Hypertension is a serious disorder that shows no symptoms even when blood pressure (BP) is elevated. If left untreated, it can lead to life-threatening diseases with atherosclerosis in humans. In rats, arteriosclerosis rather than atherosclerosis is the predominant change associated with hypertension and leads to myocardial infarction, renal failure and stroke [1, 2, 3]. Even in Japan where there is sufficient general medical care to treat hypertension, more than 300,000 patients die each year from diseases related to this condition [4, 5]. To confront this life-threatening problem, multidisciplinary knowledge of hypertension is required. Together with more effective treatments to avoid serious consequences, elucidation of the genetic background of hypertension is urgently required. Although a number of methods are available for investigating the genetic nature of hypertension, the polygenic nature of human hypertension makes it difficult to identify the responsible genes via meta-analysis of genome-wide studies [6]. The aim of this study was therefore to identify genes related to hypertension by studying rat models of human essential hypertension, namely spontaneously hypertensive rats (SHR) [7], stroke-prone SHR (SHRSP) [8] and malignant SHRSP (M-SHRSP) [9]. The two latter substrains develop stroke at high rates of 77% in SHRSP [8] and more than 96% in M-SHRSP [9].

DNA microarrays are potentially powerful tool for studying the genetics of diseases as they allow simultaneous measurement of the expression level of thousands of genes in experimental studies [10, 11, 12]. In this study, we compared gene expression in the mesenteric artery of the three SHR substrains mentioned above [7, 8, 9]. SHR was developed as an animal model for research into essential hypertension as part of a breeding program based solely on the selection of elevated BP in normotensive Wistar Kyoto (WKY) rats [7]. SHRSP were derived from SHR following selective inbreeding for susceptibility to stroke [8]. M-SHRSP were selected and established through brother–sister mating of selected SHRSP animals that showed higher BP and higher incidence rates of stroke [9]. An inbred strain of M-SHRSP has a BP of 250 mm Hg or higher before 14-weeks of age, causing brain stroke in almost all animals [9]. We previously bred three SHR substrains and families in our facility. Using these SHR substrains, we found several blood pressure-regulating genes such as Rgs2, Gja1, Uts2, Ephx2 in the adrenal glands [13], and Mapk14, Ephx2, Kcnc3 in the kidneys that could be related to hypertension [14]. The Kcnq1, Kcnh1, Ache and Chrm2 genes were identified following study of the mesenteric artery of SHRs, SHRSPs and renovascular hypertensive 2K1C (two kidneys and one clip induced hyper-renin) and 1K1C (one kidney and one clip induced hypervolemic) rats, but not M-SHRSP [15]. Other genes related to hypertension have also been reported [16], and the identify of true candidate genes for hypertension remains controversial.

The mRNA expression profile of different tissues depends on their biochemical and physiological roles and on the states of disease. Hence, it is very important to select mRNA samples from tissues that are responsive to hypertension. Resistance arteries such as the mesenteric and femoral arteries in the rats appear to be the most relevant tissues when analyzing the cause of hypertension [17]. In the present study we therefore chose the mesenteric artery to analyze mRNA in rat hereditary hypertensive models. Genes associated with hypertension were identified in the mesenteric artery of three related substrains of SHR using DNA microarray technology.

In this study, we identified hypertensive-related genes that were either up or down regulated in SHR, SHRSP and/or M-SHRSP rats compared to WKY rats. These genes were classified into four categories according to their postulated association with hypertension: (1) genes primarily pathogenic for the onset of hypertension (Group-1); (2) genes whose expression does not appear to be related to the onset of hypertension, but which may strengthen hypertensive conditions (Group-2); (3) genes whose involvement in hypertension is presently unknown, but which could in future be linked to the onset or the strengthening of hypertension after further investigation (Group-3), and (4) genes whose involvement in hypertension is less likely, but which may be associated with physiological characteristics of SHRs other than hypertension (Group-4).

Through careful measurement, we found that the increase in SBP of SHRs at 9-weeks of age was similar to that of M-SHRSPs as young as 6-weeks old (Fig. 1a). Furthermore, the SBP was significantly higher in 9-week old SHRSP and M-SHRSP, even when compared with that of 20-week old SHR from other experiments (Fig. 1b). Based on these observations, we hypothesize that most upstream genetic alterations relevant to the onset of hypertension occur in the early period, while the expressions of genes in the latter period may instead act to maintain or strengthen hypertensive conditions. Moreover, we postulate that the expression level of candidate genes would be altered in different substrains of SHRs, but not in WKY. To investigate this we employed DNA microarray technology as described previously by our group [15]. We identified several genes that may be associated with spontaneous hypertension in SHRs. At the age of 6-weeks, 13 mostly up regulated genes were selected from 31 genes listed in the upper part of Table 1. These genes were associated with increases in the SBP of SHR substrains: Abca9_predicted, Adam23_predicted, Gsg1, Padi2, Qprt, S100b, Snca, AI502837, AI715122, CO406073, TC525865, TC542387 (#1) and TC558248.

Various hypertension-related genes identified in previous reports will now be considered one by one. The functional involvement of Abca9_predicted gene [20] in hypertension has so far been uncertain, but it may be causal for hypermetabolism in SHRs through ABC-mediated proteins transport of various molecules across extra- and intracellular membranes. We, therefore, categorized Abca9_predicted as Group-4. Adam23 was originally reported to be involved in cancer and neuronal differentiation [21, 22]. More recently, Adam23 was reported to be a negative regulator of K${}_{\text{v1.1}}$/K${}_{\text{v1.4}}$ potassium currents [23]. Hence, the up regulation of Adam23_predicted observed in the present study is quite suggestive. It is well known that the opening of K${}^{+}$ permeable channels will hyperpolarize the plasma membrane potential (E${}_{\text{m}}$). The excitation of vascular smooth muscle cells (VSMC) followed by the depolarization of the E${}_{\text{m}}$ via the influx of Ca${}^{2+}$ and Na${}^{+}$ or the efflux of Cl${}^{-}$ leads to the opening of voltage-dependent Ca${}^{2+}$ channels (VDCCs). In VSMC, the opening of VDCCs causes Ca${}^{2+}$ influx, leading to a rise in intracellular Ca${}^{2+}$ concentration ([Ca${}^{2+}$]${}_{\text{i}}$) and subsequent activation of the contractile machinery. Depolarization also leads to the voltage-dependent opening of K${}_{\text{v}}$ channels, giving rise to a compensatory hyperpolarizing current that reduces the likelihood of VDCCs and inhibits vasoconstriction [24]. Although the role of Adam23 in VSMC is still unclear, its marked up-regulation may negatively regulate vasodilatation via functional suppression of vasodilative potassium currents, leading finally to increased vasocontraction. The overexpression of Adam23_predicted was therefore categorized as Group-1. The selective expression of Gsg1 in testis may cause redistribution of PAPOLB from the cytosol to the endoplasmic reticulum. This prompted us to categorize Gsg1 as Group-3, since the mechanism of action is not known. It has been reported that Padi2 mediates arginine citrullination and modulates transcription in cancer [25] as well as the progression of neurodegenerative disorders. Since the role of citrullination in hypertension remains largely unknown, we classified the overexpression of Padi2 into Group-3. There is some information regarding Qprt and elevated quinolinate levels in the brain linked to the pathogenesis of neurodegenerative disorders. However, to our knowledge there is no information in relation to SHRs. Qprt was therefore classified as Group-3. S100b [26] is expressed in the cytoplasm and/or nucleus of a wide range of cells and is a calcium binding protein involved in the regulation of various processes such as cell cycle progression and differentiation. This gene could thus be linked to hypermetabolic states in SHRs, and was therefore categorized as Group-4. According to a previous reports, Snca is preferentially expressed on neuronal cells [27], where it may serve to integrate presynaptic signaling and membrane trafficking. However, its role in arteries is not known and therefore it was categorized in Group-3. Almost no information is available for AI502837, AI715122, CO406073, TC525865, TC542387 (#1, duplication gene also in other comparisons) and TC558248. These were classified as Group-3 given they have no known role in the biochemical mechanism of hypertension.

Nine of the 24 genes that were down regulated by more than 0.25-fold in all three SHR substrains at 6-weeks of age are shown in the lower part of Table 1. Known genes were Aqp4, Cxcr3, Ephx2 (#2), Homer2 (#3), Kcnc2, Kcnq5, and Ltb4dh (#4), while the unknown genes were XM_344042 and XM_344378. Aqp4 (aquaporin 4) is expressed by astrocytes in the CNS (central nervous system) [28], and is involved in water movement, cell migration and neuroexcitation [29]. It was classified into Group-4, since down regulation of this gene may cause hydrocephaly in SHRs. Previously, it was reported that mice lacking the Cxcr3 chemokine receptor suffer from hypertension [30]. Mice deficient in Cxcr3 exhibit up regulation of angiotensin II type I receptor (ATIR) in VSMC, suggesting the importance of this gene in vascular contractility and hypertension through up regulation of AT1R expression. However, since the mechanistic relationship between down regulation of Cxcr3 and up regulation of AT1R is not yet clear, this gene was classified as Group-3. Due to the potent vasodilatory effects of epoxyeicosatrienoic acids (EETs) on VSMC, the metabolizer of EETs on the overproduction of Ephx2 leads to reduced vasorelaxation and hence to subsequent hypertension [31]. However, down regulation of Ephx2 (#2) in the mesenteric artery does not appear to be actively involved in the pathogenesis of hypertension. Based on our observations, the down regulation of Ephx2 may be compensatory for increased blood pressure and was therefore classified as Group-4. Homer2 is widely expressed in many tissue types including muscles (NCBI Gene Databank, Gene ID: 29547). Although the long form Homer 1c plays a role in synaptic changes during long-term potentiation, the biological role of Homer 2 in the circulatory system is not yet clear. Thus, Homer2 was categorized as Group-3. The Kcnc2 and Kcnq5 genes are likely to be involved in the development of hypertension. Bearing in mind the role of K${}^{+}$ channels in vasodilatation, down regulation of Kcnc2 and Kcnq5 causes spontaneous hypertension. Furthermore, Yoshimura Y previously reported that potassium-induced contraction of isolated aortic strips at concentrations of 10–100 mM were significantly greater in SHRSP than in WKY at 1.5, 3, 6 and 12 months of age [32]. This finding could indicate increased susceptibility of the vascular contractile machinery of SHR to K${}^{+}$. Therefore, the Kcnc2 and Kcnq5 genes were classified as Group-1 in the present study. Ltb4dh protein catalyzes leukotriene B4 (LTB4) to increase its activity [33]. LTB4 activates human pulmonary artery adventitial fibroblasts in pulmonary hypertension, causing increased vascular stiffness of the pulmonary artery [34]. The down regulation of Ltb4dh observed in the present study might result in prolonged activation of LTB4, thereby causing perivascular fibrosis in the mesenteric artery and increasing the vascular tone. Therefore, Ltb4dh was classified into Group-4. The biological role of the two unknown genes (XM_344042 and XM_344378) observed here is uncertain according to the NCBI ‘Gene’ information resources and were therefore classified as Group-3.

Particular attention was also paid in this study to genes that were up or down regulated in M-SHRSP and SHR at 6- and 9-weeks of age, respectively. As shown in Fig. 3, 8 genes were up regulated and 7 genes were down regulated. The known up regulated genes were AW143870, Bcas1, BG664685, BI292956, Pou3f1, and LOC679668, while the unknown genes were TC539990, and TC542387 (upper part in Table 2).

Since there is little information on AW143870, this gene was categorized as Group-3. Bcas1 is known to reside in a region of 20q13 that is amplified in several tumor types [35]. However, there is no information with regard to hypertension and hence this gene was categorized as Group-3. There is also little biochemical information available for BG664685 and BI294956. Pou3f1 is thought to be involved in early embryogenesis and neurogenesis [36]. LOC679668 encodes a leucine-rich repeat transmembrane protein that interacts with neurexins and neuroligins to modulate synaptic cell adhesion. TC539990 and TC542387 are unknown genes. Hence, these 6 genes were also classified as Group-3.

Cmah is expressed in the brain and neuronal cells and produces N-glycolylneuraminic acid [37]. However, there is no information available in relation to hypertension and therefore it was categorized as Group-3. Ephx2, Homer2 and Ltb4dh were categorized as Group-4, Group-3 and Group-4, respectively, as mentioned above. Ptprj is a member of the protein tyrosine phosphatase family and regulates a variety of cellular processes including cell growth. Since the down regulation of Ptprj might act as compensatory factor for hypermetabolism in SHRs, this gene was classified as Group-4. TC525845 and TC540893 are unknown genes and were therefore classified as Group-3.

Lastly, we focused on genes that were up regulated in both SHRSPs and M-SHRSPs at 9-weeks of age (Fig. 4). These were categorized as shown in below of Table 2. Since there is little information on BF545795, LOC502684 and RGD1309362 in relation to hypertension [38], these genes were classified as Group-3. Myo5a belongs to the Myosin V heavy-chain class of genes [39] that code for actin-based motor protein involved in cytoplasmic vesicle transport and mRNA translocation. Since this could relate to hyperactivation in hypertension, Myo5a was classified as Group-4 because there is still little information on the circulatory system. Spock2 is an extracellular heparan/chondroitin sulfate proteoglycan [40]. Vascular stiffness is an important factor for vascular resistance and is regulated by ECM [41]. Increased vascular stiffness in SHRs caused by the overproduction of Spock2 may strengthen hypertension, hence this gene was categorized as Group 2. There is little information on the biochemical pathways for TC542387 (#1), TC558814, and TC567935 and hence these were classified as Group-3.

Agtrap, Shc3 and TC525845 were markedly down regulated at 9-weeks of age. Agtrap interacts with AT1R to promote its constitutive internalization, thus inhibiting the hyperactivation of downstream signaling and augmenting the effects of angiotensin II (Ang II) [42]. Using an ex vivo system, Zhang et al. [43] recently reported that treatment of the mesenteric artery with Ang II led to the attenuation of acetylcholine (Ach)-induced vascular relaxation, thus demonstrating a direct effects of Ang II on the mesenteric artery. Agtrap is likely to counteract vasoconstriction of the mesenteric artery via the constitutive internalization of AT1R, thus inhibiting the activation of Ang II signaling. Down regulation of Agtrap in SHRs may therefore lead to increased activation of Ang II signaling in the mesenteric artery. This is at least partly causative of hypertension and Agtrap was therefore classified as Group-2. Shc3 is predicted to be involved in glutamatergic synaptic transmission and epidermal growth factor/neuregulin signaling in the tyrosine kinase-signaling pathway. Thus, Shc3 reduces hypertension and was classified as Group-4. No information was available for the down regulated gene TC525845 (#5, unknown gene) and therefore it was categorized as Group-3.

To summarize the above findings, the genes thought to be involved in the onset of hypertension (Group-1) or to promote, maintain or strengthen hypertension (Group-2) were identified as follows.

Group-1: Adam23_predicted, Kcnc2, and Kcnq5

Group-2: Spock_predicted, and Agtrap

The down regulation of K${}^{+}$ channel genes (Kcnq5 and Kcnc2) and the functional suppression of potassium currents via overexpression of Aadm23 may impair the capacity for vasodilatation and subsequently enhance vasoconstriction. These hypertensive conditions may be increased by genes belonging to Group 2 including reduced expression of Agtrap and overexpression of Spock2_predicted. This may lead to increased activation of Ang II signaling, and to increased vascular stiffness via the deposition of ECM and vascular fibrosis. Although further validation is required, the current findings provide a framework to understand the pathophysiological basis of hypertension.

Two genes thought to be directly involved in the onset of hypertension were Kcnc2, and Kcnq5. Fig. 5 shows the proposed action of these genes in more detail.

Fig. 5.

Schematic representation of the initial changes that occur during the onset of spontaneous hypertension. In WKY, the mesenteric-resistant artery recovers from contraction following the depolarization of the E${}_{\text{m}}$ for VSMC via the repolarization processes, in which the efflux of K${}^{+}$ occurs through K${}^{+}$ channels, i.e., Kcnq5 and Kcnc2. In 6-week old SHRs, SHRSPs and M-SHRSPs at the age of 6 weeks, both K${}^{+}$ channels are constitutively downregulated, resulting in inadequate vasorelaxation and consequently an increase in blood pressure (BP). WKY, Wistar Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats; M-SHRSP, malignant SHRSP.

Conceptualization—YA and HH; methodology—MSA and HH; software—TH and KK; validation—TH and KK; investigation—MSA and HH; data curation—YA and HH; writing—YA and HH; and supervision—HH.

Not applicable.

We kindly acknowledge Kozo Okamoto, who generated the SHRs and worked for a long time as the dean of Kindai University School of Medicine. We are indebted to Yuji Misaki for the design of Fig. 5.

This research received no external funding.

The authors declare no conflict of interest.