Exogenous H2S Regulates of Cystathionine Gamma-Lyase in HUVECs during Hypoxia Authors

Cystathionine gamma-lyase (CSE) is one of the essential H2S-producing enzymes, and it regulates diverse functions in connection with cardiovascular function. It is crucial how exogenous H2S regulates CSE expression of the vascular endothelial cell during hypoxia. We examined the transcription and expression of CSE in HUVECs regulated by exogenous H2S with 100 μM during hypoxia by Luciferase assay, Western blotting, and quantitative RT-qPCR. Exogenous H2S influenced on the promoter activity of CSE in HUVECs during hypoxia. The effects of 100 μM H2S on CSE mRNA expression in HUVECs is decreased compared with 0 μM H2S. The consequences of 100 μM H2S on the expression level of CSE protein in HUVECs at two h of hypoxia is reduced compared with 0 μM H2S. These findings suggest that vascular endothelial cells can respond to the signals of hypoxia in the blood, and can respond to changes in H2S concentration in the blood, thus affect the blood vessels themselves.


Introduction
The endogenous generation of H2S is mainly mediated by the enzyme cystathionine-γ-lyase (CSE) in the cardiovascular system [1]. Hydrogen sulfide (H2S) has regarded as a signaling molecule as well as a cytoprotectant, and protects various tissues and organs from oxidative stress and ischemia-reperfusion injury [2]. Endothelial CSE contributes to cardiovascular homeostasis, primarily through the production of H2S [3]. H2S is produced in the vasculature and involved in promoting vascular homeostasis, vasodilation, and endothelial cell proliferation [4].
The vascular smooth muscle cells (SMCs) from the CSE gene knockout mice are more susceptible to apoptosis induced by exogenous H2S at the physiologically relevant concentration [5]. The mechanisms of High level of homocysteine-induced endothelial dysfunction and the metabolism and physiological functions of H2S as a protective agent [6].
After inhibiting endogenous background CSE expression, direct administration of exogenous H2S at 100 μM can induce apoptosis of human aorta smooth muscle cells [7]. The mice overexpressed CSE in the heart have resistance to the ischemia-reperfusion injury, and the protection accompanied by a decrement in myocardial inflammation [8]. Endogenous H2S plays modulatory roles in hypoxia-induced cardiovascular responses, inhibiting the cardiovascular in spontaneously hypertensive rats (SHR) [9]. The bath application of 100 μM exogenous H2S can reduce the time required for the repolarization of the action potential [10].
Several studies have investigated the effects of H2S in human vessels. H2S-induced relaxation has demonstrated in internal mammary [11], pulmonary [12], mesenteric [13], and intrarenal arteries [14], as well as in perfused human placentas [15]. Up-regulation of CSE expression during hypoxia may increase the production and concentration of H2S in cells and protecting cells from hypoxia [16]. A controlled release formulation of S-propargyl-cysteine showed protective effects against myocardial infarction (MI) via the CSE/H2S pathway [17]. NADPH Oxidase 4 is a positive transcriptional regulator of CSE in endothelial cells and proposes that it may modulate the production of endogenous H2S [18]. The duration of the action potential in the healthy papillary muscles can cut down by exogenous H2S (50, 100, 200 μM), and pretreatment with glibenclamide partly blocks the effects of exogenous H2S at 100 μM [19].
It is crucial how exogenous H2S regulates CSE expression of the vascular endothelial cell during hypoxia. Therefore, we study the effects of exogenous H2S on CSE expression in HUVECs during hypoxia.

It was the construction of the reporter under the CSE promoter.
HUVECs were cultured to a confluence of 80-90%, digested with trypsin, and collected at 5000 r/min. It was extracting the genome DNA of HUVECs by using 1% agarose gel. Searching the sequence of CSE gene promoter in the GenBank database, designing upstream and downstream primers, and the target fragment DNA length was 710bp (-696~+16nt). According to the CSE (NC_000001.11), we amplified the 710 bp DNA upstream of the CSE gene by PCR using pGL4.12-HuCSE710 as the template (forward primer 5'-CGGGGTACCCATTAGGGGGAGTTTCTCTCTGT-3' and reverse

Results
Effect of exogenous H2S on CSE promoter activity during hypoxia. We analyzed the effects of exogenous H2S on the activities of the CSE promoter during hypoxia, as showed in Figure 1A.

Discussion
In this experiment, we first investigated the regulatory mechanism of exogenous H2S on CSE in HUVECs during hypoxia. We demonstrated that exogenous H2S of 100 μM is involved in the regulation of CSE expression in HUVECs during hypoxia. Exogenous H2S could affect the transcriptional activity of mouse CSE in mammalian cells [16]. As the level of free H2S is maintained at a low concentration under basal conditions [21], so CSE mainly adjusts itself through the CSE feedback inhibition at the lower level of exogenous H2S (from 10 to 80 μM).
Exogenous H2S could oppose the elevation of pulmonary arterial pressure and lessen the pulmonary vascular structure remodeling during hypoxic pulmonary hypertension (HPH) [23].
The Longchamp's group identified a requirement for CSE in vascular endothelial growth factor (VEGF) dependent angiogenesis via increased H2S production [24].
The CSE/H2S pathway has indirectly linked to hypoxia, and H2S can protect mammalian cells against hypoxia-induced injuries. Hypoxia causes apoptosis, which may play essential roles in ischemic heart disease [25], and increased tissue content of H2S protects the heart from ischemia/reperfusion damage [26]. There is no significant difference between the effects of exogenous H2S on the CSE mutation promoter activity and the CSE wild during hypoxia.
However, exogenous H2S influenced on the promoter activity of CSE.