Angiogenesis is a complex process that involves the activation of quiescent endothelial cells (ECs) to a proliferative and migratory phenotype and, subsequently, their redifferentiation to form vascular tubes. We hypothesized that NO contributes to angiogenesis by terminating the proliferative action of angiogenic growth factors and initiating a genetic program of EC differentiation. Human umbilical vein ECs (HUVECs) and calf pulmonary artery ECs (CPAECs) were grown directly on plastic dishes or on three-dimensional fibrin matrices. In the absence of fibrin, treatment with NO-donor compounds, such as S-nitroso-N-acetylpenicillamine (SNAP, 0.1 and 0.4 mmol/L), produced a dose-dependent inhibition of proliferation in both cell lines, whereas the inhibition of endogenous NO production using N G-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L) or N G-monomethyl-L-arginine (L-NMMA, 1 mmol/L) significantly increased proliferation of the CPAECs. The addition of basic fibroblast growth factor (bFGF, 30 ng/mL) increased the expression of endothelial NO synthase mRNA and the production of NO in both cell types when cultured on three-dimensional fibrin gels and produced profound morphological changes characterized by the appearance of extensive capillary-like vascular structures and the loss of EC monolayers. These changes were quantified by measuring total tube length per low-power field (100), and a differentiation index was derived using the ratio of tube length over area covered by residual EC monolayer. In the absence of additional angiogenic factors, the differentiation index was low for both HUVECs and CPAECs (control, 1.160.19 and 2.070.87, respectively). Treatment with bFGF increased the differentiation index significantly in both cell types (10.592.03 and 20.025.01 for HUVECs and CPAECs, respectively; P.05 versus control), and the addition of SNAP (0.4 mmol/L) mimicked the angiogenic response to bFGF (8.571.34 and 12.203.49 for HUVECs and CPAECs, respectively; P.05 versus control). Moreover, L-NAME inhibited EC tube formation in response to bFGF in a dose-response manner, consistent with a role of endogenous NO production in EC differentiation in this angiogenic model. These findings suggest that NO may act as a crucial signal in the angiogenic response to bFGF, terminating the proliferative actions of angiogenic growth factors and promoting EC differentiation into vascular tubes. (Circ Res. 1998;82:1007-1015.)

The constitutive expression of endothelial nitric oxide (NO) synthase (cNOS) is essential for the physiological regulation of vascular tone and structure. The mechanism of downregulation of steady state cNOS mRNA in human umbilical vein endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha) was investigated by using Northern blot analysis of total cellular RNA. TNF-alpha produced a dose- and time-dependent decrease in cNOS mRNA expression that was near maximal at 10 U/mL and 6 hours of exposure, respectively. In contrast, steady state expression of endothelin-1 and plasminogen activator inhibitor-1 (PAI-1) mRNA was upregulated by TNF-alpha. The pharmacological generation of NO using sodium nitroprusside (10 mumol/L) and S-nitroso-acetylpenicillamine (100 to 400 mumol/L) had no effect on cNOS mRNA levels, and TNF-alpha-induced downregulation of cNOS was not prevented by coincubation with the inhibitors of NO synthesis N omega-nitro-L-arginine methyl ester (1 mmol/L) and NG-monomethyl L-arginine (10 mmol/L). Under control conditions, cNOS and PAI-1 mRNA were stable after treatment with actinomycin D for periods greater than 24 hours, whereas endothelin-1 message was rapidly degraded (half-life, < 1 hour). Pretreatment with TNF-alpha (30 U/mL) selectively reduced that half-life of cNOS mRNA to less than 12 hours without altering the stability of PAI-1 message. TNF-alpha-induced destabilization of cNOS mRNA could be partially prevented by coincubation with cycloheximide (1 mumol/L) but was not reproduced by addition of sodium nitroprusside.(ABSTRACT TRUNCATED AT 250 WORDS)

Background and Purpose-Endothelin 1 (ET-1) is a potent vasoconstrictor that may play a role in cerebral vasospasm following subarachnoid hemorrhage (SAH). However, data regarding its pathogenic role in the development of vasospasm are controversial. We planned a prospective, observational clinical study to investigate the temporal relationship between increased ET-1 production and cerebral vasospasm or other neurological sequelae after SAH. Methods-ET-1 levels in cerebrospinal fluid (CSF) were measured in 20 SAH patients from admission (within 24 hours from the bleeding) until day 7. Patients received a daily transcranial Doppler study and a neurological evaluation. On day 7, angiography was performed to verify the degree and extent of vasospasm. Patients were then classified as having (1) clinical vasospasm, (2) angiographic vasospasm, (3) no vasospasm, or (4) poor neurological condition without significant vasospasm (low Glasgow Coma Scale score [GCS]). Results-On admission, ET-1 levels were increased in the low-GCS group compared with the other groups (Pϭ0.04). On day 4 ET-1 levels were not significantly different among groups, whereas on day 7 ET-1 levels were significantly increased in both the clinical vasospasm and low-GCS groups compared with the angiographic vasospasm and no vasospasm groups (PϽ0.005). Moreover, when the low-GCS group was excluded, there was a significant relationship between vasospasm grade and CSF ET-1 levels (R 2 ϭ0.73). Conclusions-CSF ET-1 levels were markedly elevated in patients with clinical manifestations of vasospasm (day 7) and with a poor neurological condition not related to vasospasm. However, ET-1 levels were low in clinical vasospasm patients before clinical symptoms were evident (day 4) and remained low in angiographic vasospasm patients throughout the study period. Thus, our data suggest that CSF ET-1 levels are increased in conditions of severe neuronal damage regardless whether this was due to vasospasm or to the primary hemorrhagic event. In addition, CSF ET-1 levels paralleled the neurological deterioration but were not predictive of vasospasm. Key Words: cerebral ischemia Ⅲ cerebral vasospasm Ⅲ endothelins Ⅲ subarachnoid hemorrhage Ⅲ ultrasonography, Doppler, transcranial D elayed cerebral vasospasm is one of the most serious consequences following aneurysmal subarachnoid hemorrhage (SAH). 1 The incidence of angiographic vasospasm is in excess of 50%, with symptomatic vasospasm occurring in 30% of patients. 2 Progression to cerebral infarction occurs in 50% of symptomatic cases. 3 Because of this high morbidity rate, considerable research efforts have been directed at identifying the mechanism of vasospasm and in identifying potential candidate factors. Endothelin (ET) is a family of 3 vasoconstrictor isopeptides with common structural features (ET-1, ET-2, ET-3) expressed by several cell types in the brain, including neurons, glial cells, and macrophages. 4,5 Experimental studies have shown that ET exerts a potent and See Editorial Comment, page 1189 long-last...