Brain-derived circulating endothelial cells in peripheral blood of newborn infants with seizures: a potential biomarker for cerebrovascular injury.

Neonatal seizures have been associated with cerebrovascular endothelial injury and neurological disabilities. In a piglet model, the long-term loss of endothelial regulation of cerebral blood flow coincides with the surge of brain-derived circulating endothelial cells (BCECs) in blood. We hypothesized that BCECs could serve as a noninvasive biomarker of cerebrovascular injury in neonates with seizures. In a prospective pilot feasibility study, we enrolled newborn infants with confirmed diagnoses of perinatal asphyxia and intraventricular hemorrhage (IVH); both are commonly associated with seizures. Infants without clinical evidence of cerebrovascular injuries were representative of the control group. BCECs were detected in the CD45-negative fraction of peripheral blood mononuclear cells by coexpression of CD31 (common endothelial antigen) and GLUT1 (blood-brain barrier antigen) via automated flow cytometry method. In Infants with asphyxia (n = 12) and those with IVH grade III/IV (n = 5), the BCEC levels were 9.9 ± 0.9% and 19.0 ± 2.0%, respectively. These levels were significantly higher than the control group (n = 27), 0.9 ± 0.2%, P < 0.001. BCECs in infants with cerebrovascular insults with documented clinical seizures (n = 10; 16.8 ± 1.3%) were significantly higher than infants with cerebrovascular insults with subclinical or no seizures (n = 7; 9.5 ± 1.2%); P < 0.001. BCEC levels decreased with seizure control. BCECs levels were elevated in infants with seizures caused by severe IVH and perinatal asphyxia. We suggest that monitoring BCEC levels in peripheral blood can potentially offer a biological marker that reflects cerebrovascular insult and recovery. Further studies with a larger number of patients are required to support these findings.

Functional role of astrocyte glutamate receptors and carbon monoxide in cerebral vasodilation response to glutamate.

In newborn pigs, vasodilation of pial arterioles in response to glutamate is mediated via carbon monoxide (CO), a gaseous messenger endogenously produced from heme degradation by a heme oxygenase (HO)-catalyzed reaction. We addressed the hypothesis that ionotropic glutamate receptors (iGluRs), including N-methyl-D-aspartic acid (NMDA)- and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA)/kainate-type receptors, expressed in cortical astrocytes mediate glutamate-induced astrocyte HO activation that leads to cerebral vasodilation. Acute vasoactive effects of topical iGluR agonists were determined by intravital microscopy using closed cranial windows in anesthetized newborn pigs. iGluR agonists, including NMDA, (±)1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD), AMPA, and kainate, produced pial arteriolar dilation. Topical L-2-aminoadipic acid, a gliotoxin that selectively disrupts glia limitans, reduced vasodilation caused by iGluR agonists, but not by hypercapnia, bradykinin, or sodium nitroprusside. In freshly isolated and cultured cortical astrocytes constitutively expressing HO-2, iGluR agonists NMDA, cis-ACPD, AMPA, and kainate rapidly increased CO production two- to threefold. Astrocytes overexpressing inducible HO-1 had high baseline CO but were less sensitive to glutamate stimulation of CO production when compared with HO-2-expressing astrocytes. Glutamate-induced astrocyte HO-2-mediated CO production was inhibited by either the NMDA receptor antagonist (R)-3C4HPG or the AMPA/kainate receptor antagonist DNQX. Accordingly, either antagonist abolished pial arteriolar dilation in response to glutamate, NMDA, and AMPA, indicating functional interaction among various subtypes of astrocytic iGluRs in response to glutamate stimulation. Overall, these data indicate that the astrocyte component of the neurovascular unit is responsible for the vasodilation response of pial arterioles to topically applied glutamate via iGluRs that are functionally linked to activation of constitutive HO in newborn piglets.

Glutamate-induced calcium signals stimulate CO production in piglet astrocytes.

Glutamate-stimulated, astrocyte-derived carbon monoxide (CO) causes cerebral arteriole dilation by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) channels. Here, we examined the hypothesis that glutamate activates heme oxygenase (HO)-2 and CO production via the intracellular Ca(2+) concentration ([Ca(2+)](i))/Ca(2+)-calmodulin signaling pathway in newborn pig astrocytes. The major findings are: 1) glutamate stimulated Ca(2+) transients and increased steady-state [Ca(2+)](i) in cerebral cortical astrocytes in primary culture, 2) in astrocytes permeabilized with ionomycin, elevation of [Ca(2+)](i) concentration-dependently increased CO production, 3) glutamate did not affect CO production at any [Ca(2+)](i) when the [Ca(2+)](i) was held constant, 4) thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase blocker, decreased basal CO production and blocked glutamate-induced increases in CO, and 5) calmidazolium, a calmodulin inhibitor, blocked CO production induced by glutamate and by [Ca(2+)](i) elevation. Taken together, our data are consistent with the hypothesis that glutamate elevates [Ca(2+)](i) in astrocytes, leading to Ca(2+)- and calmodulin-dependent HO-2 activation, and CO production.

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis.

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease.

Epileptic seizures increase circulating endothelial cells in peripheral blood as early indicators of cerebral vascular damage.

Circulating endothelial cells (CECs) are nonhematopoetic mononuclear cells in peripheral blood that are dislodged from injured vessels during cardiovascular disease, systemic vascular disease, and inflammation. Their occurrence during cerebrovascular insults has not been previously described. Epileptic seizures cause the long-term loss of cerebrovascular endothelial dilator function. We hypothesized that seizures cause endothelial sloughing from cerebral vessels and the appearance of brain-derived CECs (BCECs), possible early indicators of cerebral vascular damage. Epileptic seizures were induced by bicuculline in newborn pigs; venous blood was then sampled during a 4-h period. CECs were identified in the fraction of peripheral blood mononuclear cells by the expression of endothelial antigens (CD146, CD31, and endothelial nitric oxide synthase) and by Ulex europeaus lectin binding. In control animals, few CECs were detected. Seizures caused a time-dependent increase in CECs 2-4 h after seizure onset. Seizure-induced CECs coexpress glucose transporter-1, a blood-brain barrier-specific glucose transporter, indicating that these cells originate in the brain vasculature and are thus BCECs. Seizure-induced BCECs cultured in EC media exhibited low proliferative potential and abnormal cell contacts. BCEC appearance during seizures was blocked by a CO-releasing molecule (CORM-A1) or cobalt protoporphyrin (heme oxygenase-1 inducer), which prevented apoptosis in cerebral arterioles and the loss of cerebral vascular endothelial function during the late postictal period. These findings suggest that seizure-induced BCECs are injured ECs dislodged from cerebral microvessels during seizures. The correlation between the appearance of BCECs in peripheral blood, apoptosis in cerebral vessels, and the loss of postictal cerebral vascular function suggests that BCECs are early indicators of late cerebral vascular damage.

Cerebroprotective effects of the CO-releasing molecule CORM-A1 against seizure-induced neonatal vascular injury.

Endogenous CO, a product of heme oxygenase activity, has vasodilator and cytoprotective effects in the cerebral circulation of newborn pigs. CO-releasing molecule (CORM)-A1 (sodium boranocarbonate) is a novel, water-soluble, CO-releasing compound. We addressed the hypotheses that CORM-A1 1) can deliver CO to the brain and exert effects of CO on the cerebral microvasculature and 2) is cerebroprotective. Acute and delayed effects of topically and systemically administered CORM-A1 on cerebrovascular and systemic circulatory parameters were determined in anesthetized newborn pigs with implanted closed cranial windows. Topical application of CORM-A1 (10(-7)-10(-5) M) to the brain produced concentration-dependent CO release and pial arteriolar dilation. Systemically administered CORM-A1 (2 mg/kg ip or iv) caused pial arteriolar dilation and increased cortical cerebrospinal fluid CO concentration. Systemic CORM-A1 did not have acute or delayed effects on blood pressure, heart rate, or blood gases. Potential cerebroprotective vascular effects of CORM-A1 (2 mg/kg ip, 30 min before seizures) were tested 2 days after bicuculline-induced epileptic seizures (late postictal period). In control piglets, seizures reduced postictal cerebrovascular responsiveness to selective physiologically relevant vasodilators (bradykinin, hemin, and isoproterenol) indicative of cerebrovascular injury. In contrast, in CORM-A1-pretreated animals, no loss of postictal cerebrovascular reactivity was observed. We conclude that systemically administered CORM-A1 delivers CO to the brain, elicits the vasodilator and cytoprotective effects of CO in the cerebral circulation, and protects the neonatal brain from cerebrovascular injury caused by epileptic seizures.

Contributions of astrocytes and CO to pial arteriolar dilation to glutamate in newborn pigs.

Astrocytes can act as intermediaries between neurons and cerebral arterioles to regulate vascular tone in response to neuronal activity. Release of glutamate from presynaptic neurons increases blood flow to match metabolic demands. CO is a gasotransmitter that can be related to neural function and blood flow regulation in the brain. The present study addresses the hypothesis that glutamatergic stimulation promotes perivascular astrocyte CO production and pial arteriolar dilation in the newborn brain. Experiments used anesthetized newborn pigs with closed cranial windows, piglet astrocytes, and cerebrovascular endothelial cells in primary culture and immunocytochemical visualization of astrocytic markers. Pial arterioles and arteries of newborn pigs are ensheathed by astrocytes visualized by glial fibrillary acidic protein staining. Treatment (2 h) of astrocytes in culture with L-2-alpha-aminoadipic acid (L-AAA), followed by 14 h in toxin free medium, dose-dependently increased cell detachment, suggesting injury. Conversely, 16 h of continuous exposure to L-AAA caused no decrease in endothelial cell attachment. In vivo, topical L-AAA (2 mM, 5 h) disrupted the cortical glia limitans histologically. Such treatment also eliminated pial arteriolar dilation to the astrocyte-dependent dilator ADP and to glutamate but not to isoproterenol or CO. Glutamate stimulated CO production by the brain surface that also was abolished following L-AAA. In contrast, tetrodotoxin blocked dilation to N-methyl-D-aspartate but not to glutamate, isoproterenol, or CO or the glutamate-induced increase in CO. The concurrent loss of CO production and pial arteriolar dilation to glutamate following astrocyte injury suggests astrocytes may employ CO as a gasotransmitter for glutamatergic cerebrovascular dilation.

HO-2 provides endogenous protection against oxidative stress and apoptosis caused by TNF-alpha in cerebral vascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) causes oxidative stress and apoptosis in a variety of cell types. Heme oxygenase (HO) degrades heme to bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator, and a vasodilator. Newborn pig cerebral microvascular endothelial cells (CMVEC) highly express constitutive HO-2. We investigated the role of HO-2 in protection against TNF-alpha-induced apoptosis in cerebral vascular endothelium. In CMVEC from mice and newborn pigs, 15 ng/ml TNF-alpha alone, or with 10 microg/ml cycloheximide (CHX) caused apoptosis detected by nuclear translocation of p65 NF-kappaB, caspase-3 activation, DNA fragmentation, cell-cell contact destabilization, and cell detachment. TNF-alpha did not induce HO-1 expression in CMVEC. CMVEC from HO-2 knockout mice showed greater sensitivity to apoptosis caused by serum deprivation and TNF-alpha than did wild-type mice. TNF-alpha increased reactive oxygen species generation, including hydrogen peroxide and superoxide radicals, as detected by dihydrorhodamine-123 and dihydroethidium. The TNF-alpha response was inhibited by superoxide dismutase and catalase suggesting apoptosis is oxidative stress related. Inhibition of endogenous HO-2 in newborn pig CMVEC increased oxidative stress and exaggerated apoptosis caused by serum deprivation and TNF-alpha. In HO-1-overexpressing CMVEC (HO-1 selective induction by cobalt portophyrin), TNF-alpha did not cause apoptosis. A CO-releasing compound, CORM-A1, and bilirubin blocked TNF-alpha-induced reactive oxygen species accumulation and apoptosis consistent with the antioxidant and antiapoptotic roles of the end products of HO activity. We conclude that HO-2 is critical for protection of cerebrovascular endothelium against apoptotic changes induced by oxidative stress and cytokine-mediated inflammation.

Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection.

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1-2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-kappaB nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 microM), and by bilirubin (1 microM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium.

Epileptic seizures cause extended postictal cerebral vascular dysfunction that is prevented by HO-1 overexpression.

The extended postictal state is characterized by neurological problems in patients. Inadequate blood supply to the brain and impaired cerebral autoregulation may contribute to seizure-induced neuronal damage. Recent evidence in newborn pigs indicates that activation of the antioxidative enzyme heme oxygenase (HO) at the onset of seizures is necessary for increased cerebral blood flow during the ictal episode and for normal cerebral vascular functioning during the immediate postictal period. We hypothesized that seizures cause prolonged postictal cerebral vascular dysfunction that can be accentuated by HO inhibition and rescued by HO overexpression. Cerebral vascular responses to endothelium-dependent (hypercapnia, bradykinin) and -independent (isoproterenol, sodium nitroprusside) stimuli were assessed 48 h after bicuculline-induced seizures in: 1) saline-control newborn piglets, 2) HO-inhibited animals (HO was inhibited by tin protoporphyrin, SnPP, 3 mg/kg iv), and 3) HO-overexpressing piglets (HO-1 was upregulated by cobalt protoporphyrin, CoPP, 50 mg/kg ip). Extended alterations of HO expression in cerebral microvessels were confirmed by measuring CO production and inducible HO (HO-1) and constitutive HO (HO-2) proteins. Our data provide evidence that seizures cause a severe, sustained, postictal cerebral vascular dysfunction as reflected by impaired vascular reactivity to physiologically relevant dilators. During the delayed postictal state, vascular reactivity to all dilator stimuli was reduced in saline control and, to a greater extent, in HO-inhibited animals. In CoPP-treated piglets, no reduction in postictal cerebral vascular reactivity was observed. These findings may indicate that CoPP prevents postictal cerebral vascular dysfunction by upregulating HO-1, a finding that might have implications for preventing postictal neurological complications.

Carbon monoxide activates KCa channels in newborn arteriole smooth muscle cells by increasing apparent Ca2+ sensitivity of alpha-subunits.

Carbon monoxide (CO) is a gaseous vasodilator produced by many cell types, including endothelial and smooth muscle cells. The goal of the present study was to investigate signaling mechanisms responsible for CO activation of large-conductance Ca(2+)-activated K(+) (K(Ca)) channels in newborn porcine cerebral arteriole smooth muscle cells. In intact cells at 0 mV, CO (3 microM) or CO released from dimanganese decacarbonyl (10 microM), a novel light-activated CO donor, increased K(Ca) channel activity 4.9- or 3.5-fold, respectively. K(Ca) channel activation by CO was not blocked by 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (25 microM), a soluble guanylyl cyclase inhibitor. In inside-out patches at 0 mV, CO shifted the Ca(2+) concentration-response curve for K(Ca) channels leftward and decreased the apparent dissociation constant for Ca(2+) from 31 to 24 microM. Western blotting data suggested that the low Ca(2+) sensitivity of newborn K(Ca) channels may be due to a reduced beta-subunit-to-alpha-subunit ratio. CO activation of K(Ca) channels was Ca(2+) dependent. CO increased open probability 3.7-fold with 10 microM free Ca(2+) at the cytosolic membrane surface but only 1.1-fold with 300 nM Ca(2+). CO left shifted the current-voltage relationship of cslo-alpha currents expressed in HEK-293 cells, increasing currents 2.2-fold at +50 mV. In summary, data suggest that in newborn arteriole smooth muscle cells, CO activates low-affinity K(Ca) channels via a direct effect on the alpha-subunit that increases apparent Ca(2+) sensitivity. The optimal tuning by CO of the micromolar Ca(2+) sensitivity of K(Ca) channels will lead to preferential activation by signaling modalities, such as Ca(2+) sparks, which elevate the subsarcolemmal Ca(2+) concentration within this range.

Carbon monoxide dilates cerebral arterioles by enhancing the coupling of Ca2+ sparks to Ca2+-activated K+ channels.

Carbon monoxide (CO) is generated endogenously by the enzyme heme oxygenase. Although CO is a known vasodilator, cellular signaling mechanisms are poorly understood and are a source of controversy. The goal of the present study was to investigate mechanisms of CO dilation in porcine cerebral arterioles. Data indicate that exogenous or endogenously produced CO is a potent activator of large-conductance Ca2+-activated K+ (K(Ca)) channels and Ca2+ spark-induced transient K(Ca) currents in arteriole smooth muscle cells. In contrast, CO is a relatively poor activator of Ca2+ sparks. To understand the apparent discrepancy between potent effects on transient K(Ca) currents and weak effects on Ca2+ sparks, regulation of the coupling relationship between these events by CO was investigated. CO increased the percentage of Ca2+ sparks that activated a transient K(Ca) current (ie, the coupling ratio) from approximately 62% in the control condition to 100% and elevated the slope of the amplitude correlation between these events approximately 2.6-fold, indicating that Ca2+ sparks induced larger amplitude transient K(Ca) currents in the presence of CO. This signaling pathway for CO is physiologically relevant because ryanodine, a ryanodine-sensitive Ca2+ release channel blocker that inhibits Ca2+ sparks, abolished CO dilation of pial arterioles in vivo. Thus, CO dilates cerebral arterioles by priming K(Ca) channels for activation by Ca2+ sparks. This study presents a novel dilatory signaling pathway for CO in the cerebral circulation and appears to be the first demonstration [corrected] of a vasodilator that acts by increasing the effective coupling of Ca2+ sparks to K(Ca) channels.