Signal Transduction in Cerebral Arteries after Subarachnoid Hemorrhage—A Phosphoproteomic Approach

Abstract

After subarachnoid hemorrhage (SAH), pathologic changes in cerebral arteries contribute to delayed cerebral ischemia and poor outcome. We hypothesize such changes are triggered by early intracellular signals, targeting of which may prevent SAH-induced vasculopathy. We performed an unbiased quantitative analysis of early SAH-induced phosphorylations in cerebral arteries and evaluated identified signaling components as targets for prevention of delayed vasculopathy and ischemia. Labeled phosphopeptides from rat cerebral arteries were quantified by high-resolution tandem mass spectrometry. Selected SAH-induced phosphorylations were validated by immunoblotting and monitored over a 24-hour time course post SAH. Moreover, inhibition of key phosphoproteins was performed. Major SAH-induced phosphorylations were observed on focal adhesion complexes, extracellular regulated kinase 1/2 (ERK1/2), calcium calmodulin-dependent kinase II, signal transducer and activator of transcription (STAT3) and c-Jun, the latter two downstream of ERK1/2. Inhibition of ERK1/2 6-hour post SAH prevented increases in cerebrovascular constrictor receptors, matrix metalloprotease-9, wall thickness, and improved neurologic outcome. STAT3 inhibition partially mimicked these effects. The study shows that quantitative mass spectrometry is a strong approach to study in vivo vascular signaling. Moreover, it shows that targeting of ERK1/2 prevents delayed pathologic changes in cerebral arteries and improves outcome, and identifies SAH-induced signaling components downstream and upstream of ERK1/2.

Controversies and evolving new mechanisms in subarachnoid hemorrhage

“…Therefore, the combination of treatment with an inhibitor of RhoA, pitavastatin, and an inhibitor of Rho-kinase, fasudil, could extensively prevent cerebral vasospasm after SAH (Naraoka et al, 2013). Furthermore, phosphorylations trigger SAH-induced vasculopathy in cerebral arteries, as determined by quantitative mass spectrometry, including focal adhesion complexes, ERK1/2, calcium calmodulin-dependent kinase II, STAT3, and c-Jun (Parker et al, 2013). Tenascin-C, a matricellular protein, induces cerebral vasospasm via TLR4 and activation of JNK and p38 (Fujimoto et al, 2013; Suzuki et al, 2013).…”

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development

“…The reaction was diluted 5-fold with 100 mM TEAB and digested with trypsin (1:50 trypsin/protein) overnight at 37°C. Peptide preparation, stable isotope labeling with isobaric tags for relative and absolute quantitation (iTRAQ; AB Sciex, Framingham, MA), peptide fractionation, and nano-reverse phase liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) was performed as described previously (30). Briefly, 100 g of peptide was labeled with iTRAQ according to the manufacturer's instructions and desalted with hydrophilic-lipophilic balance solid phase extraction (Waters; Milford, MA).…”

Inhibition of mitogen‐activated protein kinase 1/2 in the acute phase of stroke improves long‐term neurological outcome and promotes recovery processes in rats

“…, Parker et al . ). In addition, it has been demonstrated that U0126 treatment in the acute phase following experimental stroke blocks the phosphorylation of ERK1/2 48 h post‐stroke (Maddahi & Edvinsson ).…”

“…It is demonstrated that U0126 specifically inhibits mitogen-activated protein kinase kinase (MEK) 1/2 and thus inhibits activation of ERK1/2 both in vitro and in vivo , Ahnstedt et al 2015. We have shown that treatment with the MEK1/2 inhibitor U0126 reduces infarct size after experimental focal ischaemic stroke (Henriksson et al 2007) and alleviates neurological deficits 2 days after experimental subarachnoid haemorrhage (Larsen et al 2011, Parker et al 2013). In addition, it has been demonstrated that U0126 treatment in the acute phase following experimental stroke blocks the phosphorylation of ERK1/2 48 h post-stroke (Maddahi & Edvinsson 2008).…”