Cytosolic Ca²⁺Oscillations in Human Cerebrovascular Endothelial Cells after Subarachnoid Hemorrhage

Abstract

Molecular mechanisms of cerebral vasospasm after subarachnoid hemorrhage (SAH) include specific modes of cell signaling like activation of nuclear factor (NF)-jB and vascular cell adhesion molecules (VCAM)-1 expression. The study's hypothesis is that cisternal cerebral spinal fluid (CSF) from patients after SAH may cause Ca 2 + oscillations which induce these modes of vascular inflammation in an in vitro model of human cerebral endothelial cells (HCECs). HCECs were incubated with cisternal CSF from 10 SAH patients with confirmed cerebral vasospasm. The CSF was collected on days 5 and 6 after hemorrhage. Cytosolic Ca 2 + concentrations and cell contraction as an indicator of endothelial barrier function were examined by fura-2 microflurometry. Activation of NF-jB and VCAM-1 expression were measured by immunocytochemistry. Incubation of HCEC with SAH-CSF provoked cytosolic Ca 2 + oscillations (0.31±0.09 per min), cell contraction, NF-jB activation, and VCAM-1 expression, whereas exposure to native CSF had no significant effect. When endoplasmic reticulum (ER) Ca 2 + -ATPase and ER inositol trisphosphate (IP3)-sensitive Ca 2 + channels were blocked by thapsigargin or xestospongin, the frequency of the Ca 2 + oscillations was reduced significantly. In analogy to the reduction of Ca 2 + oscillation frequency, the blockers impaired HCEC contraction, NF-jB activation, and VCAM-1 expression. Cisternal SAH-CSF induces cytosolic Ca 2 + oscillations in HCEC that results in cellular constriction, NF-jB activation, and VCAM-1 expression. The Ca 2 + oscillations depend on the function of ER Ca 2 + -ATPase and IP3-sensitive Ca 2 + channels.

Vascular Endothelial Growth Factor Stimulates Endothelial Colony Forming Cells Proliferation and Tubulogenesis by Inducing Oscillations in Intracellular Ca2+ Concentration

“…Because the Ca 2þ waves in adjacent cells are not synchronous, the average response recorded from a cell population will display an initial transient peak followed by a sustained plateau, which is actually due to the asynchronous oscillations in [Ca 2þ ] i in individual cells. Such variability in the Ca 2þ response to extracellular hormones or growth factors has already been reported in a number of ECs both in vitro [29,38,49] and in situ [50]. Moreover, the differential activation of jagged1 and DII4 in the Notch signaling pathway and of mitogen activated protein kinases has already been described in ECs exposed to the same VEGF concentration [51,52].…”

“…In nonstimulated cells, NF-jB is retained in the cytosol by the complex with the inhibitory protein, I jB , which masks its nuclear localization signals [34,35]. [Ca 2þ ] i oscillations induce a phosphorylation cascade which is mediated by Ca 2þ /calmodulin-dependent protein kinases and leads I jB to site-specific ubiquitination and subsequent degradation [34][35][36][37][38][39]. Thus, NF-jB is released from inhibition and translocates into the nucleus, where it activates the transcriptional program responsible for cell growth and tubulogenesis [34][35][36][37].…”

Update on vascular endothelial Ca2+signalling: A tale of ion channels, pumps and transporters

“…Indeed, when the information embedded within the [Ca 2+ ]i increase must be conveyed over a long time, both the elementary events and the global Ca 2+ sweeps can adopt an oscillatory pattern [16,72,339,396] . However, the physiological outcome of local Ca 2+ spikes depends on the Ca 2+ -sensitive decoders located within a few nanometers from the channel pore [395] , and the frequency of repetitive Ca 2+ waves encodes for the selective engagement of specific downstream targets [403][404][405] . The interspike interval of the Ca 2+ transients is inversely correlated to the strength of the stimulus until, at least at high agonist doses, they apparently fuse to generate a plateau level [16,72,406,407] .…”

“…Unlike the oscillatory Ca 2+ response to extracellular ligands [72,406,407] , the amplitude of the Ca 2+ spikes increases with the intensity of the mechanical stimulation [22][23][24][25] . Agonist-evoked Ca 2+ oscillations require PLC activation by either GPCRs or TKRs [72,339,404,405,408] . The following InsP3-dependent Ca 2+ mobilization may be amplified by adjacent RyRs via CICR [44,134] and requires Ca 2+ inflow to supply releasable Ca 2+ over time and maintain InsP3R sensitivity to the ligand [72,407,409,410] .…”

“…The following InsP3-dependent Ca 2+ mobilization may be amplified by adjacent RyRs via CICR [44,134] and requires Ca 2+ inflow to supply releasable Ca 2+ over time and maintain InsP3R sensitivity to the ligand [72,407,409,410] . Ca 2+ entering through either SOCE or the reverse-mode of NCX is sequestered by SERCA into ER lumen before being released again into the cytosol through the open InsP3Rs [72,134,339,404,405] . Some [16] , but not all [72] , microvascular ECs may be independent on Ca 2+ inflow.…”