W. Schierling

Trpv4 induces collateral vessel growth during regeneration of the arterial circulation

The development of a collateral circulation (arteriogenesis), bypassing an arterial occlusion, is important for tissue survival, but it remains functionally defective. Micro array data of growing collateral vessels, exposed to chronically elevated fluid shear stress (FSS), showed increased transcription of the transient receptor potential cation channel, subfamily V, member 4 (Trpv4). Thus, the aim of this study was to investigate the role of the shear stress sensitive Trpv4 in transmitting this physical stimulus into an active growth response. qRT‐PCR at different time points during the growth of collateral vessels after femoral artery ligature (FAL) in rats showed a strong positive correlation of Trpv4 transcription and the intensity of FSS. An increased protein expression of Trpv4 was localized in the FSS‐sensing endothelium by means of confocal immunohistochemistry. Cultured porcine endothelial cells showed a dose‐dependent expression of Trpv4 and an increased level of Ki67‐positive cells upon treatment with 4α‐Phorbol 12,13‐didecanoate (4αPDD), a specific Trpv4 activator. This was also demonstrated by flow culture experiments. These results were confirmed by in vivo application of 4αPDD in rabbit hind limb circulation via an osmotic mini‐pump after FAL. Trpv4 expression as well as Ki67‐positive staining was significantly increased in collateral vessels. Finally, 4αPDD treatment after FAL led to a 61% (215.5 ml/min/mmHg versus 350 ml/min/mmHg) recovery of conductance when compared with the non‐occluded artery. Cell culture and in vivo studies demonstrate that an FSS‐ or a 4αPDD‐induced activation of Trpv4 leads to an active proliferation of vascular cells and finally triggers collateral growth. Trpv4, a well‐known FSS‐sensitive vasodilator, has hitherto not been implicated in active growth processes of collateral arteries. This new function may lead to new therapeutic strategies for the treatment of arterial occlusive diseases.

Actin-Binding Rho Activating Protein (Abra) Is Essential for Fluid Shear Stress-Induced Arteriogenesis

Objective—Arteriogenesis, the development of a collateral circulation, is important for tissue survival but remains functionally defective because of early normalization of fluid shear stress (FSS). Using a surgical model of chronically elevated FSS we showed that rabbits exhibited normal blood flow reserve after femoral artery ligature (FAL). Inhibition of the Rho pathway by Fasudil completely blocked the beneficial effect of FSS. In a genome-wide gene profiling we identified actin-binding Rho activating protein (Abra), which was highly upregulated in growing collaterals. Methods and Results—qRT-PCR and Western blot confirmed highly increased FSS-dependent expression of Abra in growing collaterals. NO blockage by L-NAME abolished FSS-generated Abra expression as well as the whole arteriogenic process. Cell culture studies demonstrated an Abra-triggered proliferation of smooth muscle cells through a mechanism that requires Rho signaling. Local intracollateral adenoviral overexpression of Abra improved collateral conductance by 60% in rabbits compared to the natural response after FAL. In contrast, targeted deletion of Abra in CL57BL/6 mice led to impaired arteriogenesis. Conclusions—FSS-induced Abra expression during arteriogenesis is triggered by NO and leads to stimulation of collateral growth by smooth muscle cell proliferation.

The Role of Angiogenic Growth Factors in Arteriogenesis

Background/Aims: Collateral vessels restore only about 40% of the maximum dilatory reserve after femoral artery occlusion, whereas complete normalization is reached by increased fluid shear stress (FSS). We studied the role of known potent angiogenic growth factors (separately or in combination) in arteriogenesis by determining their expression in FSS-stimulated collaterals and close-to-collateral infusion of growth factor peptides in a rabbit model of femoral artery occlusion. Methods: Values of maximum collateral conductance (Cmax) and post mortem angiograms were compared to those achievable by high FSS. mRNA levels of growth factor ligands and receptors were determined in FSS-stimulated collaterals. Results: Seven days after vessel occlusion, FSS-stimulated legs showed a Cmax not significantly different from that of not occluded femoral arteries. Arteriogenesis was significantly less enhanced after growth factor treatment (MCP-1 86%, Ad5.1-FGF-4 75%, bFGF 72%, PDGF 64%, VEGF 50% of Cmax after FSS stimulation). RT-PCR showed no differential expression of FGF receptors, but an up-regulation of VEGF-receptor-2. Conclusion: The most potent known angiogenic growth factors at high pharmacological doses reach only a fraction of the maximum conductance obtained by high FSS. Arteriogenesis differs from angiogenesis, so the main focus to markedly improve arteriogenesis should be put on the underlying mechanisms of shear stress.

Effects of Endogenous Nitric Oxide and of DETA NONOate in Arteriogenesis

Previous studies showed that targeted endothelial nitric oxide synthase (eNOS) disruption in mice with femoral artery occlusion does not impede and transgenic eNOS overexpression does not stimulate collateral artery growth after femoral artery occlusion, suggesting that nitric oxide from eNOS does not play a role in arteriogenesis. However, pharmacologic nitric oxide synthase inhibition with L-NAME markedly blocks arteriogenesis, suggestive of an important role of nitric oxide. To solve the paradox, we studied targeted deletion of eNOS and of inducible nitric oxide synthase (iNOS) in mice and found that only iNOS knockout could partially inhibit arteriogenesis. However, the combination of eNOS knockout and treatment with the iNOS inhibitor L-NIL completely abolished arteriogenesis. mRNA transcription studies (reverse transcriptase-polymerase chain reaction) performed on collateral arteries of rats showed that eNOS and especially iNOS (but not neural nitric oxide synthase) become upregulated in shear stress-stimulated collateral vessels, which supports the hypothesis that nitric oxide is necessary for arteriogenesis but that iNOS plays an important part. This was strengthened by the observation that the nitric oxide donor DETA NONOate strongly stimulated collateral artery growth, activated perivascular monocytes, and increased proliferation markers. Shear stress-induced nitric oxide may activate the innate immune system and activate iNOS. In conclusion, arteriogenesis is completely dependent on the presence of nitric oxide, a large part of it coming from mononuclear cells.

Increased intravascular flow rate triggers cerebral arteriogenesis

Peripheral arteriogenesis is distinctly enhanced by increased fluid shear stress. Thus, the aim of this study was to investigate in the rat brain whether increased fluid shear stress can also stimulate cerebral arteriogenesis. To increase fluid shear stress in the cerebral circulation, we developed different shear stress models as the ligature of both common carotid arteries (Double-Ligature model), bilateral carotid ligature followed by creation of a unilateral arterio-venous fistula (two-stage protocol, Ligature-Shunt model), and unilateral arterio-venous fistula-creation alone (Solo-Shuntmodel). Blood flow changes were monitored in vivo by quantitative magnetic resonance imaging-analysis. Cerebral arteriogenesis was analyzed by magnetic resonance imaging and contrast agent-angiography. For proliferation and accumulation of mononuclear cells, immunohistochemistry was performed. During the 14 days-observation period, blood flow increased maximal by 5.5-fold in the A. basilaris and 10.3-fold in the fistula-sided A. cerebri posterior of the Ligature-Shunt model. Considerable vessel growth was found in all shear stress-stimulated arteries. Comparative analysis of vessel length and diameter versus blood flow indicated a correlation between the growth of cerebral collaterals and rising intravascular flow rates (R2 = 0.90/0.96). Immunohistochemistry showed the typical phases of arteriogenesis and accumulation of mononuclear cells. In conclusion, we provide evidence that fluid shear stress is not only the pivotal trigger of peripheral but also of cerebral arteriogenesis.

Standardized 2D ultrasound versus 3D/4D ultrasound and image fusion for measurement of aortic aneurysm diameter in follow-up after EVAR

PURPOSE To compare standardised 2D ultrasound (US) to the novel ultrasonographic imaging techniques 3D/4D US and image fusion (combined real-time display of B mode and CT scan) for routine measurement of aortic diameter in follow-up after endovascular aortic aneurysm repair (EVAR). METHOD AND MATERIALS 300 measurements were performed on 20 patients after EVAR by one experienced sonographer (3rd degree of the German society of ultrasound (DEGUM)) with a high-end ultrasound machine and a convex probe (1-5 MHz). An internally standardized scanning protocol of the aortic aneurysm diameter in B mode used a so called leading-edge method. In summary, five different US methods (2D, 3D free-hand, magnetic field tracked 3D - Curefab™, 4D volume sweep, image fusion), each including contrast-enhanced ultrasound (CEUS), were used for measurement of the maximum aortic aneurysm diameter. Standardized 2D sonography was the defined reference standard for statistical analysis. CEUS was used for endoleak detection. RESULTS Technical success was 100%. In augmented transverse imaging the mean aortic anteroposterior (AP) diameter was 4.0±1.3 cm for 2D US, 4.0±1.2 cm for 3D Curefab™, and 3.9±1.3 cm for 4D US and 4.0±1.2 for image fusion. The mean differences were below 1 mm (0.2-0.9 mm). Concerning estimation of aneurysm growth, agreement was found between 2D, 3D and 4D US in 19 of the 20 patients (95%). Definitive decision could always be made by image fusion. CEUS was combined with all methods and detected two out of the 20 patients (10%) with an endoleak type II. In one case, endoleak feeding arteries remained unclear with 2D CEUS but could be clearly localized by 3D CEUS and image fusion. CONCLUSION Standardized 2D US allows adequate routine follow-up of maximum aortic aneurysm diameter after EVAR. Image Fusion enables a definitive statement about aneurysm growth without the need for new CT imaging by combining the postoperative CT scan with real-time B mode in a dual image display. 3D/4D CEUS and image fusion can improve endoleak characterization in selected cases but are not mandatory for routine practice.

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.

Hochauflösende 3-D-Sonographie und Bildfusion mit der CT-Angiographie

ZusammenfassungHintergrundDie Ultraschalluntersuchung ist die erste Wahl zur Diagnostik von Gefäßerkrankungen an der Karotis, den peripheren Arterien und Venen sowie der abdominellen Aorta und den Nieren- und Viszeralarterien. Mittels B-Bildmodus, farbkodierter Duplexsonographie und gepulstem Doppler (pw-Doppler) können zeitgleich Morphologie und Hämodynamik beurteilt werden. In der Nachsorge nach endovaskulärer Versorgung abdomineller Aortenaneurysmen hat die Kontrastmittelsonographie zur Detektion von Endoleaks einen festen Stellenwert. Sie erfordert ebenso wie 3-D-Sonographie und Bildfusion, die eine räumliche Darstellung ermöglichen, eine spezielle Soft- und Hardware. Aufgrund des Aufbaus eines magnetischen Feldes ist die Bildfusion bei Schrittmacherpatienten kontraindiziert. Trotz dieser erweiterten sonographischen Möglichkeiten bleibt die korrekte Klassifikation von Endoleaks und die Vermessung des Aortenaneurysmas, aus der sich die weitere Therapie und Nachsorge ableitet, eine Herausforderung.SchlußfolgerungDie qualitative 3-D-Freihand-Sonographie verbessert ebenso wie die Bildfusion eines Ultraschall- und CT-Datensatzes die räumliche Darstellung und Klassifikation von Endoleaks nach EVAR. Der Stellenwert der Volumetrie der Aorta in der 3-D-Volumen-Technik ist im Vergleich zur Bestimmung des Aortendiameters, die mit der Bildfusion auch bei Verlaufsuntersuchungen sehr einfach gelingt, unklar. Der Nutzen beider Verfahren gegenüber dem bestehenden Zeit- und Kostenaufwand ist im Gegensatz zur Kontrastmitteluntersuchung bislang nicht belegt.AbstractBackgroundUltrasound examination is the method of choice for the diagnostics of vascular diseases of carotid arteries, peripheral arteries and veins as well as the abdominal aorta, renal and visceral arteries. Using the B-imaging mode, color coded duplex sonography and pulsed Doppler the morphology and hemodynamics can be simultaneously assessed. Contrast-enhanced ultraound has become the established method for detection of endoleaks in follow-up care after endovascular treatment of abdominal aortic aneurysms. This requires special software and hardware which allow a spatial representation, as do 3D sonography and image fusion. Due to the formation of a magnetic field, image fusion in patients with a pacemaker is contraindicated. Despite these extended sonographic possibilities the correct classification of endoleaks and measurement of aortic aneurysms, from which the further treatment and aftercare are derived, remain a challenge.ConclusionQualitative 3D freehand sonography improves the spatial representation and classification of endoleaks after endovascular aneurysm repair (EVAR) just as image fusion of an ultrasound and computed tomography (CT) data set. The importance of volumetric analysis of the aorta in the 3D volume technique in comparison to determination of the diameter of the aorta, which is very simple with image fusion even in follow-up investigations, is unclear. The benefits of both procedures weighed against the time and costs involved in comparison to contrast-enhanced ultrasound investigations have not yet been confirmed.

Ultraschalldiagnostik der abdominellen Aorta

ZusammenfassungHintergrund und FragestellungIdeal für Screeninguntersuchungen ist eine Methode, die möglichst nebenwirkungsfrei, leicht erlernbar und somit breit einsetzbar mit hoher Treffsicherheit ein abdominelles Aortenaneurysma (AAA) erkennt. Obwohl die Sonographie diese Kriterien erfüllt, ist die Messmethode nicht standardisiert. Sowohl in Ultraschall- als auch in CT-Studien werden unterschiedliche Messmethoden angewandt und nur in 57 % der Fälle wird überhaupt die Messmethode ausreichend beschrieben.MethodeKritisches Review der aktuellen Literatur zu Messmethodik und Validität der Sonographie bei der Durchmesserbestimmung der Aorta insbesondere beim AAA und Darstellung von Messprinzipien zur möglichst exakten Messung.Ergebnisse und SchlussfolgerungenDie exakteste Durchmesserbestimmung wird EKG-getriggert, nach der Leading-edge-Methode mit orthogonaler Schnittführung (zur Gefäßachse) durchgeführt. Im Rahmen von Screeninguntersuchungen ist eine ausreichende Messgenauigkeit bei Einhaltung der orthogonalen Schnittführung erreicht.Die Sonographie zeigt bei dieser standardisierten Messmethode auch im Methodenvergleich zum CT valide, reproduzierbare Ergebnisse und ist für Screeninguntersuchungen zum AAA die Methode der Wahl.AbstractBackground and objectivesThe ideal method for screening investigations is one which is as free as possible from side effects, is easily learnt and can therefore be broadly employed to recognize abdominal aortic aneurysms (AAA) with a high degree of certainty. Although ultrasonography fulfils these criteria, the measurement method is not standardized. Different measurement methods are used in ultrasonography as well as in computed tomography (CT) studies and the measurement method is actually described sufficiently in only 57 % of cases.MethodsThis article gives a critical review of the current literature on measurement methods and the validity of ultrasonography for determination of the diameter of the aorta, particularly for AAAs and presents the measurement principles for making measurements as precisely as possible.Results and conclusionThe most precise determination of the diameter is carried out by electrocardiogram (ECG) gating according to the leading edge method with orthogonal slicing. Within the framework of screening investigations, sufficient measurement precision can be achieved by adherence to orthogonal slicing. In these standardized measurement methods ultrasonography shows valid and reproducible results even in comparison to CT and is the method of choice in screening investigations for AAAs.

Ultrasound diagnostics of the abdominal aorta

Background and objectivesThe ideal method for screening investigations is one which is as free as possible from side effects, is easily learnt, and can therefore be broadly used to detect abdominal aortic aneurysms (AAA) with a high degree of certainty. Although ultrasonography fulfils these criteria, the measurement method is not standardized. Different measurement methods are used in ultrasonography as well as in computed tomography (CT) studies and the measurement method is actually described sufficiently in only 57 % of cases.MethodsThis article provides a critical review of the current literature on measurement methods and the validity of ultrasonography for the determination of aortic diameter, particularly in AAA, and presents the measurement principles for making measurements as precisely as possible.Results and conclusionThe most precise determination of aortic diameter is carried out by electrocardiogram (ECG) gating according to the leading-edge method with orthogonal slicing. Within the framework of screening investigations, sufficient measurement precision can be achieved by adherence to orthogonal slicing. Using these standardized measurement methods, ultrasonography shows valid and reproducible results even compared with CT and is the method of choice in screening investigations for AAA.ZusammenfassungHintergrund und FragestellungIdeal für Screeninguntersuchungen ist eine Methode, die möglichst nebenwirkungsfrei, leicht erlernbar und somit breit einsetzbar mit hoher Treffsicherheit ein abdominelles Aortenaneurysma (AAA) erkennt. Obwohl die Sonographie diese Kriterien erfüllt, ist die Messmethode nicht standardisiert. Sowohl in Ultraschall- als auch in CT-Studien werden unterschiedliche Messmethoden angewandt und nur in 57 % der Fälle wird überhaupt die Messmethode ausreichend beschrieben.MethodeKritisches Review der aktuellen Literatur zu Messmethodik und Validität der Sonographie bei der Durchmesserbestimmung der Aorta insbesondere beim AAA und Darstellung von Messprinzipien zur möglichst exakten Messung.Ergebnisse und SchlussfolgerungenDie exakteste Durchmesserbestimmung wird EKG-getriggert, nach der Leading-edge-Methode mit orthogonaler Schnittführung (zur Gefäßachse) durchgeführt. Im Rahmen von Screeninguntersuchungen ist eine ausreichende Messgenauigkeit bei Einhaltung der orthogonalen Schnittführung erreicht.Die Sonographie zeigt bei dieser standardisierten Messmethode auch im Methodenvergleich zum CT valide, reproduzierbare Ergebnisse und ist für Screeninguntersuchungen zum AAA die Methode der Wahl.