MAPK signaling pathway regulates cerebrovascular receptor expression in human cerebral arteries

Large efforts have during the last few decades been made to understand the intracellular mechanisms involved in ischemia-induced cerebral damage and to develop drugs that protect the brain from damage once a stroke has occurred. However, despite extensive research into genetics and molecular biology associated with cerebral ischemia, few acute therapies have proven effective in the clinic [1]. Investigations have revealed that cerebral ischemia is accompanied by modifications in the expression of genes regulating receptor expressions in cerebrovascular smooth muscle cell (SMC)s associated with the cerebral ischemia [2]. Thus, experimental and clinical studies of cerebral ischemia have reported increased levels of the potent vasoconstrictor substances endothelin (ET) [3, 4], 5-hydroxytryptamine (5-HT) [5, 6], angiotensin (Ang II) [7] and thromboxane (TXA₂) [8, 9]. ET-1, 5-HT, Ang II and TXA₂ are all potent vasoconstrictors of cerebral arteries that mediate effects through the family of G-protein coupled receptors (GPCRs) [10, 11]; endothelin A (ET_A), endothelin B (ET_B) [11, 12], 5-HT receptors [5], the angiotensin II type 1 (AT₁) and type 2 (AT₂) receptors [13, 14] and the thromboxane receptor (TP) [15].

Cerebral ischemia is multifactorial, involves a number of neuronal and glial mechanisms; however, several cerebrovascular receptors are in addition involved in the pathophysiology of cerebral ischemia. There is upregulation (enhanced expression) of contractile ET_B, 5-HT_1B, AT₁ and TP receptors in major cerebral arteries from experimental focal and global ischemia, via enhanced transcription and translation [16–24]. This upregulation of cerebrovascular receptors leads to enhanced vasoconstriction and correlates with reduction in regional cerebral blood flow (rCBF) and degree of neurology deficit [21]. Blockade of the individual subtypes of receptors involved might prevent or reduce the cerebral ischemia to a certain degree; we hypothesize that treatment aimed at a common signaling pathway would be more beneficial by avoiding the administration of several antagonists with circulatory consequences.

The mitogen-activated protein kinase (MAPK) pathways are implicated in neuronal death and survival after stroke. A time study of the alteration in cerebrovascular MAPKs after experimental subarachnoid hemorrhage (SAH) revealed that there was early (within minutes) and sustained activation of the specific extracellular signal-regulated kinases (ERK)1/2 pathway, while the p38 and JNK pathways were activated first at 48 hours [25]. The ERK1/2 pathway can be inhibited at various points upstream such as at ras-raf-MEK1/2; inhibition of this pathway with a specific MEK1/2 (the MAPKK of ERK1/2) inhibitor abolished the receptor upregulation as well as preventing the CBF reduction and diminishes the infarct [16, 26, 27]. ERK1/2 belongs to the family of MAPK and is phosphorylated and thereby activated by the MAP kinase/ERK kinase (MEK)1/2. Several studies have shown an involvement of the MEK/ERK1/2 signalling pathway in cerebral ischemia [26, 28].

Organ culture is an in vitro method for investigating cellular mechanisms involved in upregulation of vasocontractile G-protein coupled receptors. Organ culture is not a model for stroke, however, changes in vasoconstrictor responses after in vitro organ culture show a remarkable similarity to changes observed in animal models of ischemic and hemorrhagic stroke. Thus, there is an upregulation of contractile G-protein receptors after SAH [21] and focal ischemia [22, 23] which also is observed in organ culture [29]. This make the organ culture model an appropriate model for investigating the pharmacological characteristics and underlying molecular and cellular mechanism involved in the upregulation of vasocontractile G-protein coupled receptors. The upregulation of contractile receptors in the SMCs are prevented with MAPK inhibitor both in organ culture [29] and experimental stroke [27, 30].

In the design of future cerebrovascular therapeutics it is important that the intracellular mechanisms are characterised in human subjects. Here we hypothesize that there is an upregulation of contractile cerebrovascular receptors after 48 h of organ culture in human cerebral arteries and that this upregulation occurs via the MAPK ERK1/2 pathway and can be inhibited by the MEK1/2 inhibitor U0126.

All procedures were carried out strictly within national laws and guidelines and approved by the Ethical Committee at the University of Lund (LU-818-01) and has been performed in accordance with the Declaration of Helsinki. A consent was obtained from the participants prior to surgery.

Immunohistochemistry

For immunofluorescence the cerebral artery segments were embedded in Tissue TEK (Gibo, Invitrogen A/S, Taastrup, Denmark), frozen at -80°C and subsequently sectioned into 10 μm thick slices. Cryostat sections were fixed for 10 minutes in ice-cold acetone (−20°C) and thereafter rehydrated in phosphate buffered-saline (PBS, pH 7.2) containing 0.25% Triton X-100 (PBST), for 3×5 minutes. The sections were then permeabilized and blocked for 1 h in blocking solution containing PBS and 5% normal donkey serum and then incubated over night at 4°C with either of the following primary antibodies; rabbit anti ET_A (1:50, Santa Cruz Biotechnology, CA, U.S.A., sc-33535), rabbit anti ET_B (1:200, Abcam, Cambridge, UK, ab1921), rabbit anti AT₁ (1:100, Santa Cruz Biotechnology, sc-1173), AT₂ (1:100, Santa Cruz Biotechnology, sc-9040), 5-HT1_B (1:100, Santa Cruz Biotechnology, sc-1460), rabbit TP-receptor (1:100, Cayman Chemical company, Michigan, U.S.A., 10004452) and rabbit anti phospho-ERK p44/42 MAPK (1:50, Cell Signaling Technology, Beverly, CA, U.S.A., #4376). The primary antibodies were diluted in PBST, 1% bovine serum albumin (BSA) and 3% normal donkey serum. On the second day sections were rinsed in PBST for 3×15 minutes and incubated with the secondary antibody (1 h, room-temperature). The secondary antibody used was Cy™² conjugated donkey anti rabbit (1:200, Jackson ImmunoResearch, West Grove, PA, U.S.A, 711-165-152) diluted in PBST and 1% BSA. The sections were washed subsequently with PBST and mounted with Crystal mounting medium (Sigma, St.Louis, MO, U.S.A). Immunoreactivity was visualized with an Olympus Microscope (BX 60, Japan) at the appropriate wavelength. Negative controls for all antibodies were made by omitting primary antibodies. In all cases, no specific staining was found; only auto-fluorescence in lamina elastica interna was seen (Figure 1). To evaluate the auto-fluorescence in lamina elastica interna, controls were made with only primary antibodies.

Calculations and statistics

Data are expressed as mean ± standard error of the mean (s.e.m.), and n refers to the number of patients. Statistical analyses were performed with Kruskal-Wallis non-parametric test with Dunn’s post-hoc test, where P<0.05 was considered significant.

This study demonstrates that there is a clear association between human cerebrovascular receptor upregulation via transcription involving activation of the MAPK pathway after organ culture. This was shown by the close association between pERK1/2 activation and the enhanced expression of the contractile receptors at both protein level and at a functional level.

The study design was based on previous work that has shown that organ culture induces upregulation of cerebrovascular receptors in a manner similar as that observed in experimental SAH and MCAO [36, 37]. Organ culture is not a model for stroke, however, changes in vasoconstrictor responses after in vitro organ culture show a remarkable similarity to changes observed in animal models of ischemic and hemorrhagic stroke, which makes organ culture a useful in vitro method to study the pharmacological characteristics and underlying molecular and cellular mechanism of cerebrovascular receptor alterations.

In man cerebral vessels after a stroke contains elevated levels of several cerebrovascular receptor types ET_A, ET_B, AT_1, AT₂ and 5-HT_1B[38] and hence is in agreement with the experimental data obtained in animals. The present study revealed several novel and important findings: (i) While the contractile responses to ET-1 usually means activation of the ET_A receptors we found that there is an interaction between the ET_A and ET_B receptors in cerebral arteries following SAH both in experimental studies and in organ culture of human brain vessels [19, 35]. The present immunohistochemistry provide experimental evidence that both ET_A and ET_B receptors are upregulated after organ culture. (ii) The angiotensin II responses were increased. Contrary to animal data the responses were unaltered by specific angiotensin AT₁ blockers [22]. In animal models of stroke an upregulation of the contractile response to Ang II is observed and this contraction is mediated by the AT₁ receptor [22, 36]. In our study in the human arteries the results showed that in presence of the AT₂ receptor antagonist PD12319 there was a diminished contraction after organ culture compared to control arteries, suggesting that the AT₂ receptors are responsible for the upregulated responses induced by organ culture. Since the immunocytochemistry revealed that it was only the AT₂ receptor protein that was elevated in the cerebral artery smooth muscle cells the evidence suggests that there is de novo upregulation of AT₂ receptors producing contraction in the human brain arteries after organ culture. (iii) The 5-HT responses were reduced by organ culture, a finding in concert with what we have seen in tMCAO using two different models as well as after 24 h of organ culture.

Although the decrease in 5-HT_1B-mediated contractile responses are less pronounced after organ culture than after experimental stroke [37]. In the subarachnoid hemorrhage model and global ischemia we have demonstrated an upregulation of the 5-HT_1B receptor [20, 39]. Hence, whereas smooth muscle ET_B receptors are upregulated after all types of cerebral ischemia investigated so far (subarachnoid hemorrhage, global cerebral ischemia, and focal cerebral ischemia) as well as after organ culture, changes in the expression of the 5-HT_1B receptor appear to vary with the type of cerebral ischemia. The role of 5-HT and its receptor in ischemia are not clear; while some studies report a protective role for 5-HT receptor agonists, others show increased contractility and improvement with 5-HT receptor antagonist [40, 41]. The results from the study of TP receptors revealed an increased response to the agonist but no significant upregulation of receptor protein. In experimental SAH a similar type of reaction appears [24]. The GPCR changes that we have observed in the human cerebral arteries after organ culture show a striking similarity to the changes observed in experimental cerebral ischemia and thus complements the picture of reactions.

Early during organ culture the raf-MEK-ERK pathway is activated and remains activated during the first two days of culture [32]. Other MAPK members such as p38 and JNK may also be activated but this seems to occur later during organ culture. Detailed study of major cerebral arteries and intracerebral microvessels were performed following experimental SAH [25]; the results clearly demonstrated that the MEK/ERK1/2 pathway was activated within minutes and remained activated until end of the 48 h period. On the other hand p38 and JNK reached significance only at 48 h. This is a model of SAH, however, a similar phenomenon was observed after MCA occlusion for 2 h and then reperfusion for 48 h both in large cerebral arteries and in microvessels within the brain tissue [42]. In the present study we verified that organ culture results in enhanced expression of pERK1/2 in the smooth muscle cells. Co-administration of the specific MEK1/2 inhibitor U0126 abolished this, confirming that the MEK/ERK pathway is important.

In cultured human arteries, the specific blockade of the MAPK MEK1/2 activity abolished the vascular smooth muscle cell receptor upregulation. A number of mechanisms and receptors have been proposed to account for the late cerebral ischemia [43] but no drug exist with good effect. Clazosentan, an endothelin receptor antagonist, was in a recent clinical study shown to result in reduction in vasospasm as seen angiographically but the outcome was not altered [44]. This was taken as evidence that we may consider also other events than just arterial narrowing; early brain injury and cortical spreading depression. We propose that cerebrovascular receptor upregulation may be such a mechanism that could be of importance, the current study reveals a mechanism present in man that can be modified with inhibition of raf-MEK-ERK signaling.

In conclusion, we show that specific inhibition of the MAPK pathway using U0126 significantly attenuates the vasoconstriction mediated by ET, AT and TP receptors in human cerebral arteries and the enhanced expression of their receptors. The results indicate that MAPK inhibition might be a novel target for treatment of cerebrovascular disorders.