proteins titin and alpha-actinin. Electron microscopy of the cell organelles and immunohistochemical analysis of the cytoskeleton showed a similarity in the degree of degenerative alterations. Group 1 (n = 11) represented patients with only minor structural alterations, whereas group 2 (n = 13) showed severe morphologic degenerative changes. Wall motion abnormalities showed postoperative improvements, and nuclear imaging revealed a perfusion-metabolism mismatch without significant differences between the groups. CONCLUSIONS: Long-term hypoperfusion causes different degrees of morphologic alterations leading to degeneration. Preoperative analysis of regional contractility and perfusion-metabolism imaging does not distinguish the severity of morphologic alterations nor the functional outcome after revascularization. The insufficient act of self-preservation in hibernating myocardium may lead to a progressive structural degeneration with an incomplete and delayed recovery of function after restoration of blood flow.

Receptor-independent role of the urokinase-type plasminogen activator during arteriogenesis.

To define the role of the plasminogen activators (PAs) urokinase PA (uPA) and tissue PA (tPA) as well as the uPA receptor (uPAR) in arteriogenesis, we investigated their impact in a rabbit and mouse model of adaptive collateral artery growth. Collateral artery growth was induced by occlusion of the femoral artery in rabbit and wild-type (WT) mice and in mice with targeted inactivation of uPA (uPA-/-), tPA (tPA-/-), or uPAR (uPAR-/-). Northern blot results revealed a significant up-regulation of uPA but not uPAR or tPA in the early phase of arteriogenesis in rabbit and WT mice. This up-regulation on RNA level was followed by an increased protein level and enzymatic activity. Impaired perfusion recovery upon femoral artery ligation was observed by laser Doppler analysis in vivo in uPA-deficient mice but not in uPAR or tPA deficiency compared with WT mice. Immunohistochemical studies revealed an association of leukocyte infiltration with arteriogenesis in WT mice that was strongly reduced in uPA-/- but not in uPAR- or tPA-deficient mice. We conclude that arteriogenesis is promoted by an uPA-mediated infiltration of leukocytes that is not dependent on uPAR.

VEGF and therapeutic opportunities in cardiovascular diseases.

In the past ten years, alternative revascularization strategies have come from bench to bedside focusing on the growth of new vessels to replace the old. Hypoxia and vascular endothelial growth factor may induce capillary growth; however, atherosclerosis affects large conductance vessels, which can only be replaced by functional collateral arteries.

Therapeutic targets in cardiovascular disorders.

The feasibility of gene therapy for cardiovascular diseases related to atherosclerosis is a topic that needs to be addressed. Most recent papers have dealt with technical aspects and feasibility and most of the genes transferred were reporter genes like those for beta-galactosidase or luciferase. This may mean that the ideal vector, one that is not pathogenic or immunotolerant but is still efficient, is still not available. The results of these studies are ambiguous and it has been doubted whether the genes targeted really affect the disease. Further efforts are therefore needed to elucidate the underlying pathophysiology.

Development and role of coronary collaterals.

The collateral circulation that develops in response to gradual and progressive coronary artery occlusion can maintain the structural and functional integrity of the subtended myocardium to an astonishing degree, provided the speed of the stenosing process is not too great. Collateral development occurs by recapitulation of ontogeny: patterns of morphogenic gene expression normally active only during embryonic development are recalled for vasculogenesis (large vessel formation, as in human and canine collaterals) and angiogenesis (capillary sprouting, as in human and porcine hearts). Vascular cell proliferation is induced by ischemia-related growth factor production. However, tissue growth alone is not enough for vasculogenesis (it would by itself cause lumen reduction). Remodeling, that is, the controlled destruction and complete reconstruction of the enlarged vessel, takes place involving the concerted action of proteases. For unknown reasons, the growth-remodeling process provides only ~50% of the blood-conducting capacity of the large vessel it has replaced. This leaves room for improvement by somatic gene therapy and by drugs to be developed.

Quo vadis collateral blood flow? A commentary on a highly cited paper.

This text is a commentary on the highly cited paper by Maxwell-Hearse-Yellon describing the amount of collateral blood flow in several species of mammals after coronary artery occlusion. The measurement of collateral blood flow, an academic exercise in previous times because of its invariance and the futility of changing the degree of adaptation under chronic conditions, has reached new importance because collateral vessel growth (presently called arteriogenesis) can now be manipulated with growth factors, their genes or peptides. The early successes and failures are discussed and a plea is made for the rigorous application of goldstandard methods (like in the Maxwell-Hearse-Yellon paper) to avoid disappointments in the new science of 'therapeutic angiogenesis'.