Hypoxia Dysregulates the Transcription of Myoendothelial Junction Proteins Involved with Nitric Oxide Production in Brain Endothelial Cells

Abstract

Myoendothelial junctions (MEJs) are structures that allow chemical signals to be transmitted between endothelial cells (ECs) and vascular smooth muscle cells, which control vascular tone. MEJs contain hemoglobin alpha (Hbα) and endothelial nitric oxide synthase (eNOS) complexes that appear to control the production and scavenging of nitric oxide (NO) along with the activity of cytochrome b5 reductase 3 (CYB5R3). The aim of this study was to examine how hypoxia affected the regulation of proteins involved in the production of NO in brain ECs. In brief, human brain microvascular endothelial cells (HBMEC) were exposed to cobalt chloride (CoCl₂), a hypoxia mimetic, and a transcriptional analysis was performed using primers for eNOS, CYB5R3, and Hbα2 with ΔΔCt relative gene expression normalized to GAPDH. NO production was also measured after treatment using 4,5-diaminofluorescein diacetate (DAF-DA), a fluorescent NO indicator. When HBMEC were exposed to CoCl₂ for 48 h, eNOS and CYB5R3 messenger RNA significantly decreased (up to -17.8 ± 4.30-fold and -10.4 ± 2.8, respectively) while Hbα2 increased to detectable levels. Furthermore, CoCl₂ treatment caused a redistribution of peripheral membrane-generated NO production to a perinuclear region. To the best of our knowledge, this is the first time this axis has been studied in brain ECs and these findings imply that hypoxia may cause dysregulation of proteins that regulate NO production in brain MEJs.

Keywords: cytochrome b5 reductase 3 myoendothelial junction; endothelial nitric oxide synthase; hemoglobin alpha; nitric oxide.

Figure 1

Feedback mechanisms controlling the regulation of NO in the MEJ. Endothelial nitric oxide synthase (eNOS) production within the myoendothelial junction (MEJ) is triggered in response to Ca²⁺ derived from the connected vascular smooth muscle cell (VSMC). The process begins when Inositol 1,4,5-triphosphate (IP₃) produced by activated VSMC binds to IP₃-receptor 1 (IP₃R1) on the sarcoplasmic reticulum or transient potential receptor (TRP) at the membrane to increase cytosolic Ca²⁺ concentrations. This increase results in the phosphorylation of actin/myosin motors via a calmodulin (CaM)/myosin light chain kinase (MLCK) cascade. Concurrently, IP₃ opens connexin 43 (Cx43) channels to allow for an influx of Ca²⁺ and IP₃ into the tethered endothelial cell (EC). The resulting activated CaM then binds with eNOS and heat shock protein 90 (HSP90) to form an active complex that is released from caveolin 1 (CAV1). Subsequently produced NO can then diffuse back into VSMCs, where it activates soluble guanylyl cyclase (sGC), facilitating the conversion of GTP to cGMP, which ultimately leads to a reduction in constriction by myosin light chain phosphatase (MLCP). In the presence of oxygen, NO can be scavenged via deoxygenation reactions catalyzed by hemoglobin α (Hbα)/Fe²⁺/eNOS complexes to form both nitrate (NO₃⁻) and oxidized Hbα/Fe³⁺ complexes. This reaction is potentiated by the reduction of Hbα/Fe³⁺ back to Hbα/Fe²⁺ by cytochrome B5 reductase 3 (CyBR3). In the absence of oxygen, Hbα/Fe³⁺ can bind NO, to preserve its activity, or serve as a nitrite reductase. Figure created with Biorender.com.

Figure 2

CoCl₂ dose-dependently inhibits eNOS and CYB5R3 transcription in HBMEC. To evaluate the effects of hypoxia on the transcription of eNOS and CYB5R3, total RNA was isolated from human brain microvascular endothelial cells treated with increasing doses of CoCl₂ for 48 h. ΔΔCt relative gene expression was then calculated for eNOS and CYB5R3 and normalized to GAPDH. Data presented as mean of means ± SEM fold change in expression for 48-h CoCl₂ dose-responses (A) or 48-h 100 ng/mL IL-1β or TNFα compared to 500 μM CoCl₂ (B). * = p-value ≤ 0.05 vs. activated saline control by one sample t-test (hypothetical value = 1; α = 0.05; n = 4 for TNFα, n = 5 for 500 μM CoCl₂ and IL-1β, and n = 6 for all other treatment groups).

Figure 3

CoCl₂ dose-dependently increases Hbα transcription in HBMEC. To evaluate the effects of hypoxia on the transcription of Hbα, total RNA was isolated from human brain microvascular endothelial cells treated with increasing doses of CoCl₂ for 48 h. qRT-PCR was performed using specific primers for Hbα and GAPDH. Data presented as representative amplification curves.

Figure 4

CoCl₂ treatment of HBMEC alters the location of NO production. To evaluate the effects of hypoxia on the production of NO, human brain microvascular endothelial cells were treated with a final concentration of CoCl₂ for 48 h and then loaded with a fluorescent NO indicator (DAF-FM acetate). Overall NO was measured under an epifluorescent microscope after two hours of development at room temperature: 48-h diluent control (A) and 48-h 500 μM CoCl₂ (B).