Abstract
BackgroundChronic mild hypoxia (CMH, 8% O2) stimulates robust vascular remodelling in the brain, but it also triggers transient vascular disruption. This raises the fundamental question: is the vascular leak an unwanted side-effect of angiogenic remodelling or is it a pathological response, unrelated to endothelial proliferation, in which declining oxygen levels trigger endothelial dysfunction?MethodsTo answer this question, mice were exposed to CMH (8% O2) for periods up to 14 days, after which, brain tissue was examined by immunofluorescence (IF) to determine which type of blood vessel (arteriole, capillary or venule) was most commonly associated with endothelial proliferation and vascular leak and how this correlated with tight junction protein expression. Vascular perfusion was examined using DiI. Data were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test.ResultsThe following was observed: (1) most endothelial proliferation and extravascular fibrinogen leak occurred in capillaries and to a lesser degree in venules, (2) much to our surprise, endothelial proliferation and extravascular fibrinogen leak never colocalized, (3) interestingly however, endothelial proliferation was strongly associated with an intravascular fibrinogen staining pattern not seen in stable blood vessels, (4) DiI perfusion studies revealed that angiogenic vessels were adequately perfused, suggesting that fibrinogen retention in angiogenic vessels is not due to temporary closure of the vessel, but more likely because fibrinogen is retained within the vessel wall, (5) bromodeoxyuridine (BrdU) labelling as a means to more permanently label proliferating endothelial cells, confirmed lack of any connection between endothelial proliferation and extravascular fibrinogen leak, while (6) in contrast, proliferating microglia were detected within extravascular leaks.ConclusionsTaken together, our findings support the concept that in the short-term, hypoxia-induced endothelial proliferation triggers transient fibrinogen deposition within the walls of angiogenic blood vessels, but no overt vascular leak occurs in these vessels. Importantly, endothelial proliferation and extravascular fibrinogen leaks never co-localize, demonstrating that extravascular leak is not an unwanted side-effect of angiogenic endothelial proliferation, but rather a dysfunctional vascular response to hypoxia that occurs in a distinct group of non-angiogenic blood vessels.
Highlights
Cerebral blood vessels are unique in having high electrical resistance and low permeability, which protects sensitive cells in the brain neuropil from potentially disruptive and harmful blood components [1,2,3]
Chronic mild hypoxia (CMH) induces transient vascular leak predominantly in capillaries In this study we used a panel of markers to identify the three main types of cerebral blood vessel; the panendothelial marker CD31 which labels all vessel types, laminin-111, which has been shown to identify arterioles and venules but not capillaries [13], and the smooth muscle cell marker α-SMA which labels arterioles (Fig. 1) [14]
To determine which type of cerebral blood vessel is disrupted by chronic mild hypoxia (CMH, 8% O 2), we performed CD31/fibrinogen dual-immunofluorescence on frozen brain sections taken from mice exposed to 4 days CMH (Fig. 2A and B)
Summary
Cerebral blood vessels are unique in having high electrical resistance and low permeability, which protects sensitive cells in the brain neuropil from potentially disruptive and harmful blood components [1,2,3]. If the hypoxia state is maintained, over days to weeks, a series of chronic adaptations occur, which include enhanced hematocrit, to increase the oxygen carrying capacity of the blood, as well as active remodelling of the cerebral vasculature, which increases the vessel density and complexity, so that every cell in the brain is physically closer to a blood vessel [8, 9] In the laboratory, this point is well illustrated in the chronic mild hypoxia (CMH) model, which demonstrates that when rodents are exposed to mild hypoxia (typically 8–10% O2) for prolonged periods of time, their cerebral blood vessels mount a strong vascular remodelling response, resulting in greater than 50% enhancement of vessel density over a period of two weeks [8, 9]. This raises the fundamental question: is the vascular leak an unwanted side-effect of angiogenic remodelling or is it a pathological response, unrelated to endothelial proliferation, in which declining oxygen levels trigger endothelial dysfunction?
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