Physiological cerebrovascular remodeling in response to chronic mild hypoxia: A role for activated protein C

Abstract

Activated protein C (APC) is a serine protease that promotes favorable changes in vascular barrier integrity and post-ischemic angiogenic remodeling in animal models of ischemic stroke, and its efficacy is currently being investigated in clinical ischemic stroke trials. Interestingly, application of sub-clinical chronic mild hypoxia (CMH) (8% O2) also promotes angiogenic remodeling and increased tight junction protein expression, suggestive of enhanced blood-brain barrier (BBB) integrity, though the role of APC in mediating the influence of CMH has not been investigated. To examine this potential link, we studied CMH-induced cerebrovascular remodeling after treating mice with two different reagents: (i) a function-blocking antibody that neutralizes APC activity, and (ii) exogenous recombinant murine APC. While CMH promoted endothelial proliferation, increased vascular density, and upregulated the angiogenic endothelial integrins α5β1 and αvβ3, these events were almost completely abolished by functional blockade of APC. Consistent with these findings, addition of exogenous recombinant APC enhanced CMH-induced endothelial proliferation, expansion of total vascular area and further enhanced the CMH-induced right-shift in vessel size distribution. Taken together, our findings support a key role for APC in mediating physiological remodeling of cerebral blood vessels in response to CMH.

Keywords: Activated protein C (APC); Angiogenesis; Chronic mild hypoxia (CMH); Endothelium; Fibronectin; Integrin; Vascular biology.

Figure 1

Hypoxic-induced cerebrovascular remodeling is inhibited by functional blockade of endogenous APC. A. Dual-IF was performed on frozen sections of frontal lobe from mice exposed to 4 days normoxia or chronic mild hypoxia (CMH) that had been injected with a monoclonal anti-APC antibody (SPC-54) or heat-denatured antibody (control) using antibodies specific for CD31 (AlexaFluor-488, green) or Ki67 (Cy3, red). Scale bar = 100μm. B–C. Quantification of the number of CD31/Ki67dual-positive cells (B) or total vessel area (C). All experiments were performed with six animals per condition, and the results expressed either as the mean ± SEM of dual-positive cells (B) or as the mean ± SEM of the % change compared to normoxic conditions (C). Note that hypoxic-induced increases in endothelial cell proliferation and total vascular area were strongly inhibited by the anti-APC antibody. ** P < 0.01, ***P < 0.005.

Figure 2

Hypoxic-induced upregulation of endothelial α5 integrin is inhibited by functional blockade of endogenous APC. A. Dual-IF was performed on frozen sections of the frontal lobe from mice exposed to 4 days normoxia or chronic mild hypoxia (CMH) that had been injected with a monoclonal anti-APC antibody (SPC-54) or heat-denatured antibody (controls) using antibodies specific for CD31 (AlexaFluor-488, green) or α5 integrin (Cy3, red). Scale bar = 100μm. B. Quantification of the number of α5 integrin-positive vessels per field. All experiments were performed with six animals per condition, and the results expressed either as the mean ± SEM of α5 integrin-positive vessels per field. Note that hypoxic-induction of endothelial α5 integrin expression was strongly inhibited by the anti-APC antibody. ** P < 0.01.

Figure 3

Hypoxic-induced upregulation of endothelial β3 integrin is inhibited by functional blockade of endogenous APC. A. Dual-IF was performed on frozen sections of the frontal lobe from mice exposed to 4 days normoxia or chronic mild hypoxia (CMH) that had been injected with a monoclonal anti-APC antibody (SPC-54) or heat-denatured antibody (controls) using antibodies specific for CD31 (AlexaFluor-488, green) or β3 integrin (Cy3, red). Scale bar = 100μm. B. Quantification of the number of β3 integrin-positive vessels per field. All experiments were performed with six animals per condition, and the results expressed either as the mean ± SEM of β3 integrin-positive vessels per field. Note that the hypoxic-induction of endothelial β3 integrin expression was strongly inhibited by the anti-APC antibody. *** P < 0.005.

Figure 4

Hypoxic-induced cerebrovascular remodeling is enhanced by exogenous recombinant murine APC. A. Dual-IF was performed on frozen sections of the frontal lobe from mice exposed to 4 days normoxia or chronic mild hypoxia (CMH) that had been injected with recombinant murine APC or PBS vehicle (controls) using antibodies specific for CD31 (AlexaFluor-488, green) or Ki67 (Cy3, red). Scale bar = 100μm. B–C. Quantification of the number of CD31/Ki67dual-positive cells (B) or total vessel area (C). All experiments were performed with six animals per condition, and the results expressed either as the mean ± SEM of dual-positive cells (B) or as the mean ± SEM of the % change compared to normoxic conditions (C). Note that addition of recombinant APC enhanced the hypoxic-induced increases in endothelial cell proliferation and total vascular area. *P < 0.05, ** P < 0.01.

Figure 5

Exogenous APC enhances the right-shift in vessel size distribution following chronic mild hypoxia (CMH). Frozen sections of frontal lobe taken from mice exposed to normoxia or CMH for 2.5 or 4 days were immunostained for CD31, images captured, and vessel size distribution analysis performed using Volocity software. All points represent the mean ± SEM of three subjects. Note that 4 days CMH produced a right-shift in vessel size distribution, resulting in increased numbers of large area vessels in the 200–500 μm² and > 500μm² categories and that exogenous APC significantly increased the number of large (> 500 μm²) area vessels, while at the same time, showing a trend towards decreasing the number of small area vessels in the 0–100 μm² and 100–200 μm² categories. *P < 0.05, ** P < 0.02.