Cerebral Arteriogenesis Research Articles

Three-Dimensional Iron Oxide Nanoparticle-Based Contrast-Enhanced Magnetic Resonance Imaging for Characterization of Cerebral Arteriogenesis in the Mouse Neocortex.

Purpose: Subsurface blood vessels in the cerebral cortex have been identified as a bottleneck in cerebral perfusion with the potential for collateral remodeling. However, valid techniques for non-invasive, longitudinal characterization of neocortical microvessels are still lacking. In this study, we validated contrast-enhanced magnetic resonance imaging (CE-MRI) for in vivo characterization of vascular changes in a model of spontaneous collateral outgrowth following chronic cerebral hypoperfusion.Methods: C57BL/6J mice were randomly assigned to unilateral internal carotid artery occlusion or sham surgery and after 21 days, CE-MRI based on T2*-weighted imaging was performed using ultra-small superparamagnetic iron oxide nanoparticles to obtain subtraction angiographies and steady-state cerebral blood volume (ss-CBV) maps. First pass dynamic susceptibility contrast MRI (DSC-MRI) was performed for internal validation of ss-CBV. Further validation at the histological level was provided by ex vivo serial two-photon tomography (STP).Results: Qualitatively, an increase in vessel density was observed on CE-MRI subtraction angiographies following occlusion; however, a quantitative vessel tracing analysis was prone to errors in our model. Measurements of ss-CBV reliably identified an increase in cortical vasculature, validated by DSC-MRI and STP.Conclusion: Iron oxide nanoparticle-based ss-CBV serves as a robust, non-invasive imaging surrogate marker for neocortical vessels, with the potential to reduce and refine preclinical models targeting the development and outgrowth of cerebral collateralization.

Angiotensin-converting enzyme inhibitors stimulate cerebral arteriogenesis.

Arteriogenesis constitutes the most efficient endogenous rescue mechanism in cases of cerebral ischaemia. The aim of this work was to investigate whether angiotensin-converting enzyme inhibitors (ACEi) stimulates, and angiotensin II receptor type 1 blockers (ARB) inhibits cerebral collateral growth by applying a three-vessel occlusion (3-VO) model in rat. Cerebral collateral growth was measured post 3-VO (1) by assessing blood flow using the cerebrovascular reserve capacity (CVRC) technique, and (2) by assessing vessel diameters in the posterior cerebral artery (PCA) via the evaluation of latex angiographies. A stimulatory effect on arteriogenesis was investigated for ACEi administration±bradykinin receptor 1 (B1R) and 2 (B2R) blockers, and an inhibitory effect was analysed for ARB administration. Results were validated by immunohistochemical analysis and mechanistic data were collected by human umbilical vein endothelial cell (HUVEC) viability or scratch assay and monocyte (THP-1) migration assay. An inhibitory effect of ARB on arteriogenesis could not be demonstrated. However, collateral growth measurements demonstrated a significantly increased CVRC and PCA diameters in the ACEi group. ACEi stimulates cell viability and migration, which could be partially reduced by additional administration of bradykinin receptor 1 inhibitor (B1Ri). ACEi inhibits the degradation of pro-arteriogenic bradykinin derivatives, but combined ACEi+B1Ri+B1Ri (BRB) treatment did not reverse the stimulatory effect. Yet, co-administration of ACEi+BRB enhances arteriogenesis and cell migration. We demonstrate a potent stimulatory effect of ACEi on cerebral arteriogenesis in rats, presumable via B1R. However, results imply a pleiotropic and compensatory effect of ACEi on bradykinin receptor-stimulated arteriogenesis.

The Effect of Sympathetic Denervation on Cerebral Arteriogenesis After Chronic Cerebral Hypoperfusion

ObjectiveTo explore the effect of perivascular sympathetic nerve on cerebral collateral arteriogenesis in chronic cerebral hypoperfusion models of rats. Materials and MethodsChronic cerebral hypoperfusion model was established by right common carotid artery ligation for 8 weeks, while sympathetic denervation was performed by superior cervical ganglionectomy. The male Sprague-Dawley rats were randomly divided into 4 groups including sham group (n = 21), denervation group (n = 21), artery ligation group (n = 21) and combined group with both artery ligation and denervation (n = 21). After 8 weeks of surgery, the rats in each group were randomly divided into 3 subgroups including subgroup A (n = 7), subgroup B (n = 7) and subgroup C (n = 7). The 3 subgroups were subjected to latex perfusion, permanent right middle cerebral artery occlusion and immunohistochemical staining, respectively. ResultsThe diameters of right leptomeningeal anastomoses in artery ligation group significantly enlarged compared with sham group. When sympathetic denervation was performed in the presence of artery ligation, diameter of collateral vessel decreased, although larger than in sham group. After 8 weeks of permanent right middle cerebral artery occlusion, the cerebral perfusion over the right middle cerebral artery area in combined group was significantly lower than in artery ligation group, although both were higher than in denervation group and sham group. Triphenyltetrazolium chloride staining showed that cerebral infarct volume in combined group was significantly larger than in artery ligation group, and smaller than in denervation group and sham group. Neurologic functional scoring showed that scores in combined group were significantly higher than in artery ligation group, and lower than in denervation group and sham group. Immunohistochemical staining for α-smooth muscle actin showed that compared with sham group, tunica media thickness of right leptomeningeal anastomoses in artery ligation group increased significantly. Thickness in combined group was thinner than in artery ligation group, although thicker than in sham group. ConclusionsPerivascular sympathetic denervation can impair the cerebral collateral arteriogenesis under condition of chronic cerebral hypoperfusion.

Exercise Preconditioning and Neuroprotection: A Review of Mechanisms

Introduction: Cerebrovascular accident or stroke is the first cause of acquired disability and the third leading cause of mortality in adults. It is known that exercise modifies risk factors such as hypertension, lipid profile, diabetes that may play an important role in the prevention of cerebrovascular accidents. However, recent findings suggest that besides adjusting the risk factors of stroke, exercise may be helpful in inducing endogenous neuroprotection and neuronal survival in ischemia-reperfusion condition which is the main mechanism of ischemic stroke. The effect of previous exercises in protecting neurons against ischemic injury and inducing neuronal resistance known as exercise preconditioning, which is a relatively new field of research on the effects of exercise on the brain. Although the exact mechanisms of neuroprotection induced by exercise preconditioning have yet to be known, previous studies have shown that exercise preconditioning may be helpful through several mechanisms, such as strengthening blood-brain barrier, inducing cerebral angiogenesis and arteriogenesis, improving cerebral metabolism and decreasing neuronal metabolic disturbances following ischemic injury, upregulation of neurotrophins expression, as well as reducing inflammation, apoptosis, and oxidative stress. Conclusion: It seems that exercise preconditioning in people predisposed to brain ischemic injuries or in people with history of mild ischemic injury may help in reducing the primary damage and improve the neurological outcomes after ischemia-reperfusion injury. However, more research is needed to develop exercise protocols with appropriate time, intensity, and type to induce the optimal neuroprotection.

Inhibition of protein tyrosine phosphatases enhances cerebral collateral growth in rats.

Arteriogenesis involves the rapid proliferation of preexisting arterioles to fully functional arteries as a compensatory mechanism to overcome circulatory deficits. Stimulation of arteriogenesis has therefore been considered a treatment concept in arterial occlusive disease. Here, we investigated the impact of inhibition of protein tyrosine phosphatases (PTPs) on cerebral arteriogenesis in rats. Arteriogenesis was induced by occlusion of one carotid and both vertebral arteries (three-vessel occlusion (3-VO)). Collateral growth and functional vessel perfusion was assessed 3-35days following 3-VO. Furthermore, animals underwent 3-VO surgery and were treated with the pan-PTP inhibitor BMOV, the SHP-1 inhibitor sodium stibogluconate (SSG), or the PTP1B inhibitor AS279. Cerebral vessel diameters and cerebrovascular reserve capacity (CVRC) were determined, together with immunohistochemistry analyses and proximity ligation assays (PLA) for determination of tissue proliferation and phosphorylation patterns after 7days. The most significant changes in vessel diameter increase were present in the ipsilateral posterior cerebral artery (PCA), with proliferative markers (PCNA) being time-dependently increased. The CVRC was lost in the early phase after 3-VO and partially recovered after 21days. PTP inhibition resulted in a significant increase in the ipsilateral PCA diameter in BMOV-treated animals and rats subjected to PTP1B inhibition. Furthermore, CVRC was significantly elevated in AS279-treated rats compared to control animals, along with hyperphosphorylation of the platelet-derived growth factor-β receptor in the vascular wall in vivo. In summary, our data indicate PTPs as hitherto unrecognized negative regulators in cerebral arteriogenesis. Further, PTP inhibition leading to enhanced collateral growth and blood perfusion suggests PTPs as novel targets in anti-ischemic treatment. PTPs exhibit negative regulatory function in cerebral collateral growth in rats. Inhibition of pan-PTP/PTP1B increases vessel PDGF-β receptor phosphorylation. PTP1B inhibition enhances arteriogenesis and cerebrovascular reserve capacity.

Abstract T MP30: Cerebral Arteriogenesis and Angiogenesis: Distinct Angioarchitecture of Innate and EDAS Collaterals in Intracranial Arterial Steno-Occlusive Disease

Introduction: Patients with ICASD form various degrees of collateralization. However, those collaterals fail over time contributing to symptom progression. Indirect revascularization via encephaloduroarteriosynangiosis (EDAS) establishes collateral flow through new vessels formed from external carotid branches in both moyamoya disease (MMD) and non-moyamoya (NM) ICASD. We hypothesize that the EDAS neovascularization differs in terms of angioarchitecture when compared to spontaneously formed collaterals in ICASD. Methods: We analyze pre and post-operative digital subtraction angiograms (DSA) of patients enrolled in a prospective trial of EDAS surgery using ImageJ software (http://imagej.nih.gov/ij). Angioarchitectural differences between innate and post-EDAS collaterals were evaluated by comparing their branching angioscore (BA) and artery tortuosity index (ATI). Results: Images from 26 patients, ages 4 to 84 (mean: 37 SD+/-19.5) were evaluated. Patients included 13 MMD and 13 NM-ICASD. Postoperative mean DSA time was 14 months. Post-EDAS collaterals had significantly higher BA and lower ATI than innate collaterals (BA: 9.8 vs. 5.5 and ATI 110 vs. 167 [p<0.001] respectively). Subgroup analyses for patients with MMD and NM-MMD demonstrated the same pattern with higher BA for post-EDAS collaterals and lower ATI for innate collaterals (MMD: BA: 9.6 vs. 5.2, and ATI 107.7 vs.176.5, NM-ICASD: BA: 9.9 vs. 5.8, and ATI: 153.7 vs.115.4 p≤0.01). Type of ICASD, age, or time after angiography did not affect the indexes. Conclusions: Collaterals formed after EDAS differ in angioarchitecture to innate collaterals in MMD and NM-ICASD. Morphological differences are consistent with two distinct mechanisms of vessel formation: innate collaterals formed by arteriogenesis display high tortuosity and low branching as expected by a shear stress process acting on pre-existing conduits. Conversely, post-EDAS collaterals have higher branching and lower tortuosity as expected in a process of angiogenesis in which collaterals form through sprouting and splitting from the donor vessels.

PPARγ activation inhibits cerebral arteriogenesis in the hypoperfused rat brain

PPARγ stimulation improves cardiovascular (CV) risk factors, but without improving overall clinical outcomes. PPARγ agonists interfere with endothelial cell (EC), monocyte and smooth muscle cell (SMC) activation, function and proliferation, physiological processes critical for arterial collateral growth (arteriogenesis). We therefore assessed the effect of PPARγ stimulation on cerebral adaptive and therapeutic collateral growth. In a rat model of adaptive cerebral arteriogenesis (3-VO), collateral growth and function were assessed (i) in controls, (ii) after PPARγ stimulation (pioglitazone 2.8mgkg(-1); 10mgkg(-1) compared with metformin 62.2mgkg(-1) or sitagliptin 6.34mgkg(-1)) for 21days or (iii) after adding pioglitazone to G-CSF (40μgkg(-1) every other day) to induce therapeutic arteriogenesis for 1week. Pioglitazone effects on endothelial and SMC morphology and proliferation, monocyte activation and migration were studied. PPARγ stimulation decreased cerebrovascular collateral growth and recovery of hemodynamic reserve capacity (CVRC controls: 12±7%; pio low: -2±9%; pio high: 1±7%; metformin: 9±13%; sitagliptin: 11±12%), counteracted G-CSF-induced therapeutic arteriogenesis and interfered with EC activation, SMC proliferation, monocyte activation and migration. Pharmacologic PPARγ stimulation inhibits pro-arteriogenic EC activation, monocyte function, SMC proliferation and thus adaptive as well as G-CSF-induced cerebral arteriogenesis. Further studies should evaluate whether this effect may underlie the CV risk associated with thiazolidinedione use in patients.

Regenerative cerebral arteriogenesis is induced by GM‐CSF and inhibited by anti‐inflammatory cardiovascular medications

Restoration of blood flow in case of stenotic brain‐supplying arteries relies on restoration of arterial inflow, achieved by an outgrowth of preexistent arterioles (arteriogenesis) and depends on monocyte migration into the vesel wall. It was assumed that a stimulation of monocytic function induces collateral growth, while anti‐inflammatory substances might interfere negatively.In two animal models of cerebral hypoperfusion (3‐vessel occlusion/rats, common carotid artery occlusion/mice), administration of the pro‐arteriogenic Granulocyte Colony‐ Stimulating Factor (40ug/kg) augmented angiographic collateral diameters and restored cerebrovascular reserve capacities (CVRC) (20+ 8%, cont. −14+14%). Acetylsalicylic acid (ASA), used in patients at risk for arterial stenosis, interfered with (a) CVRC restoration (1+4%), (b) collateral vessel growth and (c) monocyte migration, while Clopidogrel did not. Current data indicate similar effects for other anti‐inflammatory drugs and other vascular beds, which may explain why their effects on outcomes may fall behind effects on cardiovascular risk factors.Anti‐inflammatory drugs should be used with care in patients with vascular occlusions until large‐scale clinical data on antiarteriogenic effects have been obtained.

Abstract TP436: Dysfunctional Cerebral Neovascularization And Remodeling Patterns In Obese And Lean Models Of Type 2 Diabetes

We previously reported intense pial cerebral collateralization and arteriogenesis in a mild and lean model of type 2 diabetes, Goto-Kakizaki (GK) rats. Further 3-dimensional fluroscein (FITC) imaging studies revealed regional differences in increased cerebral neovascularization which was associated with poor vessel wall maturity. Building upon these findings, the goals of this study were a) to compare and contrast this pathological neovascularization pattern in db/db mice and GK models of diabetes, and b) determine the effect of glycemic control on erratic cerebral neovascularization. Total vascular volume, density and surface area as well as structural parameters including microvessel/macrovessel ratio, non-FITC perfusing vessel abundance, penetrating arteriole (PA) branching density and diameter, and tortuosity were measured by 3 dimensional reconstruction of FITC stained vasculature using Z-stacked images obtained with confocal microscopy. Lean GK rats exhibited an increase in both micro and macrovessel density, non-perfusing vessel abundance, branch density, diameter and tortuosity. Glycemic control with metformin prevented these changes. Obese db/db mice, on the other hand, showed an increase in only microvascular density but this was not associated with an increase in non-FITC perfusing vessels. PA branch density was higher than controls but branch diameter was reduced. Diabetes also promoted astrogliosis. These results suggests that type 2 diabetes leads to cerebral neovascularization and remodeling but structural characteristics of newly formed vessels differ between lean and obese models that have mild or severe hyperglycemia, respectively. The prevention of dysfunctional cerebral neovascularization by early glucose control suggests that hyperglycemia is a mediator of this response. N=4-8 * p≤ 0.05, ** p≤ 0.005 *** 0.0005

Knockout of Density-Enhanced Phosphatase-1 Impairs Cerebrovascular Reserve Capacity in an Arteriogenesis Model in Mice

Collateral growth, arteriogenesis, represents a proliferative mechanism involving endothelial cells, smooth muscle cells, and monocytes/macrophages. Here we investigated the role of Density-Enhanced Phosphatase-1 (DEP-1) in arteriogenesis in vivo, a protein-tyrosine-phosphatase that has controversially been discussed with regard to vascular cell biology. Wild-type C57BL/6 mice subjected to permanent left common carotid artery occlusion (CCAO) developed a significant diameter increase in distinct arteries of the circle of Willis, especially in the anterior cerebral artery. Analyzing the impact of loss of DEP-1 function, induction of collateralization was quantified after CCAO and hindlimb femoral artery ligation comparing wild-type and DEP-1−/− mice. Both cerebral collateralization assessed by latex perfusion and peripheral vessel growth in the femoral artery determined by microsphere perfusion and micro-CT analysis were not altered in DEP-1−/− compared to wild-type mice. Cerebrovascular reserve capacity, however, was significantly impaired in DEP-1−/− mice. Cerebrovascular transcriptional analysis of proarteriogenic growth factors and receptors showed specifically reduced transcripts of PDGF-B. SiRNA knockdown of DEP-1 in endothelial cells in vitro also resulted in significant PDGF-B downregulation, providing further evidence for DEP-1 in PDGF-B gene regulation. In summary, our data support the notion of DEP-1 as positive functional regulator in vascular cerebral arteriogenesis, involving differential PDGF-B gene expression.

Granulocyte Colony-Stimulating Factor Improves Cerebrovascular Reserve Capacity by Enhancing Collateral Growth in the Circle of Willis

Background and Purpose: Restoration of cerebrovascular reserve capacity (CVRC) depends on the recruitment and positive outward remodeling of preexistent collaterals (arteriogenesis). With this study, we provide functional evidence that granulocyte colony-stimulating factor (G-CSF) augments therapeutic arteriogenesis in two animal models of cerebral hypoperfusion. We identified an effective dosing regimen that improved CVRC and stimulated collateral growth, thereby improving the outcome after experimentally induced stroke. Methods: We used two established animal models of (a) cerebral hypoperfusion (mouse, common carotid artery ligation) and (b) cerebral arteriogenesis (rat, 3-vessel occlusion). Following therapeutic dose determination, both models received either G-CSF, 40 µg/kg every other day, or vehicle for 1 week. Collateral vessel diameters were measured following latex angiography. Cerebrovascular reserve capacities were assessed after acetazolamide stimulation. Mice with left common carotid artery occlusion (CCAO) were additionally subjected to middle cerebral artery occlusion, and stroke volumes were assessed after triphenyltetrazolium chloride staining. Given the vital role of monocytes in arteriogenesis, we assessed (a) the influence of G-CSF on monocyte migration in vitro and (b) monocyte counts in the adventitial tissues of the growing collaterals in vivo. Results: CVRC was impaired in both animal models 1 week after induction of hypoperfusion. While G-CSF, 40 µg/kg every other day, significantly augmented cerebral arteriogenesis in the rat model, 50 or 150 µg/kg every day did not show any noticeable therapeutic impact. G-CSF restored CVRC in mice (5 ± 2 to 12 ± 6%) and rats (3 ± 4 to 19 ± 12%). Vessel diameters changed accordingly: in rats, the diameters of posterior cerebral arteries (ipsilateral: 209 ± 7–271 ± 57 µm; contralateral: 208 ± 11–252 ± 28 µm) and in mice the diameter of anterior cerebral arteries (185 ± 15–222 ± 12 µm) significantly increased in the G-CSF groups compared to controls. Stroke volume in mice (10 ± 2%) was diminished following CCAO (7 ± 4%) and G-CSF treatment (4 ± 2%). G-CSF significantly increased monocyte migration in vitro and perivascular monocyte numbers in vivo. Conclusion: G-CSF augments cerebral collateral artery growth, increases CVRC and protects from experimentally induced ischemic stroke. When comparing three different dosing regimens, a relatively low dosage of G-CSF was most effective, indicating that the common side effects of this cytokine might be significantly reduced or possibly even avoided in this indication.

Abstract 111: Cerebrovascular Remodeling In Diabetes: Relevance To Stroke

Background: Diabetes is a major risk factor of stroke leading to the development of both large artery and lacunar infarcts associated with increased bleeding, and neurological and functional disability. We previously reported enhanced cerebral arteriogenesis in a mild and lean model of Type 2 diabetes. Here we hypothesize that cerebral neovascularization is regulated in a spatial manner in diabetes and vascular endothelial growth factor (VEGF) and peroxynitrite signaling are the underlying key regulators of increased remodeling. Methods: Neovascularization was assessed from brain, skeletal muscles and retina of control Wistar and diabetic Goto-Kakizaki rats. 3-dimensional reconstruction of the FITC stained vasculature were obtained by confocal microscopy and stereological parameters like vascular volume, density and surface area were measured. Unperfused vasculature was measured by immunostaining with Isolectin B4. Pericyte coverage was determined by nuclear staining (DAPI). VEGF and tyrosine nitration were evaluated by immunoblot and slot blot. Results: There was a progressive increase (rostral to caudal) in both cortical and striatal angiogenic parameters in the diabetic group. Cortical vascular density was more pronounced than the striatum in the diabetic group. Difference between FITC and Isolectin co-staining revealed that unperfused new vessels were prominent in the diabetic striatum. Endothelial cell number was markedly increased and pericyte/endothelial cell ratio indicating vascular maturity was significantly decreased in diabetes. Peripheral vascularization was impaired while increased in the retina. VEGF-A was greater in the microvessels but not macrovessels or brain homogenates devoid of vessels in diabetic animals. Diabetic microvessels also exhibited greater tyrosine nitration. Conclusion: Diabetes uniquely mediates cerebral neovascularization while regresses the peripheral vasculature. There exist spatial and regional differences in the diabetic brain that have relevance to neurovascular injury in stroke. Excessive neovascularization, a large pool of unperfused microvessels and reduced pericytes compromise the blood brain barrier and lead to increased bleeding during reperfusion injury in diabetes. N=4-10 * p≤ 0.05, ** p≤ 0.005

Acetylsalicylic Acid, but Not Clopidogrel, Inhibits Therapeutically Induced Cerebral Arteriogenesis in the Hypoperfused Rat Brain

This study investigated the effects of acetylsalicylic acid (ASA) and clopidogrel, standardly used in the secondary prevention of vascular occlusions, on cerebral arteriogenesis in vivo and in vitro. Cerebral hypoperfusion was induced by three-vessel occlusion (3-VO) in rats, which subsequently received vehicle, ASA (6.34 mg/kg), or clopidogrel (10 mg/kg). Granulocyte colony-stimulating factor (G-CSF), which enhanced monocyte migration in an additional cell culture model, augmented cerebrovascular arteriogenesis in subgroups (40 μg/kg). Cerebrovascular reactivity and vessel diameters were assessed at 7 and 21 days. Cerebrovascular reserve capacity was completely abolished after 3-VO and remained severely compromised after 7 (-14±14%) and 21 (-5±11%) days in the ASA groups in comparison with controls (4±5% and 10±10%) and clopidogrel (4±13% and 10±8%). It was still significantly decreased when ASA was combined with G-CSF (1±4%) compared with G-CSF alone (20±8%). Posterior cerebral artery diameters confirmed these data. Monocyte migration into the vessel wall, improved by G-CSF, was significantly reduced by ASA. Acetylsalicylic acid, but not clopidogrel, inhibits therapeutically augmented cerebral arteriogenesis.

Manipulating Arterial Fluid-shear Stress and Arteriogenesis in the Brain

Occlusions of the brain arteries remain life-threatening diseases and are associated with devastating morbidity. The basic, physiological and spontaneous reactions of the organism to vascular occlusion are to develop adaptative collateral circulation(s) outside the ischemic region, which can potentially replace the feeding capacity of the larger arteries. From the point of view of the interventionalist, the intracranial anatomy, as well as the extreme mechanosensitivity of the brain arteries, leave few degrees of freedom to surgical actions, in contrast to situations elsewhere in the body. Moreover there are major differences between the brain arteries and their constitutive vascular cells of the brain with arteries lying outside the brain. The cerebral vasculature is made of low-resistance vessels containing neural crest-derived vascular cells opposing high-resistance vessels made of mesoderm-derived vascular smooth muscle cells in the peripheral arteries. The paper by Wilma Schierling and colleagues is one example of the recent attempts to develop new therapeutic options aimed at favoring the brain arteriogenesis by acting upon the haemodynamic properties of cerebral arteries by modifying one of the blood flow components, i.e. the fluid-shear stress which previously has been shown to be the pivotal trigger of cerebral arteriogenesis. Various experimental models have been used. The present experimental model is based upon double ligature,

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.

Nuclear contrast angiography: A simple method for morphological characterization of cerebral arteries

Visualization of the cerebral vascular tree is important in experimental stroke and cerebral vascular malformation research. We describe a simple method, nuclear contrast angiography, that enables simultaneous visualization of the arterial tree and cerebral endothelial cells in rodent brain whole mounts. A mixture of latex and black ink was injected into the arterial system of rodents, resulting in high contrast demarcation of the arterial tree of the brain. This method clearly differentiates arteries from veins. We applied this method to demonstrate that 14 days of unilateral carotid artery occlusion induces increases in the caliber of (1) bilateral anterior communicating arteries, (2) bilateral anterior cerebral arteries, and (3) ipsilateral proximal middle cerebral artery of the circle of Willis. Unlike other methods, this procedure selectively stains endothelial nuclei of arteries. Thus, cerebral endothelial nuclei can be visualized, quantitated, and morphologically characterized at the same time the cortical arterial tree is delineated. This method should be useful in studies of stroke and cerebral arteriogenesis, which require the accurate assessment of both arterial diameters and endothelial cell density.

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-Shunt model). 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.

Induction of Cerebral Arteriogenesis Leads to Early-Phase Expression of Protease Inhibitors in Growing Collaterals of the Brain

Cerebral arteriogenesis constitutes a promising therapeutic concept for cerebrovascular ischaemia; however, transcriptional profiles important for therapeutic target identification have not yet been investigated. This study aims at a comprehensive characterization of transcriptional and morphologic activation during early-phase collateral vessel growth in a rat model of adaptive cerebral arteriogenesis. Arteriogenesis was induced using a three-vessel occlusion (3-VO) rat model of nonischaemic cerebral hypoperfusion. Collateral tissue from growing posterior cerebral artery (PCA) and posterior communicating artery (Pcom) was selectively isolated avoiding contamination with adjacent tissue. We detected differential gene expression 24 h after 3-VO with 164 genes significantly deregulated. Expression patterns contained gene transcripts predominantly involved in proliferation, inflammation, and migration. By using scanning electron microscopy, morphologic activation of the PCA endothelium was detected. Furthermore, the PCA showed induced proliferation (PCNA staining) and CD68+ macrophage staining 24 h after 3-VO, resulting in a significant increase in diameter within 7 days after 3-VO, confirming the arteriogenic phenotype. Analysis of molecular annotations and networks associated with differentially expressed genes revealed that early-phase cerebral arteriogenesis is characterised by the expression of protease inhibitors. These results were confirmed by quantitative real-time reverse transcription-PCR, and in situ hybridisation localised the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and kininogen to collateral arteries, showing that TIMP-1 and kininogen might be molecular markers for early-phase cerebral arteriogenesis.