Chronic cerebral hypoxia promotes arteriogenic remodeling events that can be identified by reduced endoglin (CD105) expression and a switch in β1 integrins

Abstract

Chronic cerebral hypoxia leads to a strong vascular remodeling response, though little is known about which part of the vascular tree is modified, or whether this response includes formation of new arterial vessels. In this study, we examined this process in detail, analyzing how hypoxia (8% O(2) for 14 days) alters the size distribution of vessels, number of arteries/arterioles, and expression pattern of endoglin (CD105), a marker of angiogenic endothelial cells in tumors. We found that cerebral hypoxia promoted the biggest increase in the number of medium to large size vessels, and this correlated with increased numbers of alpha smooth muscle actin (α-SMA)-positive arterial vessels. Surprisingly, hypoxia induced a marked reduction in CD105 expression on brain endothelial cells (BECs) within remodeling arterial vessels, and these BECs also displayed an angiogenic switch in β1 integrins (from α6 to α5), previously described for developmental angiogenesis. In vitro, transforming growth factor (TGF)-β1 also promoted this switch of BEC β1 integrins. Together, these results show that cerebral hypoxia promotes arteriogenesis, and identify reduced CD105 expression as a novel marker of arteriogenesis. Furthermore, our data suggest a mechanistic model whereby BECs in remodeling arterial vessels downregulate CD105 expression, which alters TGF-β1 signaling, to promote a switch in β1 integrins and arteriogenic remodeling.

Figure 1

Differential expression profiles of CD31 and CD105 on blood vessels in the hypoxic central nervous system (CNS). (A, B) Dual-immunofluorescence (Dual-IF) was performed on frozen sections of the frontal lobe from mice exposed to normoxia or 4, 7, or 14 days hypoxia using antibodies specific for CD31 (DyLight 594, red), or CD105 (AlexaFluor-488, green). Scale bars=100 μm (A) and 20 μm (B). Note that CD31 was uniformly expressed by vessels at all stages of the hypoxic response. CD105 expression totally overlapped with CD31 on all vessels in the normoxic CNS. However in the hypoxic CNS, CD105 staining was bright on linear vessels, but fell to a much lower level on loosely aggregated vascular structures (see arrows). This point is well illustrated in (B), which displays a linear compact vessel expressing high levels of CD31 and CD105, and a loosely organized vessel expressing markedly reduced levels of CD105. (C) Quantification of low-CD105 vessels. Experiments were performed with three different animals per condition, and the results expressed as the mean±s.e.m. of the % of low-CD105 vessels per field. Note that in both the frontal lobe and medulla, hypoxia promoted the appearance of low-CD105 vessels, which peaked between 4 and 7 days hypoxia. ^**P<0.005, ^***P<0.001. The color reproduction of this figure is available on the Journal of Cerebral Blood Flow and Metabolism journal online.

Figure 2

The influence of cerebral hypoxia on vessel size distribution. Frozen sections of frontal lobe taken from mice exposed to normoxia or 4, 7, or 14 days hypoxia were immunostained for CD31, photographs taken, and vessel size distribution analysis performed using Volocity software. All points represent the mean±s.e.m. of three subjects. Note that 14 days hypoxia led to a downward trend in the number of vessels μm² but in contrast, the number of larger vessels between 200 and 500 μm² and those >500 μm² both showed a significant increase. ^*P<0.05.

Figure 3

Hypoxia stimulates cerebral arteriogenesis. (A) Dual-immunofluorescence (Dual-IF) was performed on frozen sections of the frontal lobe from mice exposed to normoxia or 4, 7, or 14 days hypoxia using antibodies specific for CD31 (AlexaFluor-488, green) or alpha smooth muscle actin (α-SMA) (Cy-3, red). Scale bar=100 μm. Note that α-SMA was predominantly expressed by larger caliber vessels, comprising ∼10% of vessels, and that cerebral hypoxia induced a marked increase in the number of α-SMA-positive vessels. (B) Quantification of the number of α-SMA+ vessels. Experiments were performed with three different animals per condition, and the results expressed as the mean±s.e.m. of the number of α-SMA+ vessels per field. Note that in both the frontal lobe and medulla, hypoxia significantly promoted the appearance of α-SMA+ vessels. ^*P<0.05, ^**P<0.005. The color reproduction of this figure is available on the Journal of Cerebral Blood Flow and Metabolism journal online.

Figure 4

Arteriogenic centers downregulate CD105 expression during cerebral hypoxia. Dual-immunofluorescence (Dual-IF) was performed on frozen sections of the frontal lobe from mice exposed to normoxia or 4, 7, or 14 days hypoxia using antibodies specific for alpha smooth muscle actin (α-SMA) (Cy-3, red) or CD105 (AlexaFluor-488, green). Scale bar=100 μm. Note that in the normoxic CNS, CD105 was expressed at equivalent levels by α-SMA-positive and α-SMA-negative vessels. However, after 4 or 7 days hypoxia, CD105 expression on α-SMA-positive vessels was markedly reduced, to the point of being barely visible on some vessels. This is well illustrated in the merged figures, which under normoxic and 14 day hypoxic conditions, which show α-SMA+ vessels as yellow, but under 4 and 7 day hypoxic conditions, as being only red. The color reproduction of this figure is available on the Journal of Cerebral Blood Flow and Metabolism journal online.

Figure 5

Cerebral hypoxia promotes a switch in endothelial β1 integrins within remodeling arterial vessels. (A). Dual-immunofluorescence (Dual-IF) was performed on frozen sections of the frontal lobe from mice exposed to normoxia or 7 days hypoxia using antibodies specific for the α5 or α6 integrin subunits (AlexaFluor-488, green) or alpha smooth muscle actin (α-SMA) (Cy-3, red). Scale bar=50 μm. Note that cerebral hypoxia induced marked changes in the relative expression of these integrins on α-SMA-positive vessels. After 7 days hypoxia, α-SMA-positive vessels showed a much weaker α6 integrin signal compared with normoxic conditions, while α5 integrin expression on α-SMA-positive vessels was noticeably enhanced (see arrows). (B) Quantification of the changes in α5/α6 integrin expression by α-SMA+ vessels. As described in Materials and methods, the Volocity software program was used to quantify fluorescent intensity of the integrin subunits, and within each experiment the fluorescent intensity of integrin staining on α-SMA+ vessels in the different conditions was expressed as the mean±s.e.m. of the percentage of fluorescent intensity on all vessels under normoxic conditions (control). Experiments were performed with three different animals per condition. Note that in the frontal lobe and medulla, after 7 days hypoxia, α-SMA+ vessels showed significant upregulation of α5 integrin and downregulation of the α6 integrin subunit. ^*P<0.05, ^**P<0.005, ^***P<0.001. The color reproduction of this figure is available on the Journal of Cerebral Blood Flow and Metabolism journal online.

Figure 6

TGF-β1 promotes a switch in endothelial cell β1 integrins. Brain endothelial cells (BECs) were isolated and cultured as described in Materials and methods, and incubated in the absence or presence of 2 or 10 ng/mL TGF-β1 for 2 days and the expression level of the α5, α6, and β1 integrin subunits quantified by flow cytometry. All points represent the mean±s.e.m. of three experiments and are expressed as the percentage change relative to control conditions. Note that both doses of TGF-β1 reduced α6 integrin expression to almost half that of control values, while concomitantly causing a threefold increase in α integrin expression. ^*P<0.05, ^**P<0.02, ^***P<0.01.