Native Collaterals Research Articles

The Potential Mechanism Behind Native and Therapeutic Collaterals in Moyamoya

Background and PurposeTo explore the genetic basis and molecular mechanism of native arteriogenesis and therapeutic synangiosis in moyamoya disease (MMD).MethodsAn angiography-based study using patients from a prospective trial of encephaloduroarteriosynangiosis (EDAS) surgery was performed. The spontaneous collaterals grades were evaluated according to the system described by a new grading system. Blood samples were collected from all the recruited patients before EDAS and during the second hospitalization 3 months post-EDAS. We performed Boolean analysis using a combination of specific cell surface markers of CD34briCD133+CD45dimKDR+. Genotyping of p.R4810K was also performed. The correlation of age, sex, initial symptoms at diagnosis, collateral grade, Suzuki stages, the RNF213 genotype, time to peak (TTP), and endothelial progenitor cell (EPC) count with good collateral circulation was evaluated.ResultsEighty-five patients with MMD were included in this study. The mutation rate of RNF213 p.R4810K in our study was 25.9% (22/85). The heterozygous mutations were occurred significantly more frequently in the cases that were presented with infarction, worse neurological status, severe posterior cerebral artery (PCA) stenosis, and longer TTP delay. Further, the heterozygous mutations occurred significantly more frequently in the poor collateral stage group. Lower grades were significantly correlated with severe ischemia symptoms, worse neurological status, and a longer TTP delay. The post-operative angiographic findings showed that a good Matsushima grade was correlated with heterozygous mutations, a lower collateral stage, and a longer TTP delay. The CD34briCD133+CD45dimKDR+ cell count in patients 3 months post-EDAS was significantly higher as compared to the count before EDAS in the good Matsushima grade group. However, this change was not observed in the poor Matsushima grade group.ConclusionsThese data imply that mutations of RNF213 p.R4810K affect the establishment of spontaneous collateral circulation, and EPCs are involved in the process of formation of new EDAS collaterals.

Collateral Arteriogenesis Involves a Sympathetic Denervation That Is Associated With Abnormal α-Adrenergic Signaling and a Transient Loss of Vascular Tone.

Stimulating collateral arteriogenesis is an attractive therapeutic target for peripheral artery disease (PAD). However, the potency of arteriogenesis-stimulation in animal models has not been matched with efficacy in clinical trials. This may be because the presence of enlarged collaterals is not sufficient to relieve symptoms of PAD, suggesting that collateral function is also important. Specifically, collaterals are the primary site of vascular resistance following arterial occlusion, and impaired collateral vasodilation could impact downstream tissue perfusion and limb function. Therefore, we evaluated the effects of arteriogenesis on collateral vascular reactivity. Following femoral artery ligation in the mouse hindlimb, collateral functional vasodilation was impaired at day 7 (17 ± 3 vs. 60 ± 8%) but restored by day 28. This impairment was due to a high resting diameter (73 ± 4 μm at rest vs. 84 ± 3 μm dilated), which does not appear to be a beneficial effect of arteriogenesis because increasing tissue metabolic demand through voluntary exercise decreased resting diameter and restored vascular reactivity at day 7. The high diameter in sedentary animals was not due to sustained NO-dependent vasodilation or defective myogenic constriction, as there were no differences between the enlarged and native collaterals in response to eNOS inhibition with L-NAME or L-type calcium channel inhibition with nifedipine, respectively. Surprisingly, in the context of reduced vascular tone, vasoconstriction in response to the α-adrenergic agonist norepinephrine was enhanced in the enlarged collateral (−62 ± 2 vs. −37 ± 2%) while vasodilation in response to the α-adrenergic antagonist prazosin was reduced (6 ± 4% vs. 22 ± 16%), indicating a lack of α-adrenergic receptor activation by endogenous norepinephrine and suggesting a denervation of the neuroeffector junction. Staining for tyrosine hydroxylase demonstrated sympathetic denervation, with neurons occupying less area and located further from the enlarged collateral at day 7. Inversely, MMP2 presence surrounding the enlarged collateral was greater at day 7, suggesting that denervation may be related to extracellular matrix degradation during arteriogenesis. Further investigation on vascular wall maturation and the functionality of enlarged collaterals holds promise for identifying novel therapeutic targets to enhance arteriogenesis in patients with PAD.

Coronary Collateral Microcirculation Reserve Becomes Vestigial with Aging

Introduction: Our previous study indicated that coronary collateral microcirculation reserve (CCMR), native collaterals, transports blood flow to an ischemic area to reduce ischemic tissue injury. This study aimed to observe the changes of CCMR in the hearts of different month-old rats. Methods: We selected 2-, 8-, 16-, and 24-month-old rats as the research objects to monitor the changes of CCMR in rats with aging. After acute myocardial infarction, lectin-FITC was injected into the femoral vein vessels of rats to mark CCMR vessels in the ischemic area. Results: Results of the lectin-FITC perfusion experiment indicated that the number and collagen IV coverage of CCMR vessels declined with aging. Moreover, data suggested a correlation between endothelial nitric oxide synthase and a decline in the number of CCMR vessels. Conclusion: Aging causes CCMR decline in rats.

Decreased inspired oxygen stimulates de novo formation of coronary collaterals in adult heart

RationaleCollateral vessels lessen myocardial ischemia when acute or chronic coronary obstruction occurs. It has long been assumed that although native (pre-existing) collaterals enlarge in obstructive disease, new collaterals do not form in the adult. However, the latter was recently shown to occur after coronary artery ligation. Understanding the signals that drive this process is challenged by the difficulty in studying collateral vessels directly and the complex milieu of signaling pathways, including cell death, induced by ligation. Herein we show that hypoxemia alone is capable of inducing collateral vessels to form and that the novel gene Rabep2 is required. ObjectiveHypoxia stimulates angiogenesis during embryonic development and in pathological states. We hypothesized that hypoxia also stimulates collateral formation in adult heart by a process that involves RABEP2, a recently identified protein required for formation of collateral vessels during development. Methods and resultsExposure of mice to reduced FiO2 induced collateral formation that resulted in smaller infarctions following LAD ligation and that reversed on return to normoxia. Deletion of Rabep2 or knockdown of Vegfa inhibited formation. Hypoxia upregulated Rabep2, Vegfa and Vegfr2 in heart and brain microvascular endothelial cells (HBMVECs). Knockdown of Rabep2 impaired migration of HBMVECs. In contrast to systemic hypoxia, deletion of Rabep2 did not affect collateral formation induced by ischemic injury caused by LAD ligation. ConclusionsHypoxia induced formation of coronary collaterals by a process that required VEGFA and RABEP2, proteins also required for collateral formation during development. Knockdown of Rabep2 impaired cell migration, providing one potential mechanism for RABEP2's role in collateral formation. This appears specific to hypoxia, since formation after acute ischemic injury was unaffected in Rabep2−/− mice. These findings provide a novel model for studying coronary collateral formation, and demonstrate that hypoxia alone can induce new collaterals to form in adult heart.

Hypoxia induces de novo formation of cerebral collaterals and lessens the severity of ischemic stroke

Pial collaterals provide protection in stroke. Evidence suggests their formation late during gestation (collaterogenesis) is driven by reduced oxygen levels in the cerebral watersheds. The purpose of this study was to determine if collaterogenesis can be re-activated in the adult to induce formation of additional collaterals (“neo-collateral formation”, NCF). Mice were gradually acclimated to reduced inspired oxygen (FIO2) and maintained at 12, 10, 8.5 or 7% for two-to-eight weeks. Hypoxemia induced “dose”-dependent NCF and remodeling of native collaterals, and decreased infarct volume after permanent MCA occlusion. In contrast, no formation occurred of addition collateral-like intra-tree anastomoses, PComs, or branches within the MCA tree. Hypoxic NCF, remodeling and infarct protection were durable, i.e. retained for at least six weeks after return to normoxia. Hypoxia increased expression of Hif2α, Vegfa, Rabep2, Angpt2, Tie2 and Cxcr4. Neo-collateral formation was abolished in mice lacking Rabep2, a novel gene involved in VEGFA→Flk1 signaling and required for formation of collaterals during development, and inhibited by knockdown of Vegfa, Flk1 and Cxcr4. Rabep2-dependent NCF was also induced by permanent MCA occlusion. This is the first report that hypoxia induces new pial collaterals to form. Hypoxia- and occlusion-induced neo-collateral formation provide models to study collaterogenesis in the adult.

Pigment Epithelium-Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction.

BackgroundWe previously found that the structural defects of the coronary collateral microcirculation reserve (CCMR) prevent these preformed collateral vessels from continuously delivering the native collateral blood and supporting the ischemic myocardium in rats. Here, we tested whether these native collaterals can be remodeled by artificially increasing pigment epithelium–derived factor (PEDF) expression and demonstrated the mechanism for this stimulation.Methods and ResultsWe performed intramyocardial gene delivery (PEDF‐lentivirus, 2×107 TU) along the left anterior descending coronary artery to artificially increase the expression of PEDF in the tissue of the region for 2 weeks. By blocking the left anterior descending coronary artery, we examined the effects of PEDF on native collateral blood flow and CCMR. The results of positron emission tomography perfusion imaging showed that PEDF increased the native collateral blood flow and significantly inhibited its decline during acute myocardial infarction. In addition, the number of CCMR vessels decreased and the size increased. Similar results were obtained from in vitro experiments. We tested whether PEDF induces CCMR remodeling in a fluid shear stress–like manner by detecting proteins and signaling pathways that are closely related to fluid shear stress. The nitric oxide pathway and the Notch‐1 pathway participated in the process of CCMR remodeling induced by PEDF.Conclusions PEDF treatment activates the nitric oxide pathway, and the Notch‐1 pathway enabled CCMR remodeling. Increasing the native collateral blood flow can promote the ventricular remodeling process and improve prognosis after acute myocardial infarction.

The impact of myogenic cell transplantation on collateral capillary arteriogenesis

Current treatments for peripheral arterial occlusive disease (PAOD), including angioplasty and bypass grafting fail in up to 25% of patients, or are not indicated in up to 50%, so there is a need to develop additional treatment options. Patients with native collaterals have a better prognosis, so a treatment such as cell transplantation that promotes collateral arteriogenesis is a potential PAOD treatment. Unfortunately, bone marrow derived stem cells, the main cell type that has been investigated, have limited clinical effectiveness in enhancing pre‐existing collateral growth. Therefore, delivering a stem cell type native to the collateral‐containing tissue, such as myogenic stem cells, could have improved outcomes. Additionally, about 20% of patients never develop collaterals, and no one, to our knowledge, has evaluated the effect of cell therapy on collateral capillary arteriogenesis (CCA), which can reperfuse ischemic tissue in animals without native arteriolar collaterals and therefore represents a potential therapeutic target in patients who lack native collaterals. To test the hypothesis that myogenic cells enhance CCA, we ligated the spinotrapezius feed artery in Balb/C mice to induce CCA, and delivered myogenic cells, thrombin (positive control), or vehicle to the muscle. Seven days post ligation, ‐‐‐‐arterialized collateral capillary (ACC) number and diameter were determined. As expected, thrombin increased ACC number, but myogenic cells had no effect, 9.3 ± 0.5 vs 7.1 ± 0.4, respectively. However, myogenic cells increased ACC diameter as compared to untreated (12.1 ± 0.8 μm vs 10.3 ± 0.3 μm, respectively) and both myogenic cells and thrombin increased the maximum ACC diameter (29.9 ± 3.4 μm and 27.9 ± 2.0 μm vs 18.8 ± 1.1 μm, respectively). These results are consistent with previous studies indicating that thrombin enhances arteriogenesis and suggest that myogenic cells have a similar effect, but likely without the deleterious pro‐inflammatory effects of thrombin. Because myogenic cells increase arteriogenesis and macrophages are an essential part of arteriogenesis, we also tested the hypothesis that myogenic cells increase macrophage content by performing CD68 staining. Although macrophage number increased following ligation, neither myoblasts nor thrombin further increased macrophage number. The lack of difference in macrophage number despite enhanced CCA could be because the day‐7 timepoint was too late, as macrophages peak around day‐3, or because a shift towards either classically‐activated macrophages or alternatively‐activated macrophages, which have a greater effect on arteriogenesis, would not be detected by the CD68 stain. Therefore, future studies may include a day‐3 timepoint and a stain for alternatively‐activated macrophages.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chloride intracellular channel 4 is required for maturation of the cerebral collateral circulation.

The number and diameter of native collaterals in tissues of healthy mice vary widely, resulting in large differences in tissue injury in occlusive diseases. Recent studies suggest similar variation may exist in humans. Collateral variation in mice is determined by genetic background-dependent differences in embryonic collateral formation, by variation in maturation of the nascent collaterals, and by environmental factors such as aging that cause collateral rarefaction in the adult. Recently, formation of the collateral circulation in the brain was found to involve a unique VEGF-A-dependent "arteriolar" angiogenic sprouting-like mechanism. Elsewhere, chloride intracellular protein 4 (CLIC4) was implicated but not investigated directly, prompting the present study. Deletion of Clic4 had no effect on embryonic collaterogenesis. However, during collateral maturation from embryonic day 18.5 to postnatal day 7, reduced mural cell investment was observed and excessive pruning of collaterals occurred. Growth in collateral diameter was reduced. This resulted in 50% fewer collaterals of smaller diameter in the adult and thus larger infarct volume after middle cerebral artery occlusion. During collateral maturation, CLIC4 deficiency resulted in reduced expression of Vegfr2, Vegfr1, Vegfc, and mural cell markers, but not notch-pathway genes. Overexpression of VEGF-A in Clic4(-/-) mice had no effect on collaterogenesis, but rescued the above defects in collateral maturation by preventing mural cell loss and collateral pruning, thus restoring collateral number and diameter and reducing stroke severity in the adult. CLIC4 is not required for collaterogenesis but is essential for perinatal maturation of nascent collaterals through a mechanism that supports VEGF signaling.

Cardiovascular risk factors cause premature rarefaction of the collateral circulation and greater ischemic tissue injury.

Collaterals lessen tissue injury in occlusive disease. However, aging causes progressive decline in their number and smaller diameters in those that remain (collateral rarefaction), beginning at 16 months of age in mice (i.e., middle age), and worse ischemic injury-effects that are accelerated in even 3-month-old eNOS(-/-) mice. These findings have found indirect support in recent human studies. We sought to determine whether other cardiovascular risk factors (CVRFs) associated with endothelial dysfunction cause collateral rarefaction, investigate possible mechanisms, and test strategies for prevention. Mice with nine different models of CVRFs of 4-12 months of age were assessed for number and diameter of native collaterals in skeletal muscle and brain and for collateral-dependent perfusion and ischemic injury after arterial occlusion. Hypertension caused collateral rarefaction whose severity increased with duration and level of hypertension, accompanied by greater hindlimb ischemia and cerebral infarct volume. Chronic treatment of wild-type mice with L-N (G)-nitro-arginine methylester caused similar rarefaction and worse ischemic injury which were not prevented by lowering arterial pressure with hydralazine. Metabolic syndrome, hypercholesterolemia, diabetes mellitus, and obesity also caused collateral rarefaction. Neither chronic statin treatment nor exercise training lessened hypertension-induced rarefaction. Chronic CVRF presence caused collateral rarefaction and worse ischemic injury, even at relatively young ages. Rarefaction was associated with increased proliferation rate of collateral endothelial cells, effects that may promote accelerated endothelial cell senescence.

Poststroke angiogenesis: blood, bloom, or brood?

Angiogenesis, the formation of new capillaries from existing blood vessels, is a naturally occurring phenomenon in adult humans, encompassing a range of normal functions from the physiological maturation of the female reproductive system to wound healing, as well as pathological functions, such as aiding unwanted tumor growth. Although ample evidence suggests that angiogenesis occurs in humans and laboratory animals after brain ischemia,1,2 it has long been debated whether angiogenesis contributes to the recovery of function after stroke and whether it can be harnessed as a therapy to treat stroke. The pro and con opinions of poststroke angiogenesis have been eloquently and pertinently discussed by Dr Greenberg, who attests to the benefit of angiogenesis and the potential for clinical application and by Drs Adamczak and Hoehn, who address the risk of angiogenesis and potential consequences in aggravating stroke progression. In light of the recent …

Abstract 115: Genetic Variation in Retinal Vascular Patterning Predicts Variation in Pial Collateral Extent and Infarct Volume After Middle Cerebral Artery Occlusion

Introduction: The presence of a native (pre-existing) collateral circulation in tissues lessens injury in stroke and other occlusive diseases. However, differences in genetic background are accompanied by wide variation in the number and diameter (extent) of native collaterals in mice, resulting in large variation in protection. Indirect evidence suggests a similar wide variation also exists in humans. However, methods of measurement in humans are indirect, invasive and not widely available. Hypothesis: We sought to determine if differences in genetic background in mice result in variation in branch-patterning of the retinal circulation, and if these differences predict differences in collateral extent and, in turn, differences in severity of ischemic stroke. Methods: Patterning metrics were obtained for the retinal arterial trees of 10 mouse strains (n=8 per strain) that differ widely in collateral extent in brain and other tissues. We also obtained pial collateral number and diameter, and infarct volume 24h after permanent middle cerebral artery occlusion. Forward- and reverse-stepwise multivariate regression analysis was conducted and model performance assessed using K-fold cross-validation. Results: Twenty-one metrics varied significantly with genetic strain (p<0.01). Ten metrics (eg, vessel caliber, bifurcation angle, lacunarity, optimality, branch length) strongly predicted collateral number and diameter across 7 regression models. The best models closely predicted (p<0.0001) collateral number (K-fold R2 =0.83-0.98), diameter (0.73-0.88) and infarct volume (0.85-0.87). Conclusions: Differences in retinal tree patterning are specified by genetic background and closely predict genetic variation in pial collateral extent and, in turn, stroke severity. If these findings can be confirmed in humans, and given that genetic variation in cerebral collaterals extends to other tissues at least in mice, a similar “retinal predictor index” could be developed as a biomarker for collateral extent in brain and other tissues. This could aid prediction of the risk-severity of tissue injury in occlusive disease as well as stratification of patients for treatment options and enrollment in clinical studies.

CXCL1 promotes arteriogenesis through enhanced monocyte recruitment into the peri-collateral space.

The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. After femoral artery ligation in Sprague-Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p < 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p < 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of peri-collateral macrophages (p < 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p < 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p < 0.05). CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.

Abstract 11511: Genetic Variation in Retinal Vascular Patterning Predicts Variation in Pial Collateral Extent and Infarct Volume After Middle Cerebral Artery Occlusion

Introduction: The presence of a native (pre-existing) collateral circulation in tissues lessens injury in stroke and other occlusive diseases. However, differences in genetic background are accompanied by wide variation in the number and diameter (extent) of native collaterals in mice, resulting in large variation in protection. Indirect evidence suggests a similar wide variation also exists in humans. However, methods of measurement in humans are indirect, invasive and not widely available. Hypothesis: We sought to determine if differences in genetic background in mice result in variation in branch-patterning of the retinal circulation, and if these differences predict differences in collateral extent and, in turn, differences in severity of ischemic stroke. Methods: Patterning metrics were obtained for the retinal arterial trees of 10 mouse strains (n=8 per strain) that differ widely in collateral extent in brain and other tissues. We also obtained pial collateral number and diameter, and infarct volume 24h after permanent middle cerebral artery occlusion. Forward- and reverse-stepwise multivariate regression analysis was conducted and model performance assessed using K-fold cross-validation. Results: Twenty-one metrics varied significantly with genetic strain (p&lt;0.01). Ten metrics (eg, vessel caliber, bifurcation angle, lacunarity, optimality, branch length) strongly predicted collateral number and diameter across 7 regression models. The best models closely predicted (p&lt;0.0001) collateral number (K-fold R 2 =0.83-0.98), diameter (0.73-0.88) and infarct volume (0.85-0.87). Conclusions: Differences in retinal tree patterning are specified by genetic background and closely predict genetic variation in pial collateral extent and, in turn, stroke severity. If these findings can be confirmed in humans, and given that genetic variation in cerebral collaterals extends to other tissues at least in mice, a similar “retinal predictor index” could be developed as a biomarker for collateral extent in brain and other tissues. This could aid prediction of the risk-severity of tissue injury in occlusive disease as well as stratification of patients for treatment options and enrollment in clinical studies.

A device for performing automated balloon catheter inflation ischemia studies.

Coronary collateral growth (arteriogenesis) is a physiological adaptive response to transient and repetitive occlusion of major coronary arteries in which small arterioles (native collaterals) with minimal to no blood flow remodel into larger conduit arteries capable of supplying adequate perfusion to tissue distal to the site of occlusion. The ability to reliably and reproducibly mimic transient, repetitive coronary artery occlusion (ischemia) in animal models is critical to the development of therapies to restore coronary collateral development in type II diabetes and the metabolic syndrome. Current animal models for repetitive coronary artery occlusion implement a pneumatic occluder (balloon) that is secured onto the surface of the heart with the suture, which is inflated manually, via a catheter connected to syringe, to effect occlusion of the left anterior descending coronary artery (LAD). This method, although effective, presents complications in terms of reproducibility and practicality. To address these limitations, we have designed a device for automated, transient inflation of balloon catheters in coronary artery occlusion models. This device allows repeated, consistent inflation (to either specified pressure or volume) and the capability for implementing very complex, month-long protocols. This system has significantly increased the reproducibility of coronary collateral growth studies in our laboratory, resulting in a significant decrease in the numbers of animals needed to complete each study while relieving laboratory personnel from the burden of extra working hours and enabling us to continue studies over periods when we previously could not. In this paper, we present all details necessary for construction and operation of the inflator. In addition, all of the components for this device are commercially available and economical (Table S1). It is our hope that the adoption of automated balloon catheter inflation protocols will improve the experimental reliability of transient ischemia studies at many research institutions.

Smooth muscle‐dependent vasodilation is impaired in immature arterialized collateral capillaries (677.17)

Native collateral vessels creates a natural bypass for blood flow around an arterial occlusion, potentially mitigating the effects of ischemic diseases, such as peripheral arterial occlusive disease in which arterial stenosis restricts blood flow to the limbs. Unfortunately, not all patients have robust collateral networks; however, animal models lacking native collaterals demonstrate the ability to adapt to the ischemic environment through arterialization of collateral capillaries, reperfusing the ischemic zone. Reactivity is important for normal muscle function in these developed vessels because increased metabolic demand is only satisfied when blood vessels can dilate to increase flow. Understanding cellular mechanisms behind impaired vasodilation is key to identifying potential therapeutic targets for improving reactivity. To assess vascular reactivity, the lateral cranial spinotrapezius feed artery was ligated 7 days prior to the application of endothelial‐dependent (acetylcholine, ACh) and endothelial‐independent (sodium nitroprusside, SNP) vasodilators. Although arterialized capillaries dilated significantly in response to ACh and SNP, dilation was impaired when compared to equivalent‐diameter terminal arterials on the sham side (19.0 ± 3.6% vs. 69.5 ± 14.0% and 20.0 ± 4.9% vs. 95.7 ± 9.2%, respectively). This impairment can be attributed to smooth muscle cell dysfunction because the responses to ACh and SNP are similar. To determine if other smooth muscle‐dependent vasodilatory pathways are also impaired, hydrogen sulfide and papaverine were applied using the same methods (31.3 ± 16.6% vs. 97.1 ± 16.2% and 49.0 ± 8.8% vs. 86.3 ± 6.1%). Future work will further characterize the signaling cascades described herein.

Genetic determinants of the native collateral circulation (19.3)

Native (pre‐existing) collaterals connect adjacent arterial trees and limit ischemic tissue injury. I will review recent findings that collaterals have distinct features, including a unique process of formation and specialized endothelial and smooth muscle cell phenotypes. Moreover, the number and diameter of collaterals (“extent”) in tissues varies widely among different mouse strains and healthy humans, associated with wide differences in the severity of ischemic damage. A single locus, Dce1, is responsible for ~80% of the variation in mouse. Ongoing work is seeking to identify the underlying gene(s) and pathway, and whether this locus links to variation in collateral extent in humans. If so, Dce1 and its up‐ and down‐stream partners could provide markers for collateral abundance to help stratify patients for treatment options, to assess risk‐severity before disease occurs, and to provide targets for therapies to stimulate new collateral formation. Recent studies in mice have found that such “neo‐collateral” formation can be induced to occur rapidly after acute MI, resulting in increased collateral‐dependent flow and reduced infarct volume. Besides genetic factors, “environmental “ factors, ie, aging and other cardiovascular risk factors, cause reduced collateral extent, resulting in more severe ischemic injury. Studies are under way to identify the underlying mechanism and strategies to prevent it.

Abstract 529: A Chloride Intracellular Channel-4 (Clic4) and VEGF-A Signaling Pathway Regulates Maturation of the Collateral Circulation

The density of native (pre-existing) cerebral collaterals in healthy tissues varies widely and is a major determinant of the wide variation in infarct volume in ischemic stroke. Collateral number in adults has been shown to be dependent on 3 processes: formation in the embryo, maturation in the perinate (i.e. slight pruning, increase in diameterand muralization) and maintenance thereafter. However, the mechanisms underlying these processes are not well understood. We previously found that collaterogenesis is led by a unique “arteriolar” angiogenic sprouting-like mechanism involving the VEGF-Notch signaling pathway. We also observed that mice lacking chloride intracellular protein 4 (Clic4), a protein which appears to reside upstream of VEGF-A in hypoxia-induced signaling, had fewer pial collaterals on postnatal day (P)1 that underwent greater pruning by P21, to yield lower collateral number and more severe stroke in adults. Therefore, herein we hypothesized that Clic4 is required for both formation and maturation of the collateral circulation. Wildtype and Clic4 -/- mice formed similar numbers of collaterals through embryonic day (E)18.5. Thus, collaterogenesis does not require Clic4. However, perinatal pruning was strongly augmented in Clic4 -/- mice, resulting in reduced collateral number and larger infarct volume in adults after MCA occlusion. Pial diameter was also reduced, consistent with our previous findings. Overexpression of VEGF-A in Clic4 -/- mice by crossing them to Vegf hi/+ mice did not affect collaterogenesis but inhibited the excessive perinatal pruning, resulting in adults with the abundant collaterals and small infarct volume seen in wildtype mice. We conclude that Clic4 is not required for embryonic collaterogenesis but is essential for perinatal stabilization of nascent collaterals through a mechanism involving VEGF signaling.

Formation of the Collateral Circulation Is Regulated by Vascular Endothelial Growth Factor-A and A Disintegrin and Metalloprotease Family Members 10 and 17

The density of native (preexisting) collaterals varies widely and is a significant determinant of variation in severity of stroke, myocardial infarction, and peripheral artery disease. However, little is known about mechanisms responsible for formation of the collateral circulation in healthy tissues. We previously found that variation in vascular endothelial growth factor (VEGF) expression causes differences in collateral density of newborn and adult mice. Herein, we sought to determine mechanisms of collaterogenesis in the embryo and the role of VEGF in this process. Pial collaterals begin forming between embryonic day 13.5 and 14.5 as sprout-like extensions from arterioles of existing cerebral artery trees. Global VEGF-A overexpressing mice (Vegf(hi/+)) formed more, and Vegf(lo/+) formed fewer, collaterals during embryogenesis, in association with differences in vascular patterning. Conditional global reduction of Vegf or Flk1 only during collaterogenesis significantly reduced collateral formation, but now without affecting vascular patterning, and the effects remained in adulthood. Endothelial-specific Vegf reduction had no effect on collaterogenesis. Endothelial-specific reduction of a disintegrin-and-metalloprotease-domain-10 (Adam10) and inhibition of γ-secretase increased collateral formation, consistent with their roles in VEGF-induced Notch1 activation and suppression of prosprouting signals. Endothelial-specific knockdown of Adam17 reduced collateral formation, consistent with its roles in endothelial cell migration and embryonic vascular stabilization, but not in activation of ligand-bound Notch1. These effects also remained in adulthood. Formation of pial collaterals occurs during a narrow developmental window via a sprouting angiogenesis-like mechanism, requires paracrine VEGF stimulation of fetal liver kinase 1-Notch signaling, and adult collateral number is dependent on embryonic collaterogenesis.

Abstract 395: Formation of New Coronary Collaterals After Acute Myocardial Infarction in Mouse

Variation in extent of the native pre-existing collateral (COL) circulation, ie, COL number and average diameter in healthy tissues, is a major determinant of the variation in severity of tissue injury after acute obstruction. Indirect evidence suggests that coronary COL extent varies widely in presumed healthy humans. Although the cause of this variation is unknown, studies in non-heart tissues of healthy mouse strains have found that differences in genetic background are a major determinant. Whether this applies to the heart is unknown, because no model exists to investigate the coronary COL circulation in mice. In addition, little is known regarding whether new COLs can be induced to form in individuals with few native COLs. To address these questions, we developed a mouse model that combines high-resolution (capillary-level) microangiography and ligation (LADX) with measurement of coronary COL conductance index and infarct volume. Adult (3-mos-old) mouse strains chosen for their large rank-order difference in native COL extent in brain and hindlimb-BLKS &gt; C57BL/6 &gt; A &gt; BALB/c-all lack native coronary COLs. After LADX, all strains formed new COLs by day-3 that reached maximum number and diameter by day-14. However, rank-order for new COL formation differed from the above, ie, C57BL/6 @ BALB/c &gt; BLKS &gt; A; COL conductance and infarct volume -1 followed this same rank-order. Moreover, the rank-order for new COL formation differed from the rank-order for COL remodeling after arterial ligation in brain and hindlimb, ie, A &gt; C57BL/6 &gt; BALB/c. Ongoing studies to determine the mechanisms of new COL formation after LADX find strongly reduced COL formation and conductance and increased infarct volume in C57BL/6 mice deficient in monocyte chemoattractant protein-1 or its receptor (CCR2)-effects partially rescued by bone marrow transplantation. Conclusions: Unlike other tissues, the mouse heart lacks native COLs. Mice thus provide a model to study coronary collaterogenesis free of complications posed by remodeling of pre-existing COLs. Gene polymorphisms underlying variation in ischemic coronary collaterogenesis differ from those underlying variation in embryonic formation of native COLs. MCP-1 and bone marrow cells play an important role in new COL formation.

Abstract 390: Cardiovascular Risk Factors and Endothelial Dysfunction Cause Rarefaction of the Collateral Circulation and Impaired Recovery from Ischemia

Aging and cardiovascular risk factors (CVRF) such as diabetes, hypertension, dyslipidemia and metabolic syndrome have been associated with collateral insufficiency in animals and humans. Long-standing CVRF as well as collateral insufficiency associate with poorer outcomes following acute ischemic stroke or myocardial infarction. Evidence suggests that endothelial dysfunction (decreased eNOS activity and NO bioavailability) is caused by and contributes to CVRF, and promotes atherosclerosis and thrombosis in patients with CVRF. Aging and eNOS deficiency have been shown to cause rarefaction of native (pre-existing) cerebral and hindlimb collaterals in mice and to reduce collateral remodeling in response to arterial occlusion. To determine whether endothelial dysfunction and CVRF cause collateral rarefaction and/or impair remodeling, we examined recovery of perfusion following femoral artery ligation (FAL) and native cerebral pial collateral extent (density and diameter) in the following mouse models (all 7-8 mos-age): chronic L-NAME treatment (endothelial dysfunction), ob/ob (obesity), LDLR -/- on high-fat diet (hyperlipidemia), LDLR -/- ; ob/ob (metabolic syndrome), RenTgMK +/- (hypertension), and Akita (type-1 diabetes). Hindlimb perfusion immediately after FAL (dependent on native collateral extent) and recovery over 21 days (dependent on collateral remodeling) were impaired by chronic endothelial dysfunction, hypertension, metabolic syndrome, and diabetes (p&lt;0.05) but not by obesity and hyperlipidemia. Similarly, native pial collateral extent was significantly reduced in models of endothelial dysfunction, hypertension, metabolic syndrome and diabetes. The functional significance of CVRF-induced collateral rarefaction was evident as more severe ischemic injury and use impairment in hindlimb and larger infarction in brain after middle cerebral artery occlusion. Investigations into the mechanisms of CVRF-induced collateral rarefaction and treatments to prevent it are ongoing. These data show that CVRF not only impair collateral remodeling but also cause significant loss of native collaterals, resulting in worse ischemic tissue injury after acute obstruction and fewer collaterals available for remodeling.