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Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models
Abstract and Figures
Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Delta-like 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic loss- and gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4(+/-) mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4(+/-) mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.
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... Inhibition of Notch signaling can affect tissue perfusion by restricting arterial structure and function, impairing the repair of angiogenesis after ischemia. [28,29]. ...
... Notch signaling limits vascular germination by limiting the formation of endothelial tip cells and maintaining arterial properties that depend on their relationship with VEGF [53]. In particular, the Notch signal is activated in endothelial cells in response to ischemic stimulation and further participates in the formation of collateral networks of ischemic stroke [29,54]. In our research, we found that the NICD, Hes1, Hey1, and related receptors, such as Jag1 and DLL4 protein levels were upregulated in the OGD/R group, and further increased in the group of overexpression of Bmal1. ...
Ischemic stroke is a major public health problem in China. Angiogenesis plays an essential role in the rehabilitation of ischemic brain injury. Recent studies demonstrated that the circadian clock is involved in the process of ischemic stroke, however, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) could increase the stroke severity and impair the ability of angiogenesis in a transient middle cerebral artery occlusion (MCAO) rat model, by detecting the rat’s infarct volume, neurological test, and western blot of angiogenesis-related protein, including vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2). We further report that the core circadian clock, aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1, also known as ARNTL1), plays an irreplaceable role in angiogenesis. Overexpression of BMAL1 promotes tube forming, migration, and wound healing capacity, and upregulate the VEGF and Notch pathway protein level. While this kind of promoting effect is reversed by the Notch pathway inhibitor, DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which BMAL1 regulates angiogenesis through the VEGF-Notch1 pathway.
... Dll4 and Notch are best known for their role in regulating sprouting angiogenesis downstream of VEGF signaling. Genetically or pharmacologically interfering with Dll4-Notch promoted formation of pre-existing collateral in part by enhancing arteriolar branching during late embryonic development [49]. However, in arterial occlusion models, the ischemic outcome did not improve. ...
... However, in arterial occlusion models, the ischemic outcome did not improve. This was attributed to a defect in capillary functionality, vessel leakage, and impaired flow-induced outward remodeling of arterioles [49]. Perhaps a more physiological-non invasive-way to promote VEGF-Kdrl signaling is to reduce the ambient oxygen tension by moving from sea level to high altitude. ...
The structure of arterial networks is optimized to allow efficient flow delivery to metabolically active tissues. Optimization of flow delivery is a continuous process involving synchronization of the structure and function of the microcirculation with the upstream arterial network. Risk factors for ischemic cardiovascular diseases, such as diabetes mellitus and hyperlipidemia, adversely affect endothelial function, induce capillary regression, and disrupt the micro- to macrocirculation cross-talk. We provide evidence showing that this loss of synchronization reduces arterial collateral network recruitment upon arterial stenosis, and the long-term clinical outcome of current revascularization strategies in these patient cohorts. We describe mechanisms and signals contributing to synchronized growth of micro- and macrocirculation in development and upon ischemic challenges in the adult organism and identify potential therapeutic targets. We conclude that a long-term successful revascularization strategy should aim at both removing obstructions in the proximal part of the arterial tree and restoring “bottom-up” vascular communication.
... Furthermore, Cristofaro et al. [39] demonstrated that an increased number of native pial collateral vessels alone without a functional recruitment or dilation of these vessels does not increase flow to ischemic territory following MCAO. Notch ligand Delta-like 4 (Dll4) improves arterial differentiation but inhibits the formation of vascular anastomoses. ...
Ischemic stroke presents a major global economic and public health burden. Although recent advances in available endovascular therapies show improved functional outcome, a good number of stroke patients are either ineligible or do not have access to these treatments. Also, robust collateral flow during acute ischemic stroke independently predicts the success of endovascular therapies and the outcome of stroke. Hence, adjunctive therapies for cerebral blood flow (CBF) enhancement are urgently needed. A very clear overview of the pial collaterals and the role of genetics are presented in this review. We review available evidence and advancement for potential therapies aimed at improving CBF during acute ischemic stroke. We identified heme-free soluble guanylate cyclase activators; Sanguinate, remote ischemic perconditioning; Fasudil, S1P agonists; and stimulation of the sphenopalatine ganglion as promising potential CBF-enhancing therapeutics requiring further investigation. Additionally, we outline and discuss the critical steps required to advance research strategies for clinically translatable CBF-enhancing agents in the context of acute ischemic stroke models.
... Dll4 is a transmembrane ligand of Notch receptors, selectively expressed in arterial and angiogenic tip cells during development. Similarly, Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis (18). DLL4 heterozygous mice show an increased number of pial collaterals compared to littermates, whereas the infarct volume upon MCA occlusion remains unchanged. ...
Ischemic diseases are the leading cause of death and disability worldwide. The main compensatory mechanism by which our body responds to reduced or blocked blood flow caused by ischemia is mediated by collateral vessels. Collaterals are present in many healthy tissues (including brain and heart) and serve as natural bypass vessels, by bridging adjacent arterial trees. This review focuses on: the definition and significance of pial collateral vessels, the described mechanism of pial collateral formation, an overview of molecular players and pathways involved in pial collateral biology and emerging approaches to prevent or mitigate risk factor-associated loss of pial collaterals. Despite their high clinical relevance and recent scientific efforts toward understanding collaterals, much of the fundamental biology of collaterals remains obscure.
Several signaling pathways are activated during hypoxia to promote angiogenesis, leading to endothelial cell patterning, interaction, and downstream signaling. Understanding the mechanistic signaling differences between normoxia and hypoxia can guide therapies to modulate angiogenesis. We present a novel mechanistic model of interacting endothelial cells, including the main pathways involved in angiogenesis. We calibrate and fit the model parameters based on well-established modeling techniques. Our results indicate that the main pathways involved in the patterning of tip and stalk endothelial cells under hypoxia differ, and the time under hypoxia affects how a reaction affects patterning. Interestingly, the interaction of receptors with Neuropilin1 is also relevant for cell patterning. Our simulations under different oxygen concentrations indicate time- and oxygen-availability-dependent responses for the two cells. Following simulations with various stimuli, our model suggests that factors such as period under hypoxia and oxygen availability must be considered for pattern control. This project provides insights into the signaling and patterning of endothelial cells under hypoxia, contributing to studies in the field.
Ischemic stroke is a major public health problem worldwide. Although the circadian clock is involved in the process of ischemic stroke, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model, by measuring the infarct volume, neurological tests, and angiogenesis-related protein. We further report that Bmal1 plays an irreplaceable role in angiogenesis. Overexpression of Bmal1 promoted tube-forming, migration, and wound healing, and upregulated the vascular endothelial growth factor (VEGF) and Notch pathway protein levels. This promoting effect was reversed by the Notch pathway inhibitor DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.
Background and purpose:
Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischemic blood flow. We previously demonstrated that the inhibition of transient receptor potential canonical 6 (TRPC6) channels facilitates contractile differentiation of VSMCs under ischemic stress. We here investigated whether TRPC6 inhibition promotes post-ischemic blood flow recovery through capillary arterialization in vivo.
Experimental approach:
Mice were subjected to hindlimb ischemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischemic left leg to non-ischemic right one. The number and diameter of blood vessels were analyzed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry.
Key results:
Although the post-ischemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production was inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by Pyrazole-2 promoted post-ischemic blood flow recovery in apolipoprotein E-knockout mice.
Conclusion and implications:
Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischemic peripheral blood circulation.
Background and purpose:
Notch1 activation mediated by γ-secretase is critical for angiogenesis. GeneCards database predicted that Caspase-4 (CASP4, with murine ortholog CASP11) interacts with presenilin-1, the catalytic core of γ-secretase. Therefore, we investigated the role of CASP4/11 in angiogenesis.
Experimental approach:
In vivo, we studied the role of Casp11 in several angiogenesis mouse models using Casp11 wild-type and knockout mice. In vitro, we detected the effects of CASP4 on endothelial functions and Notch signaling by depleting or overexpressing CASP4 in human umbilical vein endothelial cells (HUVECs). The functional domain responsible for the binding of CASP4 and presenilin-1 was detected by mutagenesis and co-immunoprecipitation.
Key results:
Casp11 deficiency remarkably impaired adult angiogenesis in ischemic hindlimbs, melanoma xenografts and Matrigel plugs, but not the developmental angiogenesis of retina. Bone marrow transplantation revealed that the pro-angiogenic effect depended on CASP11 derived from non-hematopoietic cells. CASP4 expression was induced by inflammatory factors and CASP4 knockdown decreased cell viability, proliferation, migration and tube formation in HUVECs. Mechanistically, CASP4/11 deficiency increased Notch1 activation in vivo and in vitro, while CASP4 overexpression repressed Notch1 signaling in HUVECs. Moreover, CASP4 knockdown increased γ-secretase activity. γ-Secretase inhibitor DAPT restored the effects of CASP4 siRNA on Notch1 activation and angiogenesis in HUVECs. Notably, the catalytic activity of CASP4/11 was dispensable. Instead, CASP4 directly interacted with presenilin-1 through the caspase recruitment domain (CARD).
Conclusions and implications:
These findings reveal a critical role of CASP4/11 in adult angiogenesis and make this molecule a promising therapeutic target for angiogenesis-related diseases in the future.
Here we genetically and functionally addressed potential pathways of Notch signalling in mediating vascular regeneration in mouse models. We first used transgenic adult mice with either gain‐ or loss‐of‐function Notch signalling in vascular endothelial cells and monitored perfusion in the hindlimb following ischaemia induced by femoral artery ligation. Mice deficient in Notch signalling showed defective perfusion recovery and expansion of collateral arteries. Transcriptomics analysis of arterial endothelial cells in the Notch mutants identified the guidance factor Sema3g as a candidate gene mediating reperfusion downstream of Notch. Studies in the retinal circulation showed the central role of SEMA3G downstream of Notch signalling in the orderly regulation of vascular patterning. These studies in multiple vascular beds show the primacy of Notch signalling and downstream generation of guidance peptides such as SEMA3G in promoting well‐ordered vascular regeneration. image
Key points
Notch signalling is a critical mediator of revascularization. Yet the cellular processes activated during recovery following vascular injury are incompletely understood. Here we used genetic and cellular approaches in two different vascular beds and cultured endothelial cells to address the generalizability of mechanisms.
By utilizing a highly reproducible murine model of hindlimb ischaemia in transgenic mice in which Notch signalling was inhibited at the transcriptional level, we demonstrated the centrality of Notch signalling in perfusion recovery and revascularization.
RNA‐sequencing of Notch mutants identified class 3 Semaphorins regulated by Notch signalling as downstream targets. Studies in retinal vessels and endothelial cells showed an essential role of guidance peptide Sema3g as a modulator of angiogenesis and orderly vascular patterning.
The Notch to Sema3g signalling axis functions as a feedback mechanism to sculpt the growing vasculature in multiple beds.
Pro-angiogenic cell-based therapies constitute an interesting and attractive approach to enhancing post-stroke neurogenesis and decreasing neurological deficit. However, most new stroke-induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single-cell-type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood-derived CD34(+) cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co-administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri-infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri-ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co-administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke.
Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α-deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α-dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α-dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.
In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.
Background:
Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined.
Methods and results:
We used an inhibitor of NFκB activation (IκBαSR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-κB activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-κB activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1α levels, and a marked decrease in δ-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels.
Conclusions:
These findings identify nuclear factor-κB as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-κB achieves this by regulating hypoxia-inducible factor-1α-dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating δ-like ligand 4 expression, which in turn determines the size and complexity of the network.
Rationale:
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.
Objective:
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.
Methods and results:
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.
Conclusions:
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.
Blood vessel remodeling is crucial to the formation of the definitive vasculature, but little is known about the mechanisms controlling this process. We show that Delta-like ligand 4 (Dll4)/Notch pathway regulates vessel regression in normal pathologic conditions. Genetic and pharmacologic inhibition of Dll4/Notch prevented retinal capillary regression in the oxygen-induced retinopathy (OIR) model and during normal development. Deletion of the Notch-regulated ankyrin repeat protein, a negative regulator of the Notch pathway, produced an opposite phenotype. Inhibition of Dll4/Notch reduced vessel occlusion, maintaining blood flow that is essential for survival of microvessels. Dll4/Notch inhibition up-regulated the expression of vasodilators adrenomedullin and suppressed the expression of vasoconstrictor angiotensinogen. Angiotensin II induced rapid nonperfusion and regression of developing retinal capillaries, whereas Ace1 and AT1 inhibitors and adrenomedullin attenuated vasoobliteration in OIR, indicating that both pathways are involved in modulating vessel remodeling. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) did not result in a pervasive loss of retinal capillaries, demonstrating that reduced expression of VEGF-A is not the proximate cause of capillary regression in OIR. Modulation of VEGF-A and Dll4/Notch signaling produced distinct changes in blood vessel morphology and gene expression, indicating that these pathways can have largely independent functions in vascular remodeling.
Notch signaling regulates vascular development. However, the implication of the Notch ligand Delta-like 4 (Dll4) in postischemic angiogenesis remains unclear.
We investigated the role of Dll4/Notch signaling in reparative angiogenesis using a mouse model of ischemia.
We found Dll4 weakly expressed in microvascular endothelial cells of normoperfused muscles. Conversely, Dll4 is upregulated following ischemia and localized at the forefront of sprouting capillaries. We analyzed the effect of inhibiting endogenous Dll4 by intramuscular injection of an adenovirus encoding the soluble form of Dll4 extracellular domain (Ad-sDll4). Dll4 inhibition caused the formation of a disorganized, low-perfused capillary network in ischemic muscles. This structural abnormality was associated to delayed blood flow recovery and muscle hypoxia and degeneration. Analysis of microvasculature at early stages of repair revealed that Dll4 inhibition enhances capillary sprouting in a chaotic fashion and causes excessive leukocyte infiltration of ischemic muscles. Furthermore, Dll4 inhibition potentiated the elevation of the leukocyte chemoattractant CXCL1 (chemokine [C-X-C motif] ligand 1) following ischemia, without altering peripheral blood levels of stromal cell-derived factor-1 and monocyte chemoattractant protein-1. In cultured human monocytes, Dll4 induces the transcription of Notch target gene Hes-1 and inhibits the basal and tumor necrosis factor-alpha-stimulated production of interleukin-8, the human functional homolog of murine CXCL1. The inhibitory effect of Dll4 on interleukin-8 was abolished by DAPT, a Notch inhibitor, or by coculturing activated human monocytes with Ad-sDll4-infected endothelial cells.
Dll4/Notch interaction is essential for proper reparative angiogenesis. Moreover, Dll4/Notch signaling regulates sprouting angiogenesis and coordinates the interaction between inflammation and angiogenesis under ischemic conditions.
The native (pre-existing) collateral circulation minimizes tissue injury if obstructive vascular disease develops. Evidence suggests that large differences in collateral extent exist among healthy individuals, presumably from as-yet unknown genetic and/or environmental factors. Little is known regarding when or how native collaterals form-information needed to identify these factors. We examined collateral development between the middle and anterior cerebral artery trees in BALB/c and C57BL/6 mouse embryos-strains with marked differences in adult collateral density and diameter (85% fewer, 50% smaller in BALB/c). The circulation was dilated, fixed and stained. By E15.5, a "primary collateral plexus" was beginning to form in both strains. By E18.5, plexus vessel number peaked but was 60% less and diameter smaller in BALB/c (P<0.001). Earlier time points were examined to determine if these differences correlated with differences in patterning of the general circulation. At approximately E9.0, the primary capillary plexus was similar between strains, but by E12.5 branching was less and diameter larger in BALB/c (P<0.05). Between E12.5-E18.5-during pial artery tree development-small differences in tree size, branch number and distance between branches did not correlate with the large difference in collaterogenesis. Pruning of nascent collaterals between P1-P21 was comparable in both strains, yielding the adult density, but diameter and tortuosity increased less in BALB/c. Pericyte recruitment to nascent collaterals was comparable, despite lower VEGF-A and PDGF-B expression in BALB/c mice. These findings demonstrate that collaterals form late during vascular development and undergo postnatal maturation and that differences in genetic background have dramatic effects on these processes.