Analysis of Phosphorylated MEK1 Expression Pattern in Cutaneous Dilated Vessels of Capillary Malformations

ABSTRACT

The mechanism of capillary malformation (CM) is thought to be the abnormal dilation of capillaries as a result of activation of the RAS–RAF–MEK–ERK pathway due to GNAQ/GNA11 gene mutations. However, no prior studies have actually demonstrated phosphorylation of MEK in CM. CMs were classified into three types according to clinical and histopathological findings: Flat, hypertrophic, and nodular. Comparison of the expression pattern of phosphorylated MEK1 (p‐MEK1) in these three types of CMs revealed that p‐MEK1 staining was positive in dilated capillaries in the upper dermis of the flat CMs. Double staining was performed to determine which cells of CM expressed p‐MEK1: CD34 and p‐MEK1 showed partial co‐localization, demonstrating that p‐MEK1 is expressed in the vascular endothelial cells. Both p‐MEK1‐positive and p‐MEK1‐negative vessels were observed in dilated vessels of CM. In addition, p‐MEK1‐positive vessels may be present deeper than anticipated. JNK and ERK were also phosphorylated in the dilated vessels. Furthermore, in contrast to the flat type and hypertrophic type of CM, the nodular type showed no p‐MEK1 expression in its proliferated capillaries, supporting the notion that nodular CMs arise via a distinct mechanism. Taken together, our results indicated that visualization of affected vessels of CM using p‐MEK1 immunostaining is useful not only for diagnosis but also for understanding the pathogenesis of CM.

1. Introduction

Capillary malformations (CMs), also historically termed port‐wine stains or hemangioma simplex, are common congenital slow‐flow vascular malformations that present at birth as flat, pink to reddish patches. Clinically, lesions often darken, thicken, and may develop hypertrophic or nodular lesions over time, leading to functional and psychosocial morbidity [1, 2, 3]. Histopathologically, CMs are characterized by abnormally dilated capillary vessels in the superficial dermis, without significant endothelial proliferation or atypia.

The genetic basis of CM was elucidated by the discovery of recurrent somatic activating mutations in GNAQ/GNA11. These mutations are present in a mosaic form and are enriched in endothelial cells lining the malformed vessels, suggesting that endothelial Gαq/11 activation is a primary event in the pathogenesis of CM [4]. Gαq proteins canonically couple G protein–coupled receptors to phospholipase Cβ, leading to diacylglycerol production and activation of protein kinase C (PKC), which in turn stimulates the RAS–RAF–MEK–ERK mitogen‐activated protein kinase (MAPK) cascade [3]. Experimental models of endothelial cells harboring GNAQ/GNA11 variants have demonstrated constitutive calcium signaling and enhanced MAPK pathway activity, supporting a direct link between mutant Gαq/11 and aberrant intracellular signaling in vascular endothelium [5]. Consistent with this, immunohistochemical and biochemical studies of CM tissues have shown sustained activation of JNK and ERK in lesional vessels compared with normal control tissues [6].

Despite accumulating evidence that JNK and ERK are phosphorylated in GNAQ‐mutant vascular lesions, previous histopathological studies have primarily compared lesional skin with non‐lesional normal skin and have not focused on other molecules such as MEK. To our knowledge, phosphorylation of MEK1 in human CM lesions has not been systematically examined. Moreover, the potential utility of phospho‐specific immunohistochemistry to selectively highlight abnormal CM vessels and distinguish them from normal capillaries within the same specimen has not been fully explored.

In the present study, we sought to demonstrate evidence for phosphorylated MEK1 (p‐MEK1) in abnormally dilated vessels of CM, and that p‐MEK1 immunohistochemistry can serve as a marker to distinguish affected CM vessels from normal dermal capillaries. Using phosphorylation‐specific antibodies, we examined p‐MEK1 expression in CM specimens and compared staining patterns between morphologically abnormal vessels, adjacent non‐dilated capillaries within the lesion, and normal control skin.

2. Methods

2.1. Patient Material and Ethics Statement

This research was approved by the Ethics Review Committee in Wakayama Medical University (No. 2730). Skin specimens were obtained from 9 patients with CM. Six normal skin samples were also collected from the routinely discarded skin of healthy subjects undergoing skin surgery or biopsy. Written informed consents were obtained from all patients, in accordance with the Declaration of Helsinki.

2.2. Immunohistochemical Staining

Skin specimens were fixed in 10% neutral‐buffered formalin, embedded in paraffin, and sliced [7]. Paraffin sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed by microwaving slides in a pH 6 citrate buffer for 10 min. Endogenous peroxidase activity was inhibited, and these sections were blocked with normal serum (VECTASTAIN Elite ABC Kit, Vector, Burlingame, CA) for 40 min, then incubated with a p‐MEK1 rabbit monoclonal antibody (pSer298, 1:1000) (Novus Biologicals, Centennial, CO) diluted in PBS for 1 h at 37°C: For the antibody, the immunogen was a synthetic phospho‐peptide corresponding to the residues surrounding Ser298 of human MEK1, conferring its phospho‐specificity. The antibody has actually been demonstrated that (i) the signal disappears upon competition with an immunogenic phosphorylated peptide, but not with an unphosphorylated peptide, (ii) it does not recognize the MEK1 Ser298Ala mutant; and (iii) it does not recognize MEK2 [8, 9]. The specificity and reliability of the antibody have also been rigorously validated by the manufacturer and other papers for immunohistochemistry on paraffin‐embedded tissues and immunoblot analysis [10, 11, 12].

After excess antibodies were washed out with PBS, the samples were reacted with the secondary antibodies (Biotinylated antibody from VECTASTAIN Elite ABC Kit) for 1 h at 37°C. Then, the ABC reagent from VECTASTAIN Elite ABC Kit and DAB (Dako, Denmark) was used. The slides were counterstained with Mayer's hematoxylin, and the slides were examined under a light microscope (Nikon, Tokyo, Japan) or a virtual slide system (Nanozoomer, Hamamatsu Photonics K.K, Hamamatsu, Japan).

Staining of phosphorylated JNK (p‐JNK) was performed with pH 9 citrate buffer and antibody for p‐JNK (G‐7, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA). For staining of phosphorylated ERK (p‐ERK), blocking with 5% milk and p‐ERK antibody (E‐4, 1:50, Santa Cruz Biotechnology) was used.

2.3. Immunofluorescence

Paraffin sections were deparaffinized in xylene and rehydrated in a graded ethanol series.

Antigen retrieval was performed in a pH 9 buffer by microwave heating for 10 min [13]. Slides were permeabilized with 0.5% Triton X‐100 in PBS for 5 min at room temperature, blocked with normal serum (VECTASTAIN Elite ABC Kit, Vector) for 40 min, and then incubated with the antibodies for mouse anti‐CD34 (1:50, Dako) and rabbit anti‐pMEK‐1 (pSer298, 1:50) diluted in PBS for 1 h at 37°C. After a brief wash with 0.05% Triton‐PBS, sections were incubated with Alexa Fluor 488–conjugated anti‐rabbit IgG (1:200, Invitrogen, Carlsbad, CA) and Rhodamine‐conjugated anti‐mouse IgG (1:100, Jackson Immuno‐Research, Suffolk, UK) as secondary antibodies for 1 h at room temperature. Slides were then mounted with VECTASHIELD mounting medium with DAPI (Vector). Nikon Y‐TV55 microscope (Nikon) was used for fluorescence microscopy.

2.4. Morphometry

According to the previous paper [14], we quantified staining intensity and the quantity of vessels in four randomly chosen magnification fields (×200) in all specimens. Staining intensity in all specimens was evaluated as equal (0), mildly [1], moderately [2], and strongly [3] higher than staining intensity in the control specimen. Stained vessels were counted and documented as 30% (I), 30% to 60% (II), and 60% to 100% (III) of all specimens. The staining intensity and quantity parameters were united to a score by multiplication. Two independent observers evaluated the intensity and quantity of vessels in a blinded manner, and their assessments were generally consistent.

2.5. Statistical Analysis

The Mann–Whitney U test was used for comparisons. p < 0.05 was considered statistically significant.

3. Results

3.1. P‐MEK1 Staining in Dilated Vessels of CM

In the present study, CMs were classified into 3 types with reference to previous studies: flat, hypertrophic, and nodular, according to clinical and histopathological findings [1, 2, 3]. As a result, 4 out of 9 CM patients were flat type, 3 were hypertrophic type, and 3 were nodular type: One patient (case7) exhibited both hypertrophic and nodular lesions in the same sample (Table 1).

TABLE 1.

Patients with CM included in the present study.

Case	Type	Location
1	Flat	Scalp
2	Flat	Trunk
3	Flat	Neck
4	Flat	Trunk
5	Hypertrophic	Lip
6	Hypertrophic	Face
7	Hypertrophic/Nodular	Scalp
8	Nodular	Leg
9	Nodular	Trunk

Abbreviation: CM, capillary malformation.

The expression patterns of p‐MEK1 in these 3 types of CM and normal tissue were compared. In flat CMs, p‐MEK1 staining was positive in the dilated capillaries of the upper dermis (Figure 1A–C). p‐MEK1 staining was also detected in vessels with dilated and thickened walls of hypertrophic CMs (Figure 1D–F). However, normal tissues showed only faint p‐MEK1 staining.

FIGURE 1.

p‐MEK1 staining in vessels of flat and hypertrophic CM. (A) Clinical image of a flat CM (case 1) showing reddish macules on the temporal region. (B) Hematoxylin and eosin staining of the flat CM tissue (original magnification = ×200). (C) Immunoreactivity of p‐MEK1 in dilated vessels of the flat CM (original magnification = ×200). (D) The clinical picture of a hypertrophic CM (case 5) showing a reddish bumpy lesion on the lip. (E) Hematoxylin and eosin staining of the hypertrophic CM tissue (original magnification = ×150). (F) Immunoreactivity of p‐MEK1 in dilated vessels of the hypertrophic CM (original magnification = ×150).

Notably, p‐MEK1 staining was completely absent in the proliferated capillary vessels of the nodular type CMs (Figure 2).

FIGURE 2.

p‐MEK1 staining in vessels of nodular CM. (A) Clinical image of a nodular CM (case 7) showing an erosive nodule on the temporal region. (B) Hematoxylin and eosin staining of the nodular CM tissue (original magnification = ×15). (C) Immunoreactivity of p‐MEK1 in proliferated vessels of the nodular CM (original magnification = ×15). (D) The clinical picture of a nodular CM (case 8) showing a nodule on the lower leg. (E) Hematoxylin and eosin staining of the nodular CM tissue (original magnification = ×15). (F) Immunoreactivity of p‐MEK1 in proliferated vessels of the nodular CM (original magnification = ×15).

3.2. Assessment of p‐MEK1 Staining in Dermal Blood and Lymphatic Vessels

Double immunofluorescence staining for p‐MEK1 and CD34 (an endothelial cell marker) showed partial co‐expression of the two signals in the dilated vessels of CM, indicating that the p‐MEK1 positivity is localized to vascular endothelial cells (Figure 3). We further analyzed the staining pattern of p‐MEK1 in CM. First, we evaluated p‐MEK1 expression in lymphatic vessels. No p‐MEK1 staining was detected in lymphatic vessels in any of the CM lesions or normal skin (Figure 4A,B), supporting that p‐MEK1 expression is specific to blood vessels as described above.

FIGURE 3.

Double immunofluorescence staining of p‐MEK1 and CD34. Sections of a hypertrophic CM (case 5) were stained with anti‐p‐MEK1 (green) and anti‐CD34 (red). Nuclei are stained blue with DAPI.

FIGURE 4.

Various staining patterns of p‐MEK1 in CM. (A) P‐MEK1 staining in lymphatic vessels of a flat CM (case 1) (original magnification = ×200). Asterisks indicate lymphatic vessels. (B) P‐MEK1 staining in lymphatic vessels of a hypertrophic CM (case 6) (original magnification = ×200). Asterisks indicate lymphatic vessels. (C) P‐MEK1 staining in dilated vessels of a flat CM (case 2) (original magnification = ×100). Asterisks indicate vessels without p‐MEK1 staining. (D) P‐MEK1 staining in dilated vessels of a hypertrophic CM (case 6) (original magnification = ×150). Asterisks indicate vessels without p‐MEK1 staining.

Second, p‐MEK1 is not present in every dilated blood vessel of CM. There were both dilated capillaries positive and negative for p‐MEK1 within the same tissue (Figure 4C,D). Furthermore, we noted that p‐MEK1 was occasionally detected in vessels of the deep dermis (Figure 5A,B). Therefore, there is a possibility that deeper blood vessels than anticipated have been affected.

FIGURE 5.

Staining of deeper vessels in CM and semiquantitative analysis. (A) and (B) P‐MEK1 expression in deep dermal vessels of CMs (cases 1 and 2) (original magnification = ×200). Asterisks indicate dilated vessels in the deep dermis. (C) Semiquantitative p‐MEK1 expression scores represented as a bar graph. Scores were calculated by multiplying staining intensity by the number of stained vessels in the upper dermis. *p < 0.05 vs. normal skin.

Using a semiquantitative scoring system [14], staining intensity as well as pattern and distribution of expression was assessed in comparison to control specimens. Both parameters were unified by multiplication to a score shown in Figure 5C. Eight of the 9 CM cases had higher p‐MEK1 scores in the upper dermis, which was statistically significant compared to normal skin (p < 0.05).

Lastly, to clarify the underlying pathogenesis of p‐MEK1 expression in CM vessels, immunostaining of p‐ERK and p‐JNK was performed in all CM samples (Figure 6A–D). As a result, p‐JNK was positive for almost all blood vessels within the lesion, regardless of dilation (Figure 6A,C). On the other hand, p‐ERK showed higher specificity for dilated blood vessels but was detected in more blood vessels than p‐MEK1 (Figure 6B,D).

FIGURE 6.

Staining of p‐JNK and p‐ERK in vessels of CM. (A) Immunoreactivity of p‐JNK in a flat CM tissue (case 2) (original magnification = ×100). (B) Immunoreactivity of p‐ERK in a flat CM tissue (case 2) (original magnification = ×100). Asterisks indicate vessels without p‐ERK staining. (C) Immunoreactivity of p‐JNK in a hypertrophic CM tissue (case 6) (original magnification = ×150). (D) Immunoreactivity of p‐ERK in a hypertrophic CM tissue (case 6) (original magnification = ×150). Asterisks indicate vessels without p‐ERK staining.

4. Discussion

Our study reveals three novel findings. First, by correlating vascular morphology with MEK1 activation status, we provided direct evidence of MEK activation in abnormal vessels of CM and demonstrated its potential diagnostic and pathobiological significance.

Second, not all dilated vessels in CM were positive for p‐MEK1: Both p‐MEK1‐positive and p‐MEK1‐negative vessels were observed in dilated vessels within the same lesions of CM. This is consistent with the data that the GNAQ/GNA11 mutations are not present in all affected vessels of CM as determined by next‐generation sequencing [4, 15]. In addition, some p‐MEK1‐positive vessels may lie deeper in the dermis, and there is a theoretical possibility that it contributes to lesion recurrence after laser therapy. However, the present study did not demonstrate direct evidence that these deeper vessels are truly mutated vessels; there is also the possibility that such vessels are reactive in nature and therefore show positive p‐MEK1 staining. Further studies are needed in the future.

On the other hand, p‐JNK was positive for almost all blood vessels within the lesion. p‐ERK showed higher specificity for dilated blood vessels, but was detected in more blood vessels than p‐MEK1. This is compatible with the previous paper that demonstrated that phosphorylation of JNK happened before morphological abnormalities and blood vessel dilation, even in the adjacent areas of the CM lesion [6]. Another possibility is that phosphorylation in Ser298 of MEK1 evaluated in the present study was also induced by PAK1, one of the downstream molecules of RAS. Therefore, phosphorylation of Ser298 of MEK1 is subject to regulation that differs from the standard MAPK (RAS–RAF–MEK–ERK) cascade. Further studies are required to determine the activation status in other phosphorylation sites of MEK1.

Lastly, contrary to the flat type or hypertrophic type of CMs, the nodular type lesions showed no p‐MEK1 expression in their proliferated capillary vessels. This may align with other studies suggesting that nodular CM lesions arise via distinct mechanisms—e.g., upregulated activation of PLC‐γ, PKCα, PI3K, PDPK1, and mTOR, or focal venous hypertension [1, 16, 17]. Our findings also support the notion that nodular CMs are driven by a different pathogenesis. For example, most patients with the nodular type appear to be adults. Thus, aging may induce local venous hypertension and hemodynamics (turbulent shear stress and/or pressure load as well as cellular senescence, inflammation, and oxidative stress), leading to a stepwise activation of the signaling pathways, including PI3K and PLC‐γ. However, given the small number of nodular cases, future verification is required.

The RAS–RAF–MEK–ERK axis has also emerged as a potential therapeutic target in vascular anomalies. In a mouse model expressing hyperactive GNAQ in endothelial cells, MEK inhibition, such as trametinib, reduced vascular tumor growth and coagulopathy, implicating MEK as a critical signaling node downstream of mutant Gαq [18]. Early clinical experience further supports the relevance of this pathway, as MEK inhibitors such as trametinib have shown benefit in selected patients with severe capillary malformation–arteriovenous malformation syndrome, as well as other vascular malformations driven by the MAPK pathway mutations [19]. Precise delineation of MEK activation in human CM tissue could thus contribute not only to diagnostic refinement, but also to the rational selection and monitoring of targeted therapies in these patients. In future studies, it will also be important to investigate both the phosphorylation status of upstream signaling molecules and the mechanisms responsible for the dilation of p‐MEK1–negative vessels in CM lesions. As another limitation of this study, except for only one out of the 9 patients (GNAQ Arg183Gln in case No. 7), mutation analysis was not performed. Thus, the link between mutation status and p‐MEK1 expression remains indirect in this study.

Taken together, our results indicated that immunostaining of p‐MEK1 may help distinguish affected vessels from normal capillaries or lymphatic vessels. Visualization of affected vessels could be useful not only for diagnosis, but also for understanding the pathogenesis of CM.

Disclosure

No financial conflicts of interest to disclose concerning the manuscript.

Conflicts of Interest

Masatoshi Jinnin is an Editorial Board Member of the Journal of Dermatology and a Co‐author of this Article. To minimize bias, he was excluded from all editorial decision‐making related to the acceptance of this article for publication.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.