P66 Shc Contributes to Susceptibility to Cerebrovascular Injury and Intracerebral Hemorrhages

Abstract

BackgroundBreakdown of the blood brain barrier (BBB) and loss of cerebral vessels autoregulation in response to elevated systemic pressure are closely linked to intracerebral hemorrhage and micro bleeds. Presently no established models to study the causes of intracerebral hemorrhage exist that does not rely on surgical interventions and existing models have failed to improve clinical understanding of this condition. Overproduction of reactive oxygen species (ROS) following ischaemia/reperfusion (I/R) is considered a key mechanism leading to BBB damage. It has been shown that the adaptor protein p66(Shc) is crucial regulator for ROS production in several disease states including I/R injury of the brain. p66shc when activated translocates to the mitochondria where it sequesters cytochrome C and generates hydrogen peroxide. The contribution of mitochondria to myogenic tone and autoregulation of the cerebral vasculature is not well defined.We have found previously that rats expressing constitutively active form of p66 Shc (Shc‐CA) have decreased long term survival and increased incidences of stoke despite no significant difference in systemic blood pressure.HypothesisWe hypothesis that p66Shc signaling via mitochondrial translocation contribute to vascular dysfunction of cerebral arteries and intracerebral hemorrhage in hypertensive rats by decreasing mitochondrial ATP while elevating mitochondrial ROS.ResultsVessels from WT rats, lacking p66 Shc protein (Shc‐KO), or expressing Shc‐CA were used for video microscopy. Endothelial dependent dilation to flow was assessed in vessels from Shc‐KO animals compared to their littermate controls (%Max Dilation 100 cm H2O: WT = 1.3±3 vs. Shc‐KO=39±3*; N≥3 P<0.05 one way ANOVA). Shc‐CA failed to develop tone spontaneously or to pharmacological stimuli (as a result no dilator function could be studied) suggesting a defect in vascular smooth muscle function. No difference in tone were observed in WT vs. Shc‐KO. Further evaluation revealed loss of myogenic tone in MCAs of Shc‐CA (% Tone at 150 cm H2O relative to Ca2+ free: WT = 19±6; Shc‐KO = 28±12; Shc‐CA =3±2, n≥2). In vivo cerebral vascular flow was evaluated using Doppler laser probe and revealed an inability of cerebral vessels to respond to altered systemic pressure in vivo. Shc‐CA showed decreased levels of ATP (pmol/mg Protein WT=1±0.2; Shc‐KO=0.7±0.2; Shc‐CA=0.1±0.1*; N≥3 P<0.05 one way ANOVA) and mitochondrial respiration, confirming decreased mitochondrial function in rats that express Shc‐CA.To investigate an underling physiological relevance, rats were perfused with methylene blue (MB). A substantial increase in MB staining in the brain of Shc‐CA was observed, suggesting an increase in permeability of the BBB.