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Keywords:

  • CCM;
  • hemostasis;
  • neurovascular guidance;
  • Robo4;
  • vascular stability

Abstract

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

Summary.  Neural guidance cues are essential for a growing axon to correctly course through the body and innervate target tissues. Interestingly, the vascular network follows a parallel trajectory along nerves, suggesting that guidance cues important for neural patterning may also be required for proper vascular patterning. However, while an axon arises from one cell, a blood vessel is composed of many endothelial cells. Recent evidence suggests that neural repulsive cues are usurped by multi-cellular blood vessels to ensure vascular stabilization cues. Additional clues into the signaling mechanisms that promote vascular stabilization are emerging from cerebral cavernous malformations, a disease characterized by headache, epilepsy, and stroke. Thus, neurobiology and neurology are providing insights into the concepts of vascular stability.


Mechanisms of neurovascular guidance

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

The nervous system provides a functional network whereby information can be shared between the brain and the rest of the body. For a proper network to be established, axons must navigate considerable distances to reach their target destination. The growth cone located at the tip of the axon is responsible for coordinating this directed migration [1]. This is achieved through the reaction of the growth cone to positive and negative stimuli, resulting in successful migration to target tissues and proper formation of the neural network.

The vascular system also forms a functional network that can be found throughout the body. This system of tubes permits blood flow, allowing the delivery of nutrients to target tissues. Much of the vascular plexus is formed by angiogenesis, or new blood vessel growth from pre-existing vessels. Interestingly, the vascular network follows a parallel trajectory along nerves, suggesting that guidance mechanisms that are important for neural patterning may also be required for proper vascular patterning. At the leading edge of a sprouting blood vessel is the tip cell, the endothelial analogue to the neural growth cone [2]. Like the growth cone, the tip cell uses filipodial extensions to sense the environment, react to positive and negative stimuli, and navigate the proper path of growth. A key difference, however, is that while the axon arises from one cell, a blood vessel is composed of many endothelial cells requiring orchestrated movements of not only the tip cells, but also the endothelial cells trailing behind the navigating tip cell called stalk cells. Inter-endothelial junctions, such as those formed by vascular endothelial cadherin (VE-cadherin), must be established between endothelial stalk cells to maintain vascular stability, thus further distinguishing the blood vessel from the growing axon.

Slit–Robo as a repulsive neural guidance signaling system

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

Slits are one family of neural guidance cues known for repulsive activity. This family of ligands is composed of three members, Slit1–3, and are large, secreted extracellular matrix proteins. Slits act through Roundabout (Robo) receptors. In fact, Robo was actually first discovered from a screen for mutations resulting in axon guidance defects in Drosophila [3]. In robo mutants, too many axons cross and re-cross the midline, demonstrating that robo signaling provides a repulsive cue to prevent misguided axon entry into the midline. Similarly, the growth cones of slit mutants enter the midline but never leave [4]. Embryos transheterozygous for slit and robo showed inappropriate axon crossing of the midline, indicating that Slit and Robo genetically function in the same pathway. Furthermore, two additional Robo family members have been discovered to affect axon guidance in the nervous system.

Robo4 stabilizes the vasculature

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

A fourth member of the Robo family termed Robo4, or Magic Roundabout, is unique in that its expression is endothelial-specific. Since Robo signaling in the nervous system has been demonstrated to be important in neural guidance, we hypothesized that a vascular specific Robo may be a key mechanism regulating vascular guidance. To investigate whether Robo4 is necessary for vascular guidance in vivo, we developed Robo4-null (Robo4−/−) mice. We anticipated, on the basis of this hypothesis, that removal of Robo4 expression would be lethal due to extreme defects in vascular guidance. To our surprise, these mice were viable, fertile, and demonstrated no obvious vascular patterning defects in several vascular beds [5]. To understand why Robo4 played no role in vascular guidance in vivo, we hypothesized that perhaps Robo4 expression was not found in the endothelial tip cell. Using the retinal vascular bed, we found that Robo4 expression was largely absent from tip cells, but strong expression was found in the stalk cells. This cell-specific expression led us to ask whether the function of Robo4 in vivo was that of stalk cells, that is, to strengthen vascular stability.

One aspect of vascular stabilization is the maintenance of essential inter-endothelial junctions to stabilize barrier function. Vascular barrier function can be modeled in vitro using a transwell system to assess flux of a reporter across an endothelial monolayer. Using lung endothelial cells isolated from Robo4+/+ mice, we found that Slit2 could significantly inhibit vascular endothelial growth factor (VEGF) – induced permeability. When this experiment was performed using lung endothelial cells isolated from Robo4−/− mice, the effect of Slit2 was lost, demonstrating that Robo4 is necessary for the effect of Slit2. A major effector of VEGF-induced permeability is Src kinase. In fact, Src is necessary for VEGF-induced permeability as the permeability-inducing effect of VEGF is lost in Src−/− mice [6]. We found that Slit2 could inhibit VEGF-induced Src activation, demonstrating that Slit impinges upon the Src signaling axis [5]. Next, we assessed whether Slit2 could stabilize the vasculature in vivo in the mouse dermis and retina. Using Evans Blue as a tracer for permeability, we found that Slit2 inhibited VEGF-induced permeability in the mouse dermis and retina. Again, the effect of Slit was lost in Robo4−/− mice, demonstrating that Robo4 is necessary for the effect of Slit2. Furthermore, Slit3 also inhibited VEGF-induced permeability in the mouse retina through a Robo4-dependent mechanism. This demonstrates that enhancement of the vascular barrier applies to multiple members of the Slit family.

A stabilized phenotype not only applies to the regulation of vascular barrier function, but also to the inhibition of new vascular sprouting and angiogenesis. Pro-angiogenic factors such as VEGF induce angiogenesis through enhancing vascular barrier destabilization, endothelial proliferation, and migration. While Slit2 had no effect on VEGF-induced endothelial cell proliferation, Slit2 did inhibit VEGF-induced endothelial cell migration and tube formation in vitro. We next asked whether Slit2 could inhibit neovascularization in a pathologic setting in vivo. To answer this question, we turned to oxygen-induced retinopathy (OIR), a mouse model of proliferative diabetic retinopathy. In this model, young mice are placed in a hyper-oxygen environment for several days resulting in excessive vascular pruning. When mice are placed at room oxygen, a perceived oxygen deficit in the mouse retina causes a massive release of VEGF. Using this model, Slit2 did in fact significantly inhibit neovascular tuft formation. Furthermore, when these experiments were repeated in Robo4−/− mice, not only was Slit2 ineffective, but Robo4−/− mice demonstrated a marked increase in neovascularization as compared to Robo4+/+ mice. Taken together, these data demonstrate a role for Slit/Robo4 signaling in maintaining vascular stability through maintaining vascular barrier function and inhibiting pathologic neovascularization. Furthermore, these collective data may explain why a repulsive neural cue could translate into a vascular stabilization cue when applied to the multi-cellular vascular bed.

Emerging mechanisms of maintaining vascular stability

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

Apart from the Src signaling axis upon which Slit–Robo impinges, recent evidence points to additional intracellular cell signaling systems that are important for maintaining vascular stability. Cerebral cavernous malformations (CCM) are common vascular malformations largely affecting the central nervous system. A frequent finding with these malformations is the presence of hemosiderin, a blood breakdown product. This finding is suggestive of a compromised endothelial barrier, possibly with a concurrent defect in platelet function or coagulation. Indeed, abnormal cell–cell junctions have been observed by ultrastructural analysis [7]. One gene that has been linked to the formation of CCM is osmosensing scaffold for mitogen-activated protein kinase-3 (OSM) also known as CCM2. Recent studies conducted by Whitehead et al. [8] have now demonstrated how this intracellular adaptor protein regulates vascular stability.

To understand the functional importance of CCM2 in the vasculature during development, Whitehead et al. made mice with an endothelial-specific deletion of CCM2. This mutation resulted in embryonic lethality due to lumenization defects in both the branchial arch artery and the aorta, demonstrating that CCM2 is required for lumen formation in vivo. Lumen formation can be modeled in vitro by three-dimensional culture. Using this system, endothelial cells form vacuoles that coalesce into tube-like structures. While endothelial cells treated with control siRNA formed a robust tube network, siRNA knockdown of CCM2 caused endothelial cells to form smaller lumens and decreased tube network area. These in vitro and in vivo data demonstrate that CCM2 plays a necessary role in the generation of vacuoles and the resulting vascular lumen formation.

It has been demonstrated that the cellular cytoskeleton plays a role in lumen formation [9]. To understand whether CCM2 could alter lumen formation through an effect on the cytoskeletal architecture, we performed immunofluorescence on endothelial cells lacking CCM2 expression. Indeed, endothelial cells deficient in CCM2 expression showed a marked increase in stress fiber formation. As stress fiber formation is controlled by Rho, this result was suggestive of overactive Rho signaling. When CCM2 knockdown cells were treated with a Rho inhibitor, the stress fiber phenotype was rescued. Rho signaling has also been demonstrated to play a role in vascular permeability. As previously mentioned, CCMs are characterized by hemosiderin deposits, indicative of vascular leak. Endothelial monolayers deficient in CCM2 expression demonstrated decreased electrical resistance and increased flux of macromolecules compared to control cells, indicating enhanced permeability in vitro [8]. Furthermore, this phenotype was rescued by a Rho inhibitor, further implicating the proper regulation of Rho as a key function of the CCM2 pathway.

While Ccm2-null mice are susceptible to severe developmental angiogenic defects, humans that suffer from CCM are heterozygous. We reasoned that perhaps in a clinical setting, physiologic or genetic stressors may precipitate disease pathogenesis in these heterozygous patients. To test this in mice, we injected VEGF into the dermis of Ccm2+/+ and Ccm2+/− mice. Using Evans Blue as a tracer for vascular leak, we observed that VEGF induced exaggerated vascular hyperpermeability in Ccm2+/− mice. Next, we tested whether inhibition of Rho could block the exaggerated hyperpermeability. Statins are widely used to treat patients with high blood pressure. In addition to inhibiting cholesterol synthesis, these drugs also inhibit Rho activation. By treating Ccm2+/− mice with simvastatin, we observed a significant reduction in the response of Ccm2 heterozygous mice to VEGF. These data not only demonstrate the role that CCM2 plays in maintaining correct levels of Rho activity, but also suggest that statins may be used as a therapeutic to treat patients with CCM.

With this recent discovery, the question remains, do any ligand/receptor systems utilize CCM2 as part of a signaling mechanism? Recent data from Kleaveland et al. [10] suggest that heart of glass (HEG) participates in CCM signaling. Interestingly, Heg−/− mice demonstrated severely shortened endothelial gaps and junctions between endothelial cells. Additionally, Heg−/−; CCM2+/− mice demonstrated many of the same phenotypes as a CCM2−/− mouse such as embryonic lethality and failure to form a lumen in the first branchial arch artery. These results show that HEG and CCM2 interact genetically. Furthermore, HEG binds to CCM1 and it is known that CCM1 binds to CCM2 [11]. Thus, input from HEG could be important for modulating the CCM2 pathway.

Questions remain

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

As neurobiology has informed vascular biology, so now both fields can be translated into hemostasis and thrombosis. The nexus of the Slit/Robo signaling pathways and the mechanisms behind the pathogenesis of CCMs lies in the stabilization and destabilization of cytoskeletal structures within the cell. Cytoskeletal changes in platelets are reminiscent of those involved in axon guidance and endothelial cell migration in the developing vasculature. Similarities among these cells beg the question as to whether cytoskeletal rearrangements of platelets, such as those needed for spreading, granule release and aggregation, are regulated by the same mechanisms that direct axon guidance and vascular development. The idea that molecules once thought to be primarily involved in directing axon growth are also involved in the development and function of other tissues is a rapidly emerging field [5,12]. Each of the four classes of molecules originally thought to function primarily in axon guidance (Slits, Netrins, Semaphorins and Ephrins) has been implicated in vascular and cardiac development and more recently in immune system function [12,13].

Though there have been no reports implicating a role for Slit/Robo or CCM signaling pathway in platelets, it is known that platelet function is dependent on the same cytoskeletal modulators that control these two signaling pathways. Additionally, two neural guidance cues (Semaphorins and Ephrins) have been reported to function in platelets by altering cytoskeletal remodeling in a manner similar to their respective function in neurons [14]. The question remains, which of these vascular stability mechanisms will also prove critical in the cytoskeletal stability of platelets and thus be the next target of anti or pro thrombotic drug therapy?

In this review, we have highlighted how a repulsive neural cue can translate into a vascular stabilization cue when applied to the multi-cellular vascular bed. Furthermore, it seems clear that angiogenesis, permeability and endothelial activation during inflammation are controlled by similar mechanisms including regulation of VE-cadherin function by Src [15]. The relationship between endothelial instability/stability and activation/quiescence is another emerging issue in the field of vascular biology. Is there a relationship between the concept of endothelial tip cells/vascular instability and endothelial activation? Will factors that stabilize the vasculature also inhibit endothelial activation? These signaling mechanisms may be the coalescence of vascular biology, immunology and thrombosis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

This work was funded by grants from the National Institutes of Health, Ruth L. Kirschstein National Research Service Award (N.R.L.); T-32 Hematology Training Grant (M.C.P.S); NHLBI, American Heart Association, Juvenile Diabetes Research Foundation, HA and Edna Benning Foundation, and the Burroughs Wellcome Foundation (D.Y.L.).

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References

N.R.L., M.C.P.S, and D.Y.L. are employed by the University of Utah, which has filed intellectual property surrounding the therapeutic uses of targeting Robo4 and CCM2 with the intent to license this body of intellectual property for commercialization.

References

  1. Top of page
  2. Abstract
  3. Mechanisms of neurovascular guidance
  4. Slit–Robo as a repulsive neural guidance signaling system
  5. Robo4 stabilizes the vasculature
  6. Emerging mechanisms of maintaining vascular stability
  7. Questions remain
  8. Acknowledgments
  9. Disclosure of Conflict of Interests
  10. References
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