Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura.

Dural cerebral veins (CV) are required for cerebrospinal fluid reabsorption and brain homeostasis, but mechanisms that regulate their growth and remodeling are unknown. We report molecular and cellular processes that regulate dural CV development in mammals and describe venous malformations in humans with craniosynostosis and TWIST1 mutations that are recapitulated in mouse models. Surprisingly, Twist1 is dispensable in endothelial cells but required for specification of osteoprogenitor cells that differentiate into preosteoblasts that produce bone morphogenetic proteins (BMPs). Inactivation of Bmp2 and Bmp4 in preosteoblasts and periosteal dura causes skull and CV malformations, similar to humans harboring TWIST1 mutations. Notably, arterial development appears normal, suggesting that morphogens from the skull and dura establish optimal venous networks independent from arterial influences. Collectively, our work establishes a paradigm whereby CV malformations result from primary or secondary loss of paracrine BMP signaling from preosteoblasts and dura, highlighting unique cellular interactions that influence tissue-specific angiogenesis in mammals.

Rule of 5: angiographic diameters of cervicocerebral arteries in children and compatibility with adult neurointerventional devices.

BACKGROUND AND PURPOSE: The safety of using adult-sized neuroendovascular devices in the smaller pediatric vasculature is not known. In this study we measure vessel diameters in the cervical and cranial circulation in children to characterize when adult-approved devices might be compatible in children. METHODS: For 54 children without vasculopathy (mean age 9.5±4.9 years (range 0.02-17.8), 20F/34M) undergoing catheter angiography, the diameters of the large vessels in the cervical and cranial circulation (10 locations, 611 total measurements) were assessed by three radiologists. Mean±SD diameter was calculated for the following age groups: 0-6 months, 1, 2, 3, 4, 5-9, 10-14, and 15-18 years. To compare with adult sizes, each vessel measurement was normalized to the respective region mean diameter in the oldest age group (15-18 years). Normalized measurements were compared with age and fitted to a segmented regression. RESULTS: Vessel diameters increased rapidly from 0 to 5 years of age (slope=0.069/year) but changed minimally beyond that (slope=0.005/year) (R(2)=0.2). The regression model calculated that, at 5 years of age, vessels would be 94% of the diameter of the oldest age group (compared with 59% at birth). In addition, most vessels in children under 5, while smaller, were still potentially large enough to be compatible with many adult devices. CONCLUSIONS: The growth curve of the cervicocerebral vasculature displays rapid growth until age 5, at which point most children's vessels are nearly adult size. By age 5, most neuroendovascular devices are size-compatible, including thrombectomy devices for stroke. Under 5 years of age, some devices might still be compatible.

Inhibition of protein tyrosine phosphatases enhances cerebral collateral growth in rats.

UNLABELLED: Arteriogenesis involves the rapid proliferation of preexisting arterioles to fully functional arteries as a compensatory mechanism to overcome circulatory deficits. Stimulation of arteriogenesis has therefore been considered a treatment concept in arterial occlusive disease. Here, we investigated the impact of inhibition of protein tyrosine phosphatases (PTPs) on cerebral arteriogenesis in rats. Arteriogenesis was induced by occlusion of one carotid and both vertebral arteries (three-vessel occlusion (3-VO)). Collateral growth and functional vessel perfusion was assessed 3-35 days following 3-VO. Furthermore, animals underwent 3-VO surgery and were treated with the pan-PTP inhibitor BMOV, the SHP-1 inhibitor sodium stibogluconate (SSG), or the PTP1B inhibitor AS279. Cerebral vessel diameters and cerebrovascular reserve capacity (CVRC) were determined, together with immunohistochemistry analyses and proximity ligation assays (PLA) for determination of tissue proliferation and phosphorylation patterns after 7 days. The most significant changes in vessel diameter increase were present in the ipsilateral posterior cerebral artery (PCA), with proliferative markers (PCNA) being time-dependently increased. The CVRC was lost in the early phase after 3-VO and partially recovered after 21 days. PTP inhibition resulted in a significant increase in the ipsilateral PCA diameter in BMOV-treated animals and rats subjected to PTP1B inhibition. Furthermore, CVRC was significantly elevated in AS279-treated rats compared to control animals, along with hyperphosphorylation of the platelet-derived growth factor-ß receptor in the vascular wall in vivo. In summary, our data indicate PTPs as hitherto unrecognized negative regulators in cerebral arteriogenesis. Further, PTP inhibition leading to enhanced collateral growth and blood perfusion suggests PTPs as novel targets in anti-ischemic treatment. KEY MESSAGES: PTPs exhibit negative regulatory function in cerebral collateral growth in rats. Inhibition of pan-PTP/PTP1B increases vessel PDGF-ß receptor phosphorylation. PTP1B inhibition enhances arteriogenesis and cerebrovascular reserve capacity.

Cerebral angiogenesis during development: who is conducting the orchestra?

Blood vessels provide the brain with the oxygen and the nutrients it requires to develop and function. Endothelial cells (ECs) are the principal cell type forming the vascular system and driving its development and remodeling. All vessels are lined by a single EC layer. Larger blood vessels are additionally enveloped by vascular smooth muscle cells (VSMCs) and pericytes, which increase their stability and regulate their perfusion and form the blood-brain barrier (BBB). The development of the vascular system occurs by two processes: (1) vasculogenesis, the de novo assembly of the first blood vessels, and (2) angiogenesis, the creation of new blood vessels from preexisting ones by sprouting from or by division of the original vessel. The walls of maturing vessels produce a basal lamina and recruit pericytes and vascular smooth muscle cells for structural support. Whereas the process of vasculogenesis seems to be genetically programmed, angiogenesis is induced mainly by hypoxia in development and disease. Both processes and the subsequent vessel maturation are further orchestrated by a complex interplay of inhibiting and stimulating growth factors and their respective receptors, many of which are hypoxia-inducible. This chapter intends to give an overview about the array of factors directing the development and maintenance of the brain vasculature and their interdependent actions.

A history of our understanding of cerebral vascular development and pathogenesis of perinatal brain damage over the past 30 years.

This article reviews our studies focusing on cerebral vascular development, the pathogenesis of subependymal/intraventricular hemorrhage (SEH/IVH), periventricular leukomalacia (PVL), and pontosubicular neuron necrosis (PSN). Their pathogenesis consists of predisposing developmental and causal factors. SEH/IVH may be caused by reperfusion or overperfusion following ischemia in the subependymal germinal matrix with characteristic vasculature. The cause of PVL is multifactorial (ie, ischemia and inflammation), predisposed by the maturational status of the vasculature and oligodendroglia in the white matter. Focal PVL is ischemic necrosis, and diffuse PVL or white matter injury may include cytotoxic damage. PSN has an apoptotic character, and may be induced by ischemic and oxidative stress on specific immature neurons. Further studies on preventive and therapeutic measures are necessary in clinical, pathologic, and experimental fields. The monitoring and control methods of brain hemodynamics and cellular stability should be more developed to prevent brain damages.

Granulocyte-macrophage colony-stimulating factor as an arteriogenic factor in the treatment of ischaemic stroke.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces arteriogenic growth of collateral vessels after occlusion of cardiac or peripheral arteries. Recently, evidence has been provided that arteriogenesis also occurs in the brain under conditions of reduced arterial blood supply. Hemispheric hypoperfusion induced by unilateral carotid and bilateral vertebral artery occlusion (three-vessel occlusion, [3-VO]) led to the growth of the anterior and posterior segments of the circle of Willis, which is the main collateral pathway between the origins of the anterior, middle and posterior cerebral arteries. GM-CSF applied subcutaneously at daily doses of 40 microg.kg(-1) resulted in the marked acceleration of this process. Within one week after the onset of treatment, the diameter of the posterior segment of the circle of Willis enlarged to 170% of control, blood flow and the haemodynamic reserve capacity of the brain returned to normal, and haemodynamic stroke, induced after 3-VO by systemic hypotension, was greatly alleviated. GM-CSF-induced stimulation of arteriogenesis in the hypoperfused brain thus provides powerful protection against ischaemic stroke.

Maturational modulation of endothelium-dependent vasodilatation in ovine cerebral arteries.

To address the hypothesis that maturation enhances endothelial vasodilator function in cerebral arteries, relaxant responses to ADP and A-23187 were determined in ovine carotid and cerebral arteries harvested from 25 newborn lambs (3-7 days) and 23 adult sheep. Maturation significantly increased pD(2) values for A-23187 (newborn range: 4.9 +/- 0.3 to 5.4 +/- 0.3; adult range: 6.0 +/- 0.2 to 7.1 +/- 0.2) and the maximal vasodilator response to A-23187 by 10-18%. In contrast, maturation decreased maximum responses to ADP by 5-25% with no change in pD(2). The magnitudes of endothelium-dependent relaxation were not affected by 10 microM indomethacin but were virtually abolished by 100 microM N(G)-nitro-L-arginine methyl ester/L-nitro arginine, indicating that nitric oxide (NO) is the primary endothelium-dependent vasodilator in these arteries. Maturation also modestly decreased endothelial NO synthase (eNOS) abundance in both carotid (32%) and cerebral (26%) arteries. Together, these findings reinforce the view that receptor coupling to endothelial activation is tightly regulated and may offset underlying changes in maximal endothelial vasodilator capacity. This capacity, in turn, appears to increase with postnatal age despite major growth and expansion of endothelial cell size and vascular wall volume. In ovine cerebral arteries, endothelial vasodilator capacity appears completely dependent on eNOS activity but not on cyclooxygenase activity. In turn, eNOS activity appears to be postnatally regulated by mechanisms independent of changes in eNOS abundance alone.

Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin.

There is increasing evidence that physical activity is associated with a decreased stroke risk. The purpose of this study was to determine if exercise could also reduce brain damage in rats subjected to transient middle cerebral artery (MCA) occlusion, and if the reduced brain injury is associated with angiogenesis as well as cellular expression of the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in regions supplied by the MCA. Adult male Sprague Dawley rats (n=36) exercised 30 min each day for 3 weeks on a treadmill on which repetitive locomotor movement was required. Then, stroke was induced by a 2-h MCA occlusion using an intraluminal filament, followed by 48 h of reperfusion. In addition to the two exercised groups of animals with or without MCA occlusion, there were two other groups of animals, with or without MCA occlusion, housed for the same duration and used as non-exercised controls. Brain damage in ischemic rats was evaluated by neurologic deficits and infarct volume. Exercise preconditioned and non-exercised brains were processed for immunocytochemistry to quantify the number of microvessels or NGF- and BDNF-labeled cells. Pre-ischemic motor activity significantly (P<0.01) reduced neurologic deficits and infarct volume in the frontoparietal cortex and dorsolateral striatum. Cellular expressions of NGF and BDNF were significantly (P<0.01) increased in cortex (neuron) and striatum (glia) of rats under the exercise condition. Significant (P<0.01) increases in microvessel density were found in striatum. Physical activity reduced stroke damage. The reduced brain damage may be attributable to angiogenesis and neurotrophin overexpression in brain regions supplied by the MCA following exercise.

Progenitor endothelial cell involvement in Alzheimer's disease.

There is compelling evidence that endothelial cells of the brain and periphery are dysfunctional in Alzheimer's disease. There is evidence for a fundamental defect in, or abnormal aging of, endothelial progenitor cells in atherosclerosis. The possibility that endothelial cell defects are a primary cause for Alzheimer's disease or other dementias can be researched by molecular and cell biology studies as well as cell trafficking studies using recently demonstrated molecular imaging methods. The evidence for abnormal endothelial function and the methods to explore this hypothesis are presented.

Correlation between cerebrovascular maturity and periventricular leukomalacia.

This study disclosed the close correlation between the characteristic developmental change of human cerebral vessels and the occurrence of periventricular leukomalacia (PVL), as judged by anticollagen type 6 immunohistochemical analysis. The earlier appearance of collagen type 6-positive vessels in the deep white matter supports the concept that the medullary vein, a terminal branch of the internal cerebral vein, develops earlier than the cortical and subcortical veins and the perforating artery because the latter was not stained in early gestation. In cases with an old lesion of PVL the distribution of the lesions with abnormal vessels differed with gestation age. These results suggest that its distribution correlates with the development of perforating medullary arteries. Thus a discrepancy between the arteries, revealing slow maturation, and veins, revealing early maturation, in the deep white matter may be an important predisposing factor for PVL. Furthermore, the widespread lesions of PVL may be closely related to the involvement of transcortical tract damage, in terms of specific motor or intellectual disabilities.

Age-dependent aluminum accumulation in the human aorta and cerebral artery.

The purpose of this study was to determine the extent of aluminum (Al) accumulation in the human aorta and cerebral arteries. The Al contents in the aortae and in the cerebral arteries from 23 human subjects was determined by inductively coupled plasma atomic emission spectrophotometry (ICP-AES). The subjects' age range was 45-99-yr-old; 15 of the subjects were males and 8 were females. Al was detected in twelve aortae and in six cerebral arteries, when the entire specimen was analyzed. Two specimens where Al was found in the cerebral arteries contained no Al in the aorta. No relationship to the subject's sex was found. When related to age, two groups were established. Group L (45-75 yr old) and group H (> 75 yr old), which exhibited aortal Al concentrations of 33.3 and 72.7%, respectively. When the aortic wall was dissected into the tunica intima, media, and adventitia, Al was found mainly in the tunica media. In the aorta, significant relationships were found between Al and phosphorus (P) levels (r = 0.801, p < 0.01) and between Al and calcium (Ca) (r = 0.661, p < 0.05). We have concluded that Al accumulation is age-dependent and that it occurs both in the aorta and in the cerebral artery. In the aorta, accumulation occurs mainly in the tunica media. Both P and Ca appear to enhance aortal Al accumulation.

Effects of maturation on alpha-adrenergic receptor affinity and occupancy in small cerebral arteries.

The present experiments examine the hypothesis that changes in receptor affinity and occupation mediate maturational changes in norepinephrine sensitivity in small cerebral arteries. In second-order (2B) and fourth-order (4B) branch middle cerebral artery segments from newborn and adult sheep, we first found that a stretch ratio based on artery diameter better estimated optimal prestretch than did passive tension. Next, we determined norepinephrine dose-response relations before and after prazosin, yohimbine, and benextramine. Prazosin competitively blocked contractions to norepinephrine, but yohimbine had no effect, indicating that alpha 1-adrenoceptors mediated contraction. Norepinephrine sensitivity [determined from the -log of the half-maximal effective dose (pD2)], maximal response, and binding affinity all decreased with age in 4B but not 2B segments. Receptor occupancy at the pD2 increased with age only in 2B segments. In conclusion, maturation of ovine middle cerebral arteries involves branch-specific changes in affinity and receptor occupation of the alpha 1-adrenoceptors that mediate contractile responses to norepinephrine. Age-related changes in receptor density and/or intrinsic efficacy probably are involved also.

Maturation enhances the sensitivity of ovine cerebral arteries to the ATP-sensitive potassium channel activator lemakalim.

A wide variety of previous studies have demonstrated that arterial reactivity and contractility change dramatically during maturation. In light of recent findings that binding sites for glibenclamide, a ligand for ATP-sensitive potassium (KATP) channels, become more abundant with age in many tissues, the present studies examine the hypothesis that maturational changes in vascular reactivity involve changes in arterial electrophysiologic characteristics. To test this hypothesis, we determined the dose-response relation to lemakalim, a selective activator of KATP channels, in isolated endothelium-denuded segments of the second (2B, internal diameter approximately 200 microns) and fourth (4B, internal diameter approximately 125 microns) branches of middle cerebral arteries taken from newborn (3-7 d old) and adult sheep. At 100 microM, lemakalim completely relaxed serotonin-induced tone in all arteries. However, -log ED50 values were 29 to 43 times greater in adult (2B, 7.15 +/- 0.38; 4B, 6.61 +/- 0.42) than in newborn (2B, 5.52 +/- 0.25; 4B, 5.15 +/- 0.24) segments. Correspondingly, Hill values were significantly smaller in adults (2B, 0.47 +/- 0.17; 4B, 0.71 +/- 0.30) than in newborns (2B, 1.40 +/- 0.35; 4B, 3.30 +/- 0.92). These findings demonstrate that KATP channels are less sensitive to activation in newborn than in adult cerebral arteries. Given the important influence of KATP channels on vascular tone, and their possible role in many cardiovascular responses, the present data suggest that maturational increases in the activity of KATP channels contribute significantly to age-related changes in cerebrovascular contractility.

Development of cerebral arterial innervation: synchronous development of neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-containing fibers and some observations on growth cones.

The pre- and postnatal development of sympathetic fibers containing neuropeptide Y (NPY) and parasympathetic fibers containing vasoactive intestinal polypeptide (VIP) supplying the cerebral arteries were studied with immunohistochemistry in rats. The innervation patterns and densities of NPY and VIP fibers were similar at all stages of development and similar to that previously reported for norepinephrine (NE). There was a striking reorganization of the innervation pattern of all three fiber systems between the first and second postnatal weeks. At all stages of development prior to the first postnatal week, growth cones were present on individual fibers at the distal part of major cerebral arteries and the middle segment of the basilar artery. The growth cones had a range of shapes from blunt to stellate, lanceolate or filiform. NPY and VIP immunoreactive granules were commonly present. The present results taken with our earlier developmental study of NE fibers (J. Comp. Neurol., 271 (1988) 435-444), demonstrate that: (1) both sympathetic and parasympathetic perivascular nerves on immature cerebral vessels develop with similar sequences: first longitudinal fibers and fiber bundles are present; these transform to a meshwork pattern and finally transform again into the mature, predominantly circumferential pattern; (2) both the classical (NE) and peptidergic transmitters (NPY) within the sympathetic system appear to develop identically in terms of time of appearance, innervation patterns, densities and reorganization.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical and immunoelectron microscopical characterization of cerebrovascular and senile plaque amyloid in aged dogs' brains.

Immunohistochemical and immunoelectron microscopical studies were carried out on 28 aged dogs' brains. Amyloid deposits were seen in the arteries and capillaries in the leptomeninges and in superficial areas of the cortices in 19 (67.9%) of the 28 dogs (10-22 years of age). Immunohistochemically, these amyloid deposits were reactive for anti-beta/A4 antibody. Additionally, a variable number of parenchymal deposits with diffuse beta/A4-immunoreactivity (diffuse plaques) was also noted throughout the cerebral cortex in 24/28 dogs (85.7%). However, these plaque lesions were undetectable in Congo red staining. Electron microscopically, amyloid fibrils, measuring 10 nm in width, were located mainly in the tunica media of the arteries, and in less involved vessels they tended to be present among collagen fibres in the adventitia and smooth muscle cells in the outer layer of the media. The plaque lesions appeared to contain sparse aggregations of amyloid fibrils. In immunoelectron microscopical examinations, all amyloid fibrils in both blood vessels and plaques were selectively labelled by gold particles. These findings indicate that aged dogs can provide a useful experimental model for research into the beta/A4-type of cerebral amyloidosis commonly seen in Alzheimer's disease.

Developmental aspects of endothelial function.

We believe that the mechanisms through which nitric oxide and guanylate cyclase produce relaxation are fully functional in cerebral arteries at term in the fetal sheep and probably also in the term human infant. The relation between cGMP levels and the degree of relaxation varies both with age and with the relaxant used in a vessel specific manner. The factors underlying this variability constitute a fruitful area for future research and include possible age-related changes in membrane potential, calcium channel density and currents, and the participation of cGMP-independent mechanisms, to name only a few. Between fetal and newborn life, the biotransformation of nitroglycerin appears to improve significantly, particularly in the smaller more distal cerebral arteries. This improvement may be a clue to other important vascular metabolic and enzymatic changes that occur during the perinatal period. At the endothelial level, responses to A23187, an index of maximum endothelial vasodilator capacity, are relatively stable across the perinatal period and do not change consistently with age across all arteries. More importantly, large arteries, such as the common carotid, appear to relax better than the smaller cerebral arteries, and this difference is greater in fetal than in adult arteries. Responses to ADP disappear with age in the common carotid, but remain or even become enhanced in the cerebral arteries, thus illustrating the key role played by changes in receptor type and distribution in development and maturation.