Cyp2j5-Gene Deletion Affects on Acetylcholine and Adenosine-Induced Relaxation in Mice: Role of Angiotensin-II and CYP-Epoxygenase Inhibitor

Previously, we showed vascular endothelial overexpression of human-CYP2J2 enhances coronary reactive hyperemia in Tie2-CYP2J2 Tr mice, and eNOS−/− mice had overexpression of CYP2J-epoxygenase with adenosine A2A receptor-induced enhance relaxation, but we did not see the response in CYP2J-epoxygenase knockout mice. Therefore, we hypothesized that Cyp2j5-gene deletion affects acetylcholine- and 5'-N-ethylcarboxamidoadenosine (NECA) (adenosine)-induced relaxation and their response is partially inhibited by angiotensin-II (Ang-II) in mice. Acetylcholine (Ach)-induced response was tested with N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH, CYP-epoxygenase inhibitor; 10−5M) and Ang-II (10−6M). In Cyp2j5−/− mice, ACh-induced relaxation was different from C57Bl/6 mice, at 10−5 M (76.1 ± 3.3 vs. 58.3 ± 5.2, P < 0.05). However, ACh-induced relaxation was not blocked by MS-PPOH in Cyp2j5−/−: 58.5 ± 5.0%, P > 0.05, but blocked in C57Bl/6: 52.3 ± 7.5%, P < 0.05, and Ang-II reduces ACh-induced relaxation in both Cyp2j5−/− and C57Bl/6 mice (38.8 ± 3.9% and 45.9 ± 7.8, P <0.05). In addition, NECA-induced response was tested with Ang-II. In Cyp2j5−/− mice, NECA-induced response was not different from C57Bl/6 mice at 10−5M (23.1 ± 2.1 vs. 21.1 ± 3.8, P > 0.05). However, NECA-induced response was reduced by Ang-II in both Cyp2j5−/− and C57Bl/6 mice (−10.8 ± 2.3% and 3.2 ± 2.7, P < 0.05). Data suggest that ACh-induced relaxation in Cyp2j5−/− mice depends on nitric oxide (NO) but not CYP-epoxygenases, and the NECA-induced different response in male vs. female Cyp2j5−/− mice when Ang-II treated.

EETs dilate blood vessels and also have natriuretic effects (Oltman et al., 1998;Imig et al., 2001;McGiff and Quilley, 2001), and the renal CYP-epoxygenases (CYP2C and CYP2J subfamilies) are believed to be under the regulatory control of dietary salt (Makita et al., 1994). Therefore, spontaneously hypertensive rats (SHR) have shown altered protein expression levels and activities for both renal CYP-epoxygenase and whydroxylase (Schwartzman et al., 1996;Yu et al., 2000). Athirakul et al. reported that Cyp2j5 gene disruption resulted in increased blood pressure in female mice compared to their wild-type counterparts (Athirakul et al., 2008), and the elevated blood pressure was associated with increased left ventricular mass and enhanced renal (afferent arterioles) vasoconstriction due to angiotensin II (Athirakul et al., 2008). Further, an increase in renal vascular reactivity to angiotensin II has been reported in the early stage of hypertension in both rodents and humans (Simon et al., 1995;Imig, 2000). Cyp2j5 enzyme is abundant in kidney as well as in the mouse aorta where it is very active in arachidonic acid metabolism to generate EETs (Ma et al., 2004;Nayeem et al., 2008;Nayeem et al., 2013). Mouse kidneys demonstrated higher Cyp2j5 expression in male compared to female mice after puberty, and Northern analysis also revealed that Cyp2j5 transcripts were more abundant in adult male versus adult female kidneys (Ma et al., 2004). Burgess et al. showed that Cyp2j5 is responsible for production of primarily 14,15-and 11,12-EETs in visceral adipose tissue (Burgess et al., 2012), and overexpression of Cyp2j5 and Cyp4a proteins were observed in Ephx2 −/− vs. C57Bl/6 mice with enhanced adenosine (NECA) and CGS 21680 (A 2A AR)-induced aortic relaxation . An overexpression of Cyp2j5 and Cyp4a proteins in Ephx2 −/− vs. C57Bl/6 mice may be involved as a compensatory mechanism to maintain vascular tone .
The conversion of arachidonic acid into epoxides takes place in the presence of CYP-epoxygenases, which have beneficial cardiovascular properties. Inhibition or deletion of CYPepoxygenases including Cyp2j5 may cause less EET-generation from arachidonic acid, leading to decreased vascular relaxation. Likewise, knocking out the Cyp4a10 gene caused salt-sensitive hypertension with reduced EET excretion in the urine (Nakagawa et al., 2006). Some studies revealed that single nucleotide polymorphisms (SNPs) in the CYP4A11 and CYP2J2 genes were associated with human hypertension King et al., 2005). In addition, adenosine A 2A receptor (A 2A AR) knockout mice had lower expression of CYP-epoxygenases and higher expression of w-hydroxylases with enhanced NECA (adenosine agonist) and CGS 21680 (A 2A AR-agonist)-induced vasoconstriction (aortic) compared to their respective wild-type mice (Nayeem et al., 2008;Pradhan et al., 2014;Pradhan et al., 2015). Further, Hanif et al. recently reported that A 2A AR −/− mice had less plasma EETs compared to wild-type counterparts with reduced coronary reactive hyperemic response (Hanif et al., 2017a). An increase in blood pressure has been reported with enhanced renal vasoconstriction with angiotensin II in female Cyp2j5 −/− compared to female wildtype mice (Athirakul et al., 2008). To date, there are no studies addressing the vascular (aortic) response using acetylcholine and adenosine in male and female Cyp2j5 −/− vs. C57Bl/6 mice, though Cyp2j5 enzyme is abundant in the kidneys as well as in mouse aorta where EETs get generated (Ma et al., 2004;Nayeem et al., 2008;Nayeem et al., 2013). Also, the effects of angiotensin-II in their vascular response in both Cyp2j5 −/− and wild type mice has not been investigated. Therefore, we hypothesized that Cyp2j5-gene deletion affects acetylcholine-and NECA-induced relaxation and their response is partially inhibited by Ang-II in mice. Athirakul et al. (2008) described the generation of Cyp2j5 −/− mice. Cyp2j5 −/− and their respective C57Bl/6 mice were provided by Dr. Zeldin, National Institute of Environmental Health Sciences/National Institutes of Health (NIH). We used both male and female mice (14-18 week old) equally in our study.
(1) Disruption of Cyp2j5 gene in mice (Cyp2j5 −/− ) reduced ACh-induced concentration-dependent relaxation compared to C57Bl/6 mice. Thus, it is clear that Cyp2j5 has its own role in ACh-induced vascular response. Loss of Cyp2j5 showed reduced ACh-induced relaxation, and Cyp2j5 protein is also detected in kidneys/mouse aorta/visceral adipose tissues, and is involved in the conversion of AAs into EETs (Ma et al., 2004;Burgess et al., 2012;Nayeem et al., 2013).
(3) Ang-II attenuates ACh-induced concentration dependent relaxation in Cyp2j5 −/− compared to C57Bl/6 mice. Therefore, we conclude that Cyp2j5 enzyme is involved in ACh-induced concentration dependent vascular response in Ang-II-treated mice. (4) There was no difference found between the presence and absence of Cyp2j5 in mice related to adenosine (NECA)-induced vascular response. (5) Ang-II treatment significantly attenuated NECAinduced concentration dependent relaxation in Cyp2j5 −/− (M) and C57Bl/6 (M) mice, and the attenuation of NECA-induced concentration dependent relaxation due to Ang-II infused Cyp2j5 −/− (M) is much higher than C57Bl/6 (M) mice. Therefore, presence of Cyp2j5 in C57Bl/6 (M) mice plays an important role in resisting the reduction of NECA-induced concentration dependent relaxation due to Ang-II in male mice. (6) Ang-II treatment was able to significantly attenuate NECA-induced concentration dependent relaxation in both According to our data, Cyp2j5 plays an important role in the in ACh-, NECA-, and CGS 21680-induced concentration dependent regulation of vascular response. CYP450-epoxygenase (CYP2J2) polymorphisms have been reported in different populations related to its role in cardiovascular function, including hypertension (Lee et al., 2007;Feng et al., 2008;Fava et al., 2010;Jie et al., 2010;Wang et al., 2010;Zordoky and El-Kadi, 2010;Xu et al., 2011). The Cyp2j5-epoxygenase in mouse (chromosome-4) appears to be as important as CYP2J2-epoxygenase in human (chromosome-1) and CYP2J4 in rat (chromosome-5) (Olona et al., 2018). Variations in soluble epoxide hydrolase and w-hydroxylase genes in human population also alter the risk of coronary heart disease, ischemic stroke, restenosis, diabetes heart, heart failure, ischemic stroke in Caucasians, Chinese, and in the African Americans with hypertension (Lee et al., 2006;Burdon et al., 2008;Monti et al., 2008;Kullmann et al., 2009;Fava et al., 2010;Zordoky and El-Kadi, 2010).
Role of Cyp2j5 in ACh-induced vascular response: AChinduced concentration dependent aortic relaxation in Cyp2j5 −/− was significantly different compared to C57Bl/6 mice ( Figure 1). ACh data between Cyp2j5 −/− and C57Bl/6 mice suggest that lack of Cyp2j5-epoxygenase (less EETs generation) contributes less in ACh-induced concentration dependent aortic relaxation in Cyp2j5 −/− compared to C57Bl/6 mice, and ACh-induced relaxation is almost completely dependent on NO. As Hercule, et al. indicated, CYP450-eicosanoids activates endothelial nitric oxide (NO) synthase and NO release in Ephx2 −/− (more EETs) and wild-type (WT) mouse mesenteric arteries (Hercule et al., 2009). In addition, EETs have been demonstrated to increase NO release; in bovine aortic endothelial cells (cultured) EETs can induce NO release, which may modulate vascular tone (Wang et al., 2003;Hercule et al., 2009). Therefore, according to the current study, we believe Cyp2j5 −/− mice, which lack Cyp2j5, may have reduced EET formation and subsequent ACh-induced and NO-dependent aortic relaxation compared to C57Bl/6 mice. Because, we found overexpression of Cyp2j5 protein and up-regulation of EET oxylipins (isolated heart perfusate) in Ephx2 −/− compared to C57Bl/6 mice with enhanced aortic and coronary reactive hyperemic response Hanif et al., 2016a), and endothelial vascular overexpression of human CYP2J2 had up-regulation of EET oxylipins (isolated heart perfusate) in CYP2J2 (Tie2-CYP2J2 Tr) compared to C57Bl/6 mice with enhanced coronary reactive hyperemic response (Hanif et al., 2017b).
Role of CYP-epoxygenases in ACh-induced relaxation: MS-PPOH (CYP-epoxygenases inhibitor) was able to partially block ACh-induced concentration dependent aortic relaxation in C57Bl/6 mice, but not in Cyp2j5 −/− compared to non-treated mice (Figure 2). There is no difference was found between MS-PPOH treated Cyp2j5 −/− vs. non-treated Cyp2j5 −/− mice. This suggests that Cyp2j5 is a main contributor in the formation of epoxides from AA in mouse aorta, as demonstrated previously that mouse kidneys have higher Cyp2j5 expression in male compared to female mice after puberty (Ma et al., 2004), and Northern analysis also revealed that Cyp2j5 transcripts were more abundant in adult male versus adult female kidneys (Ma et al., 2004). Burgess et al., showed that CYP2J5 is responsible for production of primarily 14,15 and 11,12 EETs in visceral adipose tissue (Burgess et al., 2012), and the overexpression of Cyp2j5 and Cyp4a proteins were observed in Ephx2 −/− vs. C57Bl/6 mice with enhanced adenosine (NECA) and CGS 21680 (A 2A AR)induced aortic relaxation . Relative loss of ACh-induced concentration dependent aortic relaxation in Cyp2j5 −/− vs. C57Bl/6 mice and no change between MS-PPOH treated Cyp2j5 −/− vs. non-treated Cyp2j5 −/− mice indicates a central role of CYP-epoxygenases like Cyp2j5 in ACh-induced concentration dependent aortic relaxation. This is similar to that reported in mouse mesenteric arteries (Hercule et al., 2009).
Effect of Ang-II in ACh-induced relaxation: Ang-II was able to partially block ACh-induced concentration dependent aortic relaxation in both Cyp2j5 −/− and C57Bl/6 mice compared to non-treated mice (Figure 3), and a significant difference was found between the Ang-II-treated C57Bl/6 vs. non-treated C57Bl/6 mice and Ang-II-treated Cyp2j5 −/− vs. non-treated Cyp2j5 −/− mice (Figure 3). Attenuation of ACh-induced concentration dependent aortic relaxation was greater in Ang-II-treated Cyp2j5 −/− vs. non-treated C57Bl/6 mice compared to Ang-II-treated C57Bl/6 vs. non-treated C57Bl/6 mice. This shows that the lack of Cyp2j5 gene in Cyp2j5 −/− mice has less advantage over C57Bl/6 to block the action of Ang-II in AChinduced concentration dependent aortic relaxation.
Deletion of Cyp2j5 (Cyp2j5 −/− ) or inhibition (through MS-PPOH) of CYP-epoxygenases activity causes less EET generation from arachidonic acid (AA) metabolism and leads to a decrease in ACh-concentration dependent relaxation. Ang-II blocked ACh-induced concentration dependent relaxation in both Cyp2j5 −/− and C57Bl/6 mice. However, in the absence of Cyp2j5, Ang-II blocking action is significantly more than the blocking action in C57Bl/6 mice. Therefore, we conclude that inhibition or deletion of CYP-epoxygenases in mice may enhance the toxic action of Ang-II in ACh-induced concentration dependent relaxation in Cyp2j5 −/− compared to C57Bl/6 mice. Also, deletion or inhibition of CYP-epoxygenases activity causes less EETs to form and involved in vascular contraction, reduced coronary reactive hyperemic response with the association of vascular inflammation (Nayeem et al., 2008;Nayeem et al., 2009;Nayeem et al., 2010;Nayeem et al., 2011;Ponnoth et al., 2012b;Hanif et al., 2016b;Hanif et al., 2017b).
Role of Cyp2j5 in NECA-induced vascular response and effect of Ang-II in NECA-induced vascular response: there was no difference between the presence or absence of Cyp2j5 in both male vs. female or male vs. female C57Bl/6 mice regarding NECA-induced concentration dependent relaxation ( Figures  4A-D), which is not similar to ACh-induced concentration dependent vascular response where Cyp2j5 −/− had lesser relaxation compared C57Bl/6 mice ( Figure 1). But, Ang-II treatment was able to block NECA-induced concentration dependent aortic relaxation in both male Cyp2j5 −/− and male C57Bl/6 mice compared to non-treated male mice (Figure 5A), and a large difference was found between the Ang-II-treated male C57Bl/6 vs. non-treated male C57Bl/6 mice and a significant difference was observed between Ang-II-treated male Cyp2j5 −/− vs. non-treated male Cyp2j5 −/− mice ( Figure 5A). Attenuation of NECA-induced concentration dependent aortic relaxation was greater in Ang-II-treated male Cyp2j5 −/− vs. Ang-II-treated male C57Bl/6 mice ( Figure 5A). In addition, there was significant difference observed between Ang-II-treated male Cyp2j5 −/− vs. Ang-II-treated male C57Bl/6 mice ( Figure 5A). This shows that the lack of Cyp2j5 gene in Cyp2j5 −/− mice has less advantage over C57Bl/6 to block the action of Ang-II in NECA-induced concentration dependent aortic relaxation. However, Ang-II blocking action on the NECA-induced concentration dependent relaxation is stronger in male Cyp2j5 −/− than the blocking action in male C57Bl/6 mice. Therefore, we conclude that inhibition or deletion of CYP-epoxygenases in mice may have stronger toxic action of Ang-II in NECA-induced concentration dependent relaxation in male Cyp2j5 −/− compared to male C57Bl/6 mice, because deletion or inhibition of CYP-epoxygenases activity causes less EETs to form and involved in vasoconstriction, reduced coronary reactive hyperemic response with the association of vascular inflammation (Nayeem et al., 2008;Nayeem et al., 2009;Nayeem et al., 2010;Nayeem et al., 2011;Ponnoth et al., 2012b;Hanif et al., 2016b;Hanif et al., 2017b).
There were no significant differences observed between female Cyp2j5 −/− vs. female C57Bl/6 mice, similar to male Cyp2j5 −/− vs. male C57Bl/6 mice. Lower NECA-induced relaxation was observed in Ang-II infused female C57Bl/6/Ang-II infused female Cyp2j5 −/− mice compared to non-treated female Cyp2j5 −/− /female C57Bl/6 mice ( Figure 5B). However, no significant difference in NECA-induced concentration dependent vascular response was observed between Ang-II infused female C57Bl/6 vs. Ang-II infused female Cyp2j5 −/− mice ( Figure 5B), and there was no significant difference in NECA-induced concentration dependent vascular response observed between Ang-II infused male C57Bl/6 vs. Ang-II infused female C57Bl/6 mice ( Figure 5C). In contrast, Ang-II blocking action on the NECA-induced concentration dependent relaxation is stronger in male Cyp2j5 −/− vs. Ang-II + male C57Bl/ 6 mice compared to Ang-II + female Cyp2j5 −/− vs. Ang-II + C57Bl/6 female mice ( Figures 5A, B). Also, Ang-II blocking action on the NECA-induced concentration dependent relaxation is more dominant in male Cyp2j5 −/− vs. Ang-II + female Cyp2j5 −/− mice compared to Ang-II + female Cyp2j5 −/− vs. female Cyp2j5 −/− mice ( Figure 5D). This disparity between Ang-II treated male Cyp2j5 −/− vs. Ang-II treated female Cyp2j5 −/− mice were prominent in NECA-induced vascular response. However, this type of difference in NECA-induced vascular response was not observed between non-treated male vs. female Cyp2j5 −/− mice. This observation is in agreement with Ma et al. where mouse kidneys demonstrated higher Cyp2j5 expression in male compared to female mice after puberty, and Northern analysis also revealed that Cyp2j5 transcripts were more abundant in adult male versus adult female kidneys (Ma et al., 2004). However, an increased in blood pressure reported with enhanced renal (afferent arterioles) vasoconstriction with angiotensin II in female Cyp2j5 −/− compared to its respective female wild-type mice (Athirakul et al., 2008). In contrast, we demonstrated in the current study that an enhanced adenosine (NECA)-induced aortic vasoconstriction in male vs. female Cyp2j5 −/− mice when Ang-II infused. This difference may be due to different blood vessels (renal afferent arterioles vs. aorta) and different media (Ang-II alone vs. NECA + Ang-II). Before Ang-II treatment, there was no significant difference noticed between male vs. female Cyp2j5 −/− and between C57Bl/6 vs. Cyp2j5 −/− mice in NECA-induced aortic response. However, there was no significant difference observed in acetylcholineconcentration dependent aortic response between male vs. female Cyp2j5 −/− mice when Ang-II infused, but the significant difference was observed between C57Bl/6 vs. Cyp2j5 −/− mice in acetylcholine-concentration dependent aortic response without Ang-II treatment. Also, Burgess et al., showed that Cyp2j5 is responsible for production of primarily 14,15-and 11,12-EETs in visceral adipose tissue (Burgess et al., 2012), and the overexpression of Cyp2j5 and Cyp4a proteins were observed in Ephx2 −/− vs. C57Bl/6 mice with enhanced adenosine (NECA) and CGS 21680 (A 2A AR)-induced aortic relaxation . Any deletion or inhibition of CYP-epoxygenases activity causes less EETs to form and participate in vasoconstriction and reduced coronary reactive hyperemic response with the association of vascular inflammation (Nayeem et al., 2008;Nayeem et al., 2009;Nayeem et al., 2010;Nayeem et al., 2011;Ponnoth et al., 2012b;Hanif et al., 2016b;Hanif et al., 2017b).
In summary, the result reported here provide new insights that inhibition or absence of Cyp2j5 enzyme through deletion of Cyp2j5 gene appears to be critical for acetylcholine-or NECA-or CGS 21680-induced concentration dependent vascular response while challenged by angiotensin-II or MS-PPOH ( Figure 6). When the Cyp2j5 gene was absent (Cyp2j5 −/− mice), AChinduced concentration dependent relaxation was significantly lower than C57Bl/6 mice. However, when the ACh-induced concentration dependent relaxation was challenged by MS-PPOH, absence of Cyp2j5 made no significant difference in the ACh-induced concentration dependent relaxation compared to the non-treated Cyp2j5 −/− mice. In contrast MS-PPOH-treated C57Bl/6 mice made a significant difference compared to nontreated C57Bl/6 mice. When the ACh-induced concentration dependent relaxation was challenged by Ang-II in Cyp2j5 −/− mice, a significant difference was observed in the blocking action on ACh-induced concentration dependent relaxation compared to C57Bl/6 mice. Similarly, when the NECA-induced concentration dependent relaxation was challenged by Ang-II in male Cyp2j5 −/− vs. female Cyp2j5 −/− mice and male Cyp2j5 −/− vs. male C57Bl/6 mice, a significantly huge difference in the blocking of NECA-induced concentration dependent relaxation was observed. However, NECA-induced concentration dependent vascular response itself did not make any significant difference between the Cyp2j5 −/− vs. C57Bl/6 mice. In addition, DDMS enhanced CGS 21680-induced concentration dependent relaxation in DDMS treated Cyp2j5 −/− vs. non-treated Cyp2j5 −/− mice, and there was no difference was observed between DDMS + Ang-II + Cyp2j5 −/− vs. non-treated Cyp2j5 −/− mice. Therefore, we conclude that inhibition or deletion of Cyp2j5 in mice may have an increase in toxic action of both Ang-II and MS-PPOH in ACh/NECA-induced concentration dependent relaxation in Cyp2j5 −/− compared to C57Bl/6 mice, and DDMS may have rescued from the toxic effect of Ang-II treatment in Cyp2j5 −/− mice.
Note: Unfortunately, we lost Cyp2j5 −/− mouse colony, and this colony is not available anywhere except frozen embryos, therefore, some areas of this manuscript has some limitations. In future, we will revive this colony as the grant gets funded.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ supplementary material.

ETHICS STATEMENT
West Virginia University Institutional Animal Care and Use Committee approved all animal care and experimentation protocols, which were in accordance with the principles and guidelines of the NIH's Guide for the Care and Use of Laboratory Animals.

AUTHOR CONTRIBUTIONS
MN: conception, design of research, performing experiments, analysis drafting and editing. SA and AH cooperated in the experimentations, reading, correction, editing and input. ME helped in correction, editing and input and DZ advised, read, corrected, edited, provided transgenic mice and input.

FUNDING
This work was supported by a National Institutes of Health Grant HL-114559 to MN and a National Institute of Environmental Health Sciences Grant Z01 ES025034 to DZ.