Large differences in collateral blood vessel abundance among individuals arise from multiple genetic variants.

Collateral blood flow varies greatly among humans for reasons that remain unclear, resulting in significant differences in ischemic tissue damage. A similarly large variation has also been found in mice that is caused by genetic background-dependent differences in the extent of collateral formation, termed collaterogenesis-a unique angiogenic process that occurs during development and determines collateral number and diameter in the adult. Previous studies have identified several quantitative trait loci (QTL) linked to this variation. However, understanding has been hampered by the use of closely related inbred strains that do not model the wide genetic variation present in the "outbred" human population. The Collaborative Cross (CC) multiparent mouse genetic reference panel was developed to address this limitation. Herein we measured the number and average diameter of cerebral collaterals in 60 CC strains, their 8 founder strains, 8 F1 crosses of CC strains selected for abundant versus sparse collaterals, and 2 intercross populations created from the latter. Collateral number evidenced 47-fold variation among the 60 CC strains, with 14% having poor, 25% poor-to-intermediate, 47% intermediate-to-good, and 13% good collateral abundance, that was associated with large differences in post-stroke infarct volume. Collateral number in skeletal muscle and intestine of selected high- and low-collateral strains evidenced the same relative abundance as in brain. Genome-wide mapping demonstrated that collateral abundance is a highly polymorphic trait. Subsequent analysis identified: 6 novel QTL circumscribing 28 high-priority candidate genes harboring putative loss-of-function polymorphisms (SNPs) associated with low collateral number; 335 predicted-deleterious SNPs present in their human orthologs; and 32 genes associated with vascular development but lacking protein coding variants. Six additional suggestive QTL (LOD > 4.5) were also identified in CC-wide QTL mapping. This study provides a comprehensive set of candidate genes for future investigations aimed at identifying signaling proteins within the collaterogenesis pathway whose variants potentially underlie genetic-dependent collateral insufficiency in brain and other tissues.

Role of Vascular Smooth Muscle Cell Phenotype Switching in Arteriogenesis.

Arteriogenesis is one of the primary physiological means by which the circulatory collateral system restores blood flow after significant arterial occlusion in peripheral arterial disease patients. Vascular smooth muscle cells (VSMCs) are the predominant cell type in collateral arteries and respond to altered blood flow and inflammatory conditions after an arterial occlusion by switching their phenotype between quiescent contractile and proliferative synthetic states. Maintaining the contractile state of VSMC is required for collateral vascular function to regulate blood vessel tone and blood flow during arteriogenesis, whereas synthetic SMCs are crucial in the growth and remodeling of the collateral media layer to establish more stable conduit arteries. Timely VSMC phenotype switching requires a set of coordinated actions of molecular and cellular mediators to result in an expansive remodeling of collaterals that restores the blood flow effectively into downstream ischemic tissues. This review overviews the role of VSMC phenotypic switching in the physiological arteriogenesis process and how the VSMC phenotype is affected by the primary triggers of arteriogenesis such as blood flow hemodynamic forces and inflammation. Better understanding the role of VSMC phenotype switching during arteriogenesis can identify novel therapeutic strategies to enhance revascularization in peripheral arterial disease.

The impact of native leptomeningeal collateralization on rapid blood flow recruitment following ischemic stroke.

The leptomeningeal collateral status is an independent predictor of stroke outcome. By means of optical coherent tomography angiography to compare two mouse strains with different extent of native leptomeningeal collateralization, we determined the spatiotemporal dynamics of collateral flow and downstream hemodynamics following ischemic stroke. A robust recruitment of leptomeningeal collateral flow was detected immediately after middle cerebral artery (MCA) occlusion in C57BL/6 mice, with continued expansion over the course of seven days. In contrast, little collateral recruitment was seen in Balb/C mice during- and one day after MCAO, which coincided with a greater infarct size and worse functional outcome compared to C57BL/6, despite a slight improvement of cortical perfusion seven days after MCAO. Both strains of mice experienced a reduction of blood flow in the penetrating arterioles (PA) by more than 90% 30-min after dMCAO, although the decrease of PA flow was greater and the recovery was less in the Balb/C mice. Further, Balb/C mice also displayed a prolonged greater heterogeneity of capillary transit time after dMCAO in the MCA territory compared to C57BL/6 mice. Our data suggest that the extent of native leptomeningeal collaterals affects downstream hemodynamics with a long lasting impact in the microvascular bed after cortical stroke.

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.

Background We 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 Results We 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.

Requirement of ß1 integrin for endothelium-dependent vasodilation and collateral formation in hindlimb ischemia.

An acute increase in blood flow triggers flow-mediated dilation (FMD), which is mainly mediated by endothelial nitric oxide synthase (eNOS). A long-term increase in blood flow chronically enlarges the arterial lumen, a process called arteriogenesis. In several common human diseases, these processes are disrupted for as yet unknown reasons. Here, we asked whether ß1 integrin, a mechanosensory protein in endothelial cells, is required for FMD and arteriogenesis in the ischemic hindlimb. Permanent ligation of the femoral artery in C57BL/6 J mice enlarged pre-existing collateral arteries and increased numbers of arterioles in the thigh. In the lower leg, the numbers of capillaries increased. Notably, injection of ß1 integrin-blocking antibody or tamoxifen-induced endothelial cell-specific deletion of the gene for ß1 integrin (Itgb1) inhibited both arteriogenesis and angiogenesis. Using high frequency ultrasound, we demonstrated that ß1 integrin-blocking antibody or endothelial cell-specific depletion of ß1 integrin attenuated FMD of the femoral artery, and blocking of ß1 integrin function did not further decrease FMD in eNOS-deficient mice. Our data suggest that endothelial ß1 integrin is required for both acute and chronic widening of the arterial lumen in response to hindlimb ischemia, potentially via functional interaction with eNOS.

Collateral Vessels Have Unique Endothelial and Smooth Muscle Cell Phenotypes.

Collaterals are unique blood vessels present in the microcirculation of most tissues that, by cross-connecting a small fraction of the outer branches of adjacent arterial trees, provide alternate routes of perfusion. However, collaterals are especially susceptible to rarefaction caused by aging, other vascular risk factors, and mouse models of Alzheimer's disease-a vulnerability attributed to the disturbed hemodynamic environment in the watershed regions where they reside. We examined the hypothesis that endothelial and smooth muscle cells (ECs and SMCs, respectively) of collaterals have specializations, distinct from those of similarly-sized nearby distal-most arterioles (DMAs) that maintain collateral integrity despite their continuous exposure to low and oscillatory/disturbed shear stress, high wall stress, and low blood oxygen. Examination of mouse brain revealed the following: Unlike the pro-inflammatory cobble-stoned morphology of ECs exposed to low/oscillatory shear stress elsewhere in the vasculature, collateral ECs are aligned with the vessel axis. Primary cilia, which sense shear stress, are present, unexpectedly, on ECs of collaterals and DMAs but are less abundant on collaterals. Unlike DMAs, collaterals are continuously invested with SMCs, have increased expression of Pycard, Ki67, Pdgfb, Angpt2, Dll4, Ephrinb2, and eNOS, and maintain expression of Klf2/4. Collaterals lack tortuosity when first formed during development, but tortuosity becomes evident within days after birth, progresses through middle age, and then declines-results consistent with the concept that collateral wall cells have a higher turnover rate than DMAs that favors proliferative senescence and collateral rarefaction. In conclusion, endothelial and SMCs of collaterals have morphologic and functional differences from those of nearby similarly sized arterioles. Future studies are required to determine if they represent specializations that counterbalance the disturbed hemodynamic, pro-inflammatory, and pro-proliferative environment in which collaterals reside and thus mitigate their risk factor-induced rarefaction.

IRF3 and IRF7 mediate neovascularization via inflammatory cytokines.

OBJECTIVE: To elucidate the role of interferon regulatory factor (IRF)3 and IRF7 in neovascularization. METHODS: Unilateral hind limb ischaemia was induced in Irf3-/- , Irf7-/- and C57BL/6 mice by ligation of the left common femoral artery. Post-ischaemic blood flow recovery in the paw was measured with laser Doppler perfusion imaging. Soleus, adductor and gastrocnemius muscles were harvested to investigate angiogenesis and arteriogenesis and inflammation. RESULTS: Post-ischaemic blood flow recovery was decreased in Irf3-/- and Irf7-/- mice compared to C57BL/6 mice at all time points up to and including sacrifice, 28 days after surgery (t28). This was supported by a decrease in angiogenesis and arteriogenesis in soleus and adductor muscles of Irf3-/- and Irf7-/- mice at t28. Furthermore, the number of macrophages around arterioles in adductor muscles was decreased in Irf3-/- and Irf7-/- mice at t28. In addition, mRNA expression levels of pro-inflammatory cytokines (tnfα, il6, ccl2) and growth factor receptor (vegfr2), were decreased in gastrocnemius muscles of Irf3-/- and Irf7-/- mice compared to C57BL/6 mice. CONCLUSION: Deficiency of IRF3 and IRF7 results in impaired post-ischaemic blood flow recovery caused by attenuated angiogenesis and arteriogenesis linked to a lack of inflammatory components in ischaemic tissue. Therefore, IRF3 and IRF7 are essential regulators of neovascularization.

The Embryologic Origin of Vieussens' Ring.

Vieussens' ring is an embryologic remnant that acquires clinical significance as an intercoronary collateral vessel in advanced coronary artery disease. Its origin as a peritruncal structure early in embryologic development, and its association with congenital pulmonary artery fistula, provides a crucial insight into the early stages of the coronary circulation. This review describes the embryologic basis of Vieussens' ring in relation to the formation of the coronary arteries, which explains its location, appearance, and clinical importance.

Genetic and Environmental Contributions to Variation in the Posterior Communicating Collaterals of the Circle of Willis.

Variation in blood flow mediated by the posterior communicating collateral arteries (PComs) contributes to variation in the severity of tissue injury in obstructive disease. Evidence in animals and humans indicates that differences in the extent of PComs, i.e., their anatomic lumen diameter and whether they are present bilaterally, unilaterally, or absent, are a major factor. These differences arise during development since they are present at birth. However, the causal mechanisms are unknown. We used angiography after maximal dilation to examine involvement of genetic, environmental, and stochastic factors. The extent of PComs varied widely among seven genetically diverse strains of mice. Like pial collaterals in the microcirculation, aging and hypertension reduced PCom diameter, while in contrast, obesity, hyperlipidemia, metabolic syndrome, and diabetes mellitus had no effect. Naturally occurring intrauterine growth restriction had no effect on extent of PCom or pial collaterals in the adult. The number and diameter of PComs evidenced much larger apparent stochastic-dependent variation than pial collaterals. In addition, both PComs underwent flow-mediated outward remodeling after unilateral permanent MCA occlusion that varied with genetic background and was greater on the ipsilesional side. These findings indicate that variation in the number and diameter of PCom collateral arteries arises from stochastic factors and naturally occurring genetic variants that differ from those that cause variation in pial collateral arterioles. Environmental factors also contribute: aging and hypertension reduce PCom diameter. Our results suggest possible sources of variation of PComs in humans and provide information relevant when studying mouse models of occlusive cerebrovascular disease.

Mouse models of Alzheimer's disease cause rarefaction of pial collaterals and increased severity of ischemic stroke.

Vascular dysfunction contributes to the progression and severity of Alzheimer's disease (AD). Patients with AD also sustain larger infarctions after ischemic stroke; however, the responsible mechanisms are unknown. Pial collaterals are the primary source of protection in stroke. Unfortunately, natural aging and other vascular risk factors cause a decline in collateral number and diameter (rarefaction) and an increase in stroke severity. Herein, we tested the hypothesis that AD accelerates age-induced collateral rarefaction and examined potential underlying mechanisms. Triple and double transgenic mouse models of AD both sustained collateral rarefaction by 8 months of age, well before the onset of rarefaction caused by aging alone (16 months of age). Rarefaction, which did not progress further at 18 months of age, was accompanied by a twofold increase in infarct volume after MCA occlusion. AD did not induce rarefaction of similarly sized pial arterioles or penetrating arterioles. Rarefaction was minimal and occurred only at 18 months of age in a parenchymal vascular amyloid-beta model of AD. Rarefaction was not associated with amyloid-beta deposition on collaterals or pial arteries, nor was plaque burden or CD11b+ cell density greater in brain underlying the collateral zones versus elsewhere. However, rarefaction was accompanied by increased markers of oxidative stress, inflammation, and aging of collateral endothelial and mural cells. Moreover, rarefaction was lessened by deletion of CX3CR1 and prevented by overexpression of eNOS. These findings demonstrate that mouse models of AD promote rarefaction of pial collaterals and implicate inflammation-induced accelerated aging of collateral wall cells. Strategies that reduce vascular inflammation and/or increase nitric oxide may preserve collateral function.

22q11.2 Deletion Status and Perioperative Outcomes for Tetralogy of Fallot with Pulmonary Atresia and Multiple Aortopulmonary Collateral Vessels.

Deletion of 22q11.2 (del22q11) is associated with adverse outcomes in patients with tetralogy of Fallot (TOF). We sought to investigate its contribution to perioperative outcome in patients with a severe form of TOF characterized by pulmonary atresia (PA) or severe pulmonary stenosis (PS) and major aortopulmonary collateral arteries (MAPCAS). We conducted a retrospective review of patients with TOF/MAPCAS who underwent staged surgical reconstruction between 1995 and 2006. Groups were compared according to 22q11.2 deletion status using t-tests or the Wilcoxon Rank sum test. We included 26 subjects, 24 of whom survived the initial operation. Of those, 21 subjects had known deletion status and constitute the group for this analysis [15 with no deletion present (ND) and 6 del22q11 subjects]. There was no difference with respect to occurrence of palliative procedure prior to initial operation, or to timing of closure of the ventricular septal defect (VSD). Other than higher prevalence of prematurity (50%) in the del22q11 group versus no prematurity in the ND, the groups were comparable in terms of pre-operative characteristics. The intra- and post-operative course outcomes (length of cardiopulmonary bypass, use of vasopressors, duration of intensive care and length of hospital stay, tube-feeding) were also comparable. Although the del22q11 had longer mechanical ventilation than the ND, this difference was not significant [68 h (range 4-251) vs. 45 h (range 3-1005), p = 0.81]. In this detailed comparison of a small patient cohort, 22q11.2 deletion syndrome was not associated with adverse perioperative outcomes in patients with TOF, PA, and MAPCAS when compared to those without 22q11.2 deletion syndrome. These results are relevant to prenatal and neonatal pre-operative counseling and planning.

Coronary collateralization shows sex and racial-ethnic differences in obstructive artery disease patients.

BACKGROUND: Coronary collateral circulation protects cardiac tissues from myocardial infarction damage and decreases sudden cardiac death. So far, it is unclear how coronary collateralization varies by race-ethnicity groups and by sex. METHODS: We assessed 868 patients with obstructive CAD. Patients were assessed for collateral grades based on Rentrop grading system, as well as other covariates. DNA samples were genotyped using the Affymetrix 6.0 genotyping array. To evaluate genetic contributions to collaterals, we performed admixture mapping using logistic regression with estimated local and global ancestry. RESULTS: Overall, 53% of participants had collaterals. We found difference between sex and racial-ethnic groups. Men had higher rates of collaterals than women (P-value = 0.000175). White Hispanics/Latinos showed overall higher rates of collaterals than African Americans and non-Hispanic Whites (59%, 50% and 48%, respectively, P-value = 0.017), and especially higher rates in grade 1 and grade 3 collateralization than the other two populations (P-value = 0.0257). Admixture mapping showed Native American ancestry was associated with the presence of collaterals at a region on chromosome 17 (chr17:35,243,142-41,251,931, ß = 0.55, P-value = 0.000127). African ancestry also showed association with collaterals at a different region on chromosome 17 (chr17: 32,266,966-34,463,323, ß = 0.38, P-value = 0.00072). CONCLUSIONS: In our study, collateralization showed sex and racial-ethnic differences in obstructive CAD patients. We identified two regions on chromosome 17 that were likely to harbor genetic variations that influenced collateralization.

Aldehyde Dehydrogenase 2 (ALDH2) Glu504Lys Polymorphism Affects Collateral Circulation and Short-Term Prognosis of Acute Cerebral Infarction Patients.

BACKGROUND Acute cerebral infarction is a major clinical subtype of ischemic stroke that has become a leading cause of death and disability worldwide. Aldehyde dehydrogenase 2 (ALDH2) is an important oxidative enzyme in alcohol metabolism. The polymorphism of ALDH2 Glu504Lys polymorphism modifies the activity of this enzyme. However, the potential association between the allelic variation of ALDH2 Glu504Lys with collateral circulation and short-term prognosis of acute cerebral infarction remains unclear. MATERIAL AND METHODS A total of 394 patients with acute cerebral infarction were recruited for ALDH2 genotyping using direct sequencing. Cerebrovascular stenosis and collateral circulation were evaluated by digital subtraction angiography (DSA). Short-term prognosis was assessed in accordance with the modified Ranking Scale (mRS). RESULTS We identified 297 as EAS and 394 as IAS. There were more patients with occluded blood vessel in the opened group and far fewer in the unopened group. ALDH2 polymorphism was significantly different among the primary, secondary, and tertiary opened groups. ALDH2 gene Glu504Lys was significantly associated with short-term prognosis. The genotype GA+AA of ALDH2 gene Glu504Lys locus was an independent risk factor of poor 90-day prognosis. CONCLUSIONS ALDH2 Glu504Lys could be a risk factor for collateral circulation and a negative predictor for short-term prognosis in acute cerebral infarction in Han Chinese. ALDH2 Glu504Lys could be a new therapeutic target for patients with acute cerebral infarction.

CCR7-CCL19/CCL21 Axis is Essential for Effective Arteriogenesis in a Murine Model of Hindlimb Ischemia.

BACKGROUND: In order to identify factors that stimulate arteriogenesis after ischemia, we followed gene expression profiles in two extreme models for collateral artery formation over 28 days after hindlimb ischemia, namely "good-responding" C57BL/6 mice and "poor-responding" BALB/c mice. METHODS AND RESULTS: Although BALB/c mice show very poor blood flow recovery after ischemia, most known proarteriogenic genes were upregulated more excessively and for a longer period than in C57BL/6 mice. In clear contrast, chemokine genes Ccl19, Ccl21a, and Ccl21c and the chemokine receptor CCR7 were upregulated in C57BL/6 mice 1 day after hindlimb ischemia, but not in BALB/C mice. CCL19 and CCL21 regulate migration and homing of T lymphocytes via CCR7. When subjecting CCR7-/-/LDLR-/- mice to hindlimb ischemia, we observed a 20% reduction in blood flow recovery compared with that in LDLR-/- mice. Equal numbers of α-smooth muscle actin-positive collateral arteries were found in the adductor muscles of both mouse strains, but collateral diameters were smaller in the CCR7-/-/LDLR-/-. Fluorescence-activated cell sorter analyses showed that numbers of CCR7+ T lymphocytes (both CD4+ and CD8+) were decreased in the spleen and increased in the blood at day 1 after hindlimb ischemia in LDLR-/- mice. At day 1 after hindlimb ischemia, however, numbers of activated CD4+ T lymphocytes were decreased in the draining lymph nodes of LDLR-/- mice compared with CCR7-/-/LDLR-/- mice. CONCLUSIONS: These data show that CCR7-CCL19/CCL21 axis facilitates retention CD4+ T lymphocytes at the site of collateral artery remodeling, which is essential for effective arteriogenesis.

Role of Genetic Variation in Collateral Circulation in the Evolution of Acute Stroke: A Multimodal Magnetic Resonance Imaging Study.

BACKGROUND AND PURPOSE: No studies have determined the effect of differences in pial collateral extent (number and diameter), independent of differences in environmental factors and unknown genetic factors, on severity of stroke. We examined ischemic tissue evolution during acute stroke, as measured by magnetic resonance imaging and histology, by comparing 2 congenic mouse strains with otherwise identical genetic backgrounds but with different alleles of the Determinant of collateral extent-1 (Dce1) genetic locus. We also optimized magnetic resonance perfusion and diffusion-deficit thresholds by using histological measures of ischemic tissue. METHODS: Perfusion, diffusion, and T2-weighted magnetic resonance imaging were performed on collateral-poor (congenic-Bc) and collateral-rich (congenic-B6) mice at 1, 5, and 24 hours after permanent middle cerebral artery occlusion. Magnetic resonance imaging-derived penumbra and ischemic core volumes were confirmed by histology in a subset of mice at 5 and 24 hours after permanent middle cerebral artery occlusion. RESULTS: Although perfusion-deficit volumes were similar between strains 1 hour after permanent middle cerebral artery occlusion, diffusion-deficit volumes were 32% smaller in collateral-rich mice. At 5 hours, collateral-rich mice had markedly restored perfusion patterns showing reduced perfusion-deficit volumes, smaller infarct volumes, and smaller perfusion-diffusion mismatch volumes compared with the collateral-poor mice (P<0.05). At 24 hours, collateral-rich mice had 45% smaller T2-weighted lesion volumes (P<0.005) than collateral-poor mice, with no difference in perfusion-diffusion mismatch volumes because of penumbral death occurring 5 to 24 hours after permanent middle cerebral artery occlusion in collateral-poor mice. CONCLUSIONS: Variation in collateral extent significantly alters infarct volume expansion, transiently affects perfusion and diffusion magnetic resonance imaging signatures, and impacts salvage of ischemic penumbra after stroke onset.

Variants of Rab GTPase-Effector Binding Protein-2 Cause Variation in the Collateral Circulation and Severity of Stroke.

BACKGROUND AND PURPOSE: The extent (number and diameter) of collateral vessels varies widely and is a major determinant, along with arteriogenesis (collateral remodeling), of variation in severity of tissue injury after large artery occlusion. Differences in genetic background underlie the majority of the variation in collateral extent in mice, through alterations in collaterogenesis (embryonic collateral formation). In brain and other tissues, ≈80% of the variation in collateral extent among different mouse strains has been linked to a region on chromosome 7. We recently used congenic (CNG) fine mapping of C57BL/6 (B6, high extent) and BALB/cByJ (BC, low extent) mice to narrow the region to a 737 Kb locus, Dce1. Herein, we report the causal gene. METHODS: We used additional CNG mapping and knockout mice to narrow the number of candidate genes. Subsequent inspection identified a nonsynonymous single nucleotide polymorphism between B6 and BC within Rabep2 (rs33080487). We then created B6 mice with the BC single nucleotide polymorphism at this locus plus 3 other lines for predicted alteration or knockout of Rabep2 using gene editing. RESULTS: The single amino acid change caused by rs33080487 accounted for the difference in collateral extent and infarct volume between B6 and BC mice attributable to Dce1. Mechanistically, variants of Rabep2 altered collaterogenesis during embryogenesis but had no effect on angiogenesis examined in vivo and in vitro. Rabep2 deficiency altered endosome trafficking known to be involved in VEGF-A→VEGFR2 signaling required for collaterogenesis. CONCLUSIONS: Naturally occurring variants of Rabep2 are major determinants of variation in collateral extent and stroke severity in mice.

Arginase inhibition attenuates arteriogenesis and interferes with M2 macrophage accumulation.

l-Arginine is the common substrate for nitric oxide synthases (NOS) and arginase. Whereas the contribution of NOS to collateral artery growth (arteriogenesis) has been demonstrated, the functional role of arginase remains to be elucidated and was topic of the present study. Arteriogenesis was induced in mice by ligation of the femoral artery. Laser Doppler perfusion measurements demonstrated a significant reduction in arteriogenesis in mice treated with the arginase inhibitor nor-NOHA (N(ω)-hydroxy-nor-arginine). Accompanying in vitro results on murine primary arterial endothelial cells and smooth muscle cells revealed that nor-NOHA treatment interfered with cell proliferation and resulted in increased nitrate/nitrite levels, indicative for increased NO production. Immuno-histological analyses on tissue samples demonstrated that nor-NOHA administration caused a significant reduction in M2 macrophage accumulation around growing collateral arteries. Gene expression studies on isolated growing collaterals evidenced that nor-NOHA treatment abolished the differential expression of Icam1 (intercellular adhesion molecule 1). From our data we conclude that arginase activity is essential for arteriogenesis by promoting perivascular M2 macrophage accumulation as well as arterial cell proliferation.

786T/c endothelial nitric oxide synthase gene polymorphism and coronary collateral circulation.

INTRODUCTION: In this study, we investigated the association between -786T/C polymorphism of the endothelial nitric oxide (NOS3) gene in which thymidine is replaced by a cytosine at nucleotide -786 (rs 2070744) and coronary collateral circulation (CCC) in patients with stable coronary artery disease. MATERIALS AND METHODS: 286 patients having a critical stenosis (> 95%) in at least one major epicardial coronary vessel were included in the study. CCC was defined according to the Rentrop classification (R). Patients with R0-1 CCC were included in the poor CCC group and subjects with R2-3 CCC were assigned to the good CCC group. The polymerase chain reaction method was used for genotyping. 152 patients with poor CCC and 134 patients with good CCC were examined. RESULTS: The frequency of cytosine-cytosine (CC) and thymidine-cytosine (TC) genotypes and allele C were higher in the poor CCC group, but the difference did not reach statistical significance. In the dominant model, the frequency of CC+TC vs. thymidine-thymidine (TT) genotypes was significantly higher in the poor CCC group (67.1% vs. 54.5%, respectively; χ²=4.78; p=0.02). In multivariate regression analysis, the dominant model for -786T/C polymorphism of the NOS3 gene remained as an independent correlate of poor CCC. DISCUSSION: -786T/C polymorphism of the NOS3 gene (rs 2070744) may be associated with poor angiogenesis and the development of CCC in stable coronary artery disease.

Genetic lineage tracing discloses arteriogenesis as the main mechanism for collateral growth in the mouse heart.

AIMS: Capillary and arterial endothelial cells share many common molecular markers in both the neonatal and adult hearts. Herein, we aim to establish a genetic tool that distinguishes these two types of vessels in order to determine the cellular mechanism underlying collateral artery formation. METHODS AND RESULTS: Using Apln-GFP and Apln-LacZ reporter mice, we demonstrate that APLN expression is enriched in coronary vascular endothelial cells. However, APLN expression is reduced in coronary arterial endothelial cells. Genetic lineage tracing, using an Apln-CreER mouse line, robustly labelled capillary endothelial cells, but not arterial endothelial cells. We leveraged this differential activity of Apln-CreER to study collateral artery formation following myocardial infarction (MI). In a neonatal heart MI model, we found that Apln-CreER-labelled capillary endothelial cells do not contribute to the large collateral arteries. Instead, these large collateral arteries mainly arise from pre-existing, infrequently labelled coronary arteries, indicative of arteriogenesis. Furthermore, in an adult heart MI model, Apln-CreER activity also distinguishes large and small diameter arteries from capillaries. Lineage tracing in this setting demonstrated that most large and small coronary arteries in the infarcted myocardium and border region are derived not from capillaries, but from pre-existing arteries. CONCLUSION: Apln-CreER-mediated lineage tracing distinguishes capillaries from large arteries, in both the neonatal and adult hearts. Through genetic fate mapping, we demonstrate that pre-existing arteries, but not capillaries, extensively contribute to collateral artery formation following myocardial injury. These results suggest that arteriogenesis is the major mechanism underlying collateral vessel formation.