Coordinated interaction of the vascular and nervous systems: from molecule- to cell-based approaches

Endothelial cells (ECs) build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. Blood vessel/nerve interactions, ultimately, play essential roles for the neurovascular network and brain function. With conventional molecular approaches, such coordinated interaction is likely due to complex interplay of neuroangiogenic factors and receptors. Aside from molecular regulation of neuroangiogenic factors, currently, cell-based approaches to investigate how blood vessels (or nerves) respond to nerves (or blood vessels) appropriately in the pathophysiological situation are gradually emerging. In order to define responsiveness and flexibility of the neurovascular network in response to the local need, the intercellular communication and coordinated interaction between the vascular and nervous systems need to be thought as a working unit. Based on the scale of the working unit which is in the millimeter range with respect to the physical distance of the neurovascular network, we propose to use a rather conceptual term "Millibiology". The millibiological approach for the coordinated interaction might bring us new paradigm to define neurovascular functions in the pathophysiological state.     

Selective requirements for NRP1 ligands during neurovascular patterning

Blood vessels and neurons share several types of guidance cues and cell surface receptors to control their behaviour during embryogenesis. The transmembrane protein NRP1 is present on blood vessels and nerves. NRP1 binds two structurally diverse ligands, the semaphorin SEMA3A and the VEGF164 isoform of vascular endothelial growth factor. SEMA3A was originally identified as a repulsive cue for developing axons that acts by signalling through receptor complexes containing NRP1 and plexins. In vitro, SEMA3A also inhibits integrin function and competes with VEGF164 for binding to NRP1 to modulate the migration of endothelial cells. These observations resulted in a widely accepted model of vascular patterning in which the balance of VEGF164 and SEMA3A determines endothelial cell behaviour. However, we now demonstrate that SEMA3A is not required for angiogenesis in the mouse, which instead is controlled by VEGF164. We find that SEMA3A, but not VEGF164, is required for axon patterning of limb nerves, even though the competition between VEGF164 and SEMA3A for NRP1 affects the migration of neuronal progenitor cells in vitro and has been hypothesised to control axon guidance. Moreover, we show that there is no genetic interaction between SEMA3A and VEGF164 during vasculogenesis, angiogenesis or limb axon patterning, suggesting that ligand competition for NRP1 binding cannot explain neurovascular congruence, as previously suggested. We conclude that NRP1 contributes to both neuronal and vascular patterning by preferentially relaying SEMA3A signals in peripheral axons and VEGF164 signals in blood vessels.     

The developing neurovascular anatomy of the embryo: a technique of simultaneous evaluation using fluorescent labeling, confocal microscopy, and three-dimensional reconstruction.

The close spatial relationship between peripheral nerves and blood vessels in the adult is well known. However, evidence supporting the congruent development of these structures in embryos remains anecdotal. Neurovascular relationships also have been shown to be conserved in other vertebrates. This homology suggests that either peripheral nerves or blood vessels, or both, might have fundamental morphogenetic roles during embryologic development. Both peripheral nerves and blood vessels have been independently implicated as etiologic agents in the pathogenesis of congenital disabilities, and several congenital anomalies fit their distribution patterns. This article presents a technique for the simultaneous visualization of peripheral nerves and blood vessels at different stages in the developing embryo. The forelimbs of 310 quail embryos were dissected over a 1-year period. Peripheral nerves were labeled with the neural crest and axon antibody, HNK-1, followed by fluorescein-conjugated secondary antibodies. Blood vessels were labeled by a perfusion technique using the fluorescent dye, dioctadecyl-tetramethylindocarbocyanine. Specimens were processed and imaged in whole-mount with confocal microscopy, and images were reconstructed using three-dimensional modeling software. Both nerves and blood vessels seem to undergo a highly stereotypic sequence of development in the embryonic quail forelimb. Furthermore, the existence of a close spatial relationship between nerves and blood vessels suggests either a high degree of developmental interdependence or shared patterning mechanisms. This technique permits further evaluation of the possible role peripheral nerves and blood vessels might play in the pathogenesis of congenital disabilities and provides a starting point for further studies aimed at elucidating the means by which peripheral nerves and blood vessels are patterned in the forelimb of the avian embryo.     

The pattern of neurovascular development in the forelimb of the quail embryo

Peripheral nerve and vascular patterns are congruent in the adult vertebrate, but this has been disputed in vertebrate embryos. The most detailed of these studies have used the avian forelimb as a model system, yet neurovascular anatomical relationships and critical vascular remodeling events remain inadequately characterized in this model. To address this, we have used a combination of intravascular marker injection, multilabel fluorescent stereomicroscopy, and confocal microscopy to analyze the spatiotemporal relationships between peripheral nerves and blood vessels in the forelimb of 818 quail embryos from E2 (HH13) to E15 (HH41). We find that the neurovascular anatomical relationships established during development are highly stereotypic and congruent. Blood vessels typically arise before their corresponding nerves, but there are several critical exceptions to this rule. The vascular pattern is extensively remodeled from the earliest stage examined (E2; HH13), whereas the peripheral nerves, the first of which enter the forelimb at E3.5-E4 (HH21-HH24), have a progressively unfolding pattern that, once formed, remains essentially unchanged. The adult neurovascular pattern is not established until E8 (HH34). Peripheral nerves are always found to track close and parallel to the vasculature. As they track distally, peripheral nerves always lie on the side of the vasculature away from the center of the forelimb. Neurovascular patterns have a hierarchy of congruence that is highest in the dorsoventral plane, followed by the anteroposterior, and lastly the proximodistal planes.     

Blood vessels and nerves: common signals,pathways and diseases

Both blood vessels and nerves are vital channels to and from tissues. Recent genetic insights show that they have much more in common than was originally anticipated. They use similar signals and principles to differentiate, grow and navigate towards their targets. Moreover, the vascular and nervous systems cross-talk and, when dysregulated, this contributes to medically important diseases. The realization that both systems use common genetic pathways should not only form links between vascular biology and neuroscience, but also promises to accelerate the discovery of new mechanistic insights and therapeutic opportunities.     

Neurovascular congruence results from a shared patterning mechanism that utilizes Semaphorin3A and Neuropilin-1

Peripheral nerves and blood vessels have similar patterns in quail forelimb development. Usually, nerves extend adjacent to existing blood vessels, but in a few cases, vessels follow nerves. Nerves have been proposed to follow vascular smooth muscle, endothelium, or their basal laminae. Focusing on the major axial blood vessels and nerves, we found that when nerves grow into forelimbs at E3.5-E5, vascular smooth muscle was not detectable by smooth muscle actin immunoreactivity. Additionally, transmission electron microscopy at E5.5 confirmed that early blood vessels lacked smooth muscle and showed that the endothelial cell layer lacks a basal lamina, and we did not observe physical contact between peripheral nerves and these endothelial cells. To test more generally whether lack of nerves affected blood vessel patterns, forelimb-level neural tube ablations were performed at E2 to produce aneural limbs; these had completely normal vascular patterns up to at least E10. To test more generally whether vascular perturbation affected nerve patterns, VEGF(165), VEGF(121), Ang-1, and soluble Flt-1/Fc proteins singly and in combination were focally introduced via beads implanted into E4.5 forelimbs. These produced significant alterations to the vascular patterns, which included the formation of neo-vessels and the creation of ectopic avascular spaces at E6, but in both under- and overvascularized forelimbs, the peripheral nerve pattern was normal. The spatial distribution of semaphorin3A protein immunoreactivity was consistent with a negative regulation of neural and/or vascular patterning. Semaphorin3A bead implantations into E4.5 forelimbs caused failure of nerves and blood vessels to form and to deviate away from the bead. Conversely, semaphorin3A antibody bead implantation was associated with a local increase in capillary formation. Furthermore, neural tube electroporation at E2 with a construct for the soluble form of neuropilin-1 caused vascular malformations and hemorrhage as well as altered nerve trajectories and peripheral nerve defasciculation at E5-E6. These results suggest that neurovascular congruency does not arise from interdependence between peripheral nerves and blood vessels, but supports the hypothesis that it arises by a shared patterning mechanism that utilizes semaphorin3A.     

Effects of L1 blockade on sensory axon outgrowth and pathfinding in the chick hindlimb

In the developing chick hindlimb, sensory axons, which grow together in bundles as they extend distally, and the motoneuron axons they encounter express the cell adhesion molecule L1. Following injection of function-blocking anti-L1 antibodies into the limb at stage 25, some sensory axons choose inappropriate peripheral nerves even though motoneuron pathfinding is unaffected. Here, to further elucidate L1's role, we assessed the effects of this perturbation using pathway tracing, immune labeling, confocal microscopy, and electron microscopy. After L1 blockade, sensory axons were still bundled and closely apposed. However, clear signs of decreased adhesion were detectable ultrastructurally. Further, sensory axons grew into the limb more slowly than normal, wandering more widely, branching more frequently, and sometimes extending along inappropriate peripheral nerves. Sensory axons that ultimately projected along different cutaneous nerves showed increased intermixing in the spinal nerves, due to errors in pathfinding and also to a decreased ability to segregate into nerve-specific fascicles. These results suggest that, in the highly complex in vivo environment, as in tissue culture, L1 stimulates axon growth and enhances fasciculation, and that these processes contribute to the orderly, timely, and specific growth of sensory axons into the limb.     

Immunohistochemical localization and vascular effects of vasoactive intestinal polypeptide in skeletal muscle of the cat

Summary Scattered vasoactive intestinal polypeptide (VIP) — immunoreactive nerves were found in the striated muscle of the hind limb of the cat, where they usually were associated with small blood vessels. VIP-immunoreactive nerves were also demonstrated in the sciatic nerve; after nerve ligation an abundance of intensely immunoreactive VIP fibres were seen proximal to the ligation. Intraarterial infusion of VIP into the isolated hind limb of the cat had dramatic effects on different sections of the vascular bed. Thus, VIP dilated the resistance vessels leading to a marked increment in muscle blood flow. VIP also relaxed the capacitance vessels causing regional pooling of blood; it increased the capillary surface area available for fluid exchange. Infusions of VIP at a dose of 8 g/min significantly inhibited the vasoconstriction induced by electrical stimulation of the regional sympathetic nerves. It is suggested that local nervous release of VIP may act as a modulator of vascular tone in skeletal muscle.     

Perivascular nerves and the regulation of cerebrovascular tone.

Brain perfusion is tightly coupled to neuronal activity, is commonly used to monitor normal or pathological brain function, and is a direct reflection of the interactions that occur between neuronal signals and blood vessels. Cerebral blood vessels at the surface and within the brain are surrounded by nerve fibers that originate, respectively, from peripheral nerve ganglia and intrinsic brain neurons. Although of different origin and targeting distinct vascular beds, these "perivascular nerves" fulfill similar roles related to cerebrovascular functions, a major one being to regulate their tone and, therein, brain perfusion. This utmost function, which underlies the signals used in functional neuroimaging techniques and which can be jeopardized in pathologies such as Alzheimer's disease, stroke, and migraine headache, is thus regulated at several levels. Recently, new insights into our understanding of how neural input regulate cerebrovascular tone resulted in the rediscovery of the functional "neurovascular unit." These remarkable advances suggest that neuron-driven changes in vascular tone result from interactions that involve all components of the neurovascular unit, transducing neuronal signals into vasomotor responses not only through direct interaction between neurons and vessels but also indirectly via the perivascular astrocytes. Neurovascular coupling is thus determined by chemical signals released from activated perivascular nerves and astrocytes that alter vascular tone to locally adjust perfusion to the spatial and temporal changes in brain activity.     

Neurovascular signalling defects in neurodegeneration

It is anticipated that by 2040 neurodegeneration will affect 40 million people worldwide, more than twice as many as today. The traditional neurocentric view holds that neurodegeneration is caused primarily by intrinsic neuronal defects. However, recent evidence indicates that the millions of blood vessels that criss-cross the nervous system might not be the silent bystanders they were originally considered. Indeed, recent genetic studies reveal that insufficient production of angiogenic signals, which stimulate the growth of blood vessels, can cause neurodegeneration. Remarkably, some angiogenic factors can also regulate neuroregeneration, and have direct neuroprotective and other effects on various neural cell types. Here we provide an overview of the molecules that affect both neural and vascular cell processes--to underline their duality, we term them angioneurins. Unravelling the molecular mechanisms by which these angioneurins act might create opportunities for developing new neurovascular medicine.     

Vascularization,innervation, and ultrastructure of the endocrine cell types of stannius corpuscles in the teleost Gasterosteus aculeatus

The blood circulation of the Stannius corpuscles, like that of the kidneys to which the corpuscles are attached, represents a portal system. The corpuscles receive blood from the dorsal caudal vein and from a vein coming from the hypaxial musculature. They are drained by veins which enter the caudal parts of the kidneys and therefore endocrine substances released by the corpuscles pass through the kidneys before they enter the general body circulation. The corpuscles are penetrated by sympathetic nerves coming from a small subvertebral ganglion. It is likely that these nerves innervate the muscular coat around the blood vessels. The muscular coat surrounding the renal blood vessels, the collecting tubules and part of the ureters, is innervated by nerves from the same ganglion. The secretory activity of the gland cells appears to be controlled by blood borne factors, because neither synaptic contacts with these cells, nor gap junctions among the cells, have been found in thin sections and freeze-etch replicas of the corpuscles. The corpuscles contain two cell types, both presumed to have endocrine function. Histochemical and ultrastructural data indicate that the gland cells produce glycoproteins. It is likely that the contents of the secretory granules are released by exocytosis. One cell type is structurally similar to the cells described in many other teleosts and thought to be engaged in the synthesis of a hypocalcemic hormone. The ultrastructure of the second cell type resembles cells described only in other migratory species: salmonids and eels. It may be involved in the control of monovalent ions.     

Cutaneous nerves of the embryonic chick wing do not develop in regions denuded of ectoderm

Peripheral nerves travel to their targets along precise routes, and it is likely that different cues provide guidance at different stages of the journey. In a developing chick limb, the cutaneous nerve fibres follow at first deep mixed nerve trunks, in company with motor axons; they branch from these trunks at predictable points and approach the skin; they then ramify profusely to form a plexus at a precisely defined depth beneath the ectoderm, at exactly the same level as the blood vascular plexus. To analyse the role of signals from the target patch of skin in regulating cutaneous nerve development, we have ablated patches of dorsal wing ectoderm using short-wave ultraviolet irradiation at E4 (embryonic day 4), approximately one day before nerves grow into the limb bud. The irradiated patches remain denuded of ectoderm for more than a week, by which time the cutaneous nerve plexus on the contralateral control side is well developed and can be revealed by whole-mount silver staining. Where the ectoderm has been ablated, no cutaneous nerve plexus forms, and the nerve branches that normally would have diverged from the neighbouring mixed nerve trunk to innervate the missing patch of skin are absent - ab initio, apparently. The routes of the mixed nerve trunks are not affected. Partial ablation of the territory of a cutaneous nerve branch often leads to loss of the whole nerve branch; the intact skin territory thus left vacant is invaded by ramifications from the remaining cutaneous branches, as expected if the normal extent of a cutaneous nerve's territory is regulated by competition. Where there is an ectodermal lesion, cutaneous innervation stops precisely at its boundary, even though the vascular plexus extends for some distance beyond this margin, beneath the denuded surface. The data suggest that the embryonic skin is required firstly to trigger divergence of cutaneous nerve branches from the mixed nerve trunks, and secondly, once the nerve fibres have reached the skin, to supply a trophic cue (probably NGF) encouraging growth of a plexus; at the same time, the embryonic skin generates a signal inhibiting nerves from approaching closer than about 70 microns to the surface.     

Nerve-dependent and -independent tenascin expression in the developing chick limb bud.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.     

Neuropeptide Y-containing nerves in rat gonads: sex difference and development

The objectives of the present study were 1) to evaluate for a sex difference in innervation of adult rat gonads by neuropeptide Y-immunoreactive (NPY-I) nerves and 2) to examine the development of innervation of rat gonads by NPY-I nerves during the fetal and neonatal periods. With fluorescence immunocytochemistry, NPY-I nerves were profuse in adult ovarian tissues. Ovarian blood vessels were particularly well innervated by NPY-I nerves, and nerves were also detected in interstitial gland tissues. No nerves were found within the testis, and NPY-I nerves were only rarely located within the tunica albuginea. During fetal life, ovaries were devoid of NPY-I nerves; however, nerves were visualized within the connective tissue immediately peripheral to the ovary on fetal Day 22. As early as postnatal Day 2, NPY-I nerves were observed in connective tissue septa of the developing ovary. By postnatal Day 12, NPY-I nerves surrounded developing follicles and blood vessels of the ovarian cortex. In the developing testis after postnatal Day 5, NPY-I nerves were limited to the tunica albuginea and surrounding large subcapsular blood vessels. Structures within the testis lacked innervation by NPY-I nerves. These anatomical studies suggest that NPY-I nerves are absent in the gonads during fetal life and grow into the ovary and not the testis during the perinatal period and that NPY-I nerves may play a role in the functioning of the rat ovary, but may not be important in control of testicular function.     

The fine structure of limb tissues of the adult newt, Diemictylus viridescens

The limb tissues of the adult newt investigated for their fine structure include epidermis, subcutaneous glands, dermis, striated muscle, peripheral nerves and blood vessels. This survey complements and extends previous observations, emphasizing intercellular junctions, and the ubiquitous “glycocalyx” (= polysaccharide-protein lamella, around cells and adjacent to epithelia). Our survey touches on the characteristic tonofilaments, intercellular desmosomes and basal hemidesmosomes of the epidermis. The subcutaneous glands consist of secretory cells with a granular product, and myoepithelial cells; intercellular desmosomes are present. The adepidermal reticulum of collagen fibrils reveals periodic regions of intersecting fibrils ( = nodules), and fibril continuity with the underlying dermis: a striking feature is the adipose tissue closely applied to the adepidermal reticulum. The limb striated muscle displays typical banded myofibrils, and a triad system with centrotubules in the I-band close to the Z-band: terminal sacs of sarcoplasmic reticulum complete the triad system. A particularly prominent glycocalyx is applied to the surface of the sarcolemma. The peripheral nerves of the limb possess connective tissue sheaths with prominent vesiculation of the cell membranes, and an occasional intercellular desmosomal junction. Blood vessels typically have endothelial cells with prominently vesiculated plasma membranes. This investigation serves as the basis for recognizing the fine structure of tissue responses to trauma, their repair, and regeneration.     

Blood Vessels in the Hind Limb of the Mallard (Anas platyrhynchos): Anatomical Evidence for a Sphincteric Action of Shunt Vessels in Connection with the Arterio-venous Heat Exchange System

Midtgård, U. 1980. Blood vessels in the hind limb of the Mallard (Anas platyrhynchos): anatomical evidence for a sphincteric action of shunt vessels in connection with the arterio-venous heat exchange system. (Institute of Comparative Anatomy, University of Copenhagen, Denmark.) — Acta zool. (Stockh.) 61(1): 39–49. The rete tibiotarsale is the main arterio-venous heat exchange system in the hind limb of the Mallard. A large arterial shunt and a venous shunt allow the blood to by-pass the rete. These shunt vessels must be able to constrict so as to direct the blood to the rete when heat conservation is needed. Using ordinary histological methods and the technique of Falck and Hillarp for demonstration of biogenic monoamines, it was shown that the arterial shunt is more muscular and receives a more dense adrenergic innervation than adjacent segments of the same vessel. Perfusion with noradrenaline before fixation revealed that the arterial shunt was able to reduce its lumen to near closure. No structure, in the ordinary sense of a sphincter, was found in the shunt vein but adrenergic nerves were scattered throughout the tunica media at the base of venous valves, suggesting that a sphincteric action at these sites is possible.     

Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation

Summary The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation.Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern.The altered proportion of sensory peptide-and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.     

Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation

The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation. Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern. The altered proportion of sensory peptide- and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.