Positive correlation between YULINK expression and cell migration in PASMCs

To evaluate the role of YULINK in PASMC migration in either YULINK knockdown (KD) or overexpression (OE), PASMCs were analyzed by Western blot analysis, with the efficiency shown in Fig. 1A, B, respectively. To further examine whether diminished YULINK expression could inhibit PASMC migration, PASMCs with or without YULINK knockdown were seeded on the permeable upper layer of the cell culture with a dose-dependent PDGF (0, 5, 20 ng/ml) treatment in the culture medium on the bottom layer of the well for cell migration analysis. As shown in Fig. 1C, PASMC migration increased in a dose-dependent manner. Cell migration was significantly reduced in YULINK-deficient PASMCs treated with PDGF, while in contrast, cell migration was enhanced in PASMCs overexpressing YULINK with or without PDGF treatment (Fig. 1D).

Fig. 1

Positive correlation between YULINK expression and cell migration in PASMCs. YULINK expression in PASMCs with A YULINK knockdown (KD) or B overexpression (OE) was examined by western blot analysis. SC stands for scramble control. The numbers labeled below the respective blot lanes indicate the relative fold normalized with the internal control. C, D Micrographs (magnification 200×) together with bar graphs depict PDGF (5 and 20 ng/ml)-triggered cell migration in PASMCs with C YULINK KD or D YULINK OE using Transwell analysis. The values represent the mean of three independent experiments ± standard deviation. *p < 0.05 compared to scramble control without PDGF. #p < 0.05 between compared groups. E PASMCs with or without YULINK KD or OE were treated with PDGF in a dose-dependent manner (0, 5, and 20 ng/ml) for 24 h and subjected to western blot analysis for the indicated proteins. β-actin served as an internal control

Furthermore, focal adhesion kinase (FAK), a protein that is well recognized as a marker of cell migration, was also determined by Western blot analysis. In addition, FAK is widely expressed as a cytoplasmic protein tyrosine kinase and plays an important key step in cell migration when activated by phosphorylation at Tyr397 [17]. As shown in Fig. 1E, the administration of PDGF induced either YULINK or FAK phosphorylation at Tyr397 in a dose-dependent manner; however, this PDGF-triggered enhancement of FAK phosphorylation was inhibited in PASMCs with YULINK knockdown. In contrast, FAK Tyr397 phosphorylation in response to PDGF treatment was enhanced in PASMCs overexpressing YULINK (Fig. 1E). These data illustrate that YULINK could be a positive regulator in PASMCs under PDGF treatment.

Positive correlation between YULINK expression and cell proliferation in PASMCs

To investigate whether the YULINK expression level could affect cell proliferation, PASMCs with YULINK knockdown (KD) or overexpression (OE) in response to PDGF (20 ng/ml) at 0 and 5 days were subjected to MTT proliferation/viability analysis (Fig. 2A), with increased cell proliferation being noted in PASMCs with PDGF for 5 days compared to those without PDGF treatment. Cell proliferation was inhibited in PASMCs with YULINK KD with or without PDGF treatment. In contrast, cell proliferation was enhanced in PASMCs with YULINK OE with or without PDGF treatment. Similarly, the colony formation assay provided additional evidence of the relationship between YULINK expression and cell proliferation. In the presence of PDGF treatment or in PAH-PASMCs, numerous and distinct colonies formed. However, when YULINK was preemptively suppressed, the colony formations were noticeably reduced (Additional file 1: Fig. S1).

Fig. 2

Positive correlation between YULINK expression and cell proliferation in PASMCs. A PASMCs with YULINK KD or OE were treated with or without PDGF (20 ng/ml) for 5 days before harvest for MTT analysis. B PASMCs with the same treatment for 24 h were stained with PI and subjected to flow cytometry. The cell cycle profiles are presented with bar graphs indicating the distributions of the cells in the cell cycle. The values represent the mean of three independent experiments ± standard deviation. *p < 0.05 compared to scramble control without PDGF. #p < 0.05 between compared groups

As shown in Fig. 2B, these results were further confirmed by cell cycle profile analysis. Both floating and attached cells were collected and stained with propidium iodide (PI) for DNA content detection. In comparison to the scramble control, treatment with PDGF triggered a significant increase in the S-phase population, indicating further DNA synthesis for cell proliferation; however, this upregulated S-phase population was not found in PASMCs with YULINK knockdown. Even without PDGF treatment, PASMCs with YULINK knockdown or overexpression alone slightly suppressed or increased the S-phase population, respectively, accompanied by an increased or decreased G1 phase population, respectively.

Furthermore, existing researches have indicated that PAH pathogenesis involves genomic instability, the production of free radicals, and elevated levels of DNA damage [13, 18], which can lead to apoptosis. However, this effect is likely transient, as cells eventually tend to exhibit increased proliferation during PAH pathogenesis. It is evident that in PASMCs treated with PDGF, there was an increase in the subG1 population, which was further elevated in cells with YULINK overexpression. However, when YULINK was inhibited, this elevation in the subG1 population was significantly mitigated. These results align with the notion that YULINK overexpression, particularly in conjunction with PDGF treatment, leads to genome instability and cell death associated with PAH. This finding also suggests that the inhibition of YULINK could be advantageous in suppressing the pathogenesis of PAH. Taken together, the cell proliferation of PASMCs was positively correlated with YULINK under PDGF treatment.

YULINK colocalized with GLUT1 and participated in PDGF-triggered glucose uptake and glycolysis

Increased glucose uptake and glycolysis in both preclinical studies and PAH patients were noted in hyperproliferative PASMCs, which have increased GLUT1 1 expression [19,20,21]. Therefore, one of our goals was to investigate whether YULINK expression was related to either glucose uptake or glycolysis in PASMCs. PASMCs were treated with the fluorescent glucose analog 2-deoxy-d-glucose (2-DG) to determine the levels of glucose uptake in response to PDGF treatment. Since the 2-hydroxyl group of 2-DG is replaced by hydrogen, it would be transported and thereby accumulate in cells without entering glycolysis, so the fluorescence of accumulated 2-DG would indicate the level of glucose uptake. As shown in Fig. 3A, glucose uptake was inhibited in PASMCs with YULINK knockdown compared to the scramble control. In contrast, glucose uptake was enhanced in PASMCs with YULINK overexpression, while PDGF administration promoted glucose uptake in PASMCs, which was inhibited in PASMCs with YULINK knockdown. On the other hand, glucose uptake was further enhanced in PASMCs overexpressing YULINK under PDGF treatment (Fig. 3A).

Fig. 3

YULINK interacted with GLUT1 and participated in PDGF-triggered glucose uptake and glycolysis. The bar graphs illustrate A glucose uptake and B pyruvate production in PASMCs with YULINK KD or OE treated with or without PDGF (20 ng/ml) for 24 h. The values represent the mean of three independent experiments ± standard deviation. *p < 0.05 compared to scramble control without PDGF. #p < 0.05 between compared groups. C Western blot analysis indicates the expression of GLUT1 and HK-2 in normal PASMCs, PASMCs with YULINK KD, and PASMCs with YULINK OE treated with or without PDGF for 24 h. The numbers labeled below the respective blot lanes indicate the relative fold normalized with the internal control. D Representative images from immunofluorescence analysis indicate the expression of YULINK (green) and GLUT1 (red), and DAPI (blue) indicates nuclear staining. Magnification 200×. E Whole-cell lysates and F membrane fractions derived from PASMCs or PAH-PASMCs with or without 20 ng/ml PDGF treatment and PAH-PASMCs were subjected to Western blot analysis for YULINK expression. β-Actin and Na/K ATPase served as internal controls. G Membrane protein lysates obtained from PASMCs subjected to indicated treatments were used for immunoprecipitation with YULINK antibody-conjugated beads. The proteins pulled down were subsequently analyzed via Western blot to assess the expression of YULINK and GLUT1. While Input serves as positive control, Isotype IgG serves as negative control

We next examined whether YULINK expression was correlated with glycolysis in PASMCs. PASMCs with YULINK knockdown or overexpression were collected for pyruvate production under PDGF treatment for 48 h, and as shown in Fig. 3B, pyruvate production was significantly increased in PASMCs under PDGF treatment, which indicated an upregulation of glycolysis. Furthermore, pyruvate production was further enhanced in PASMCs with YULINK overexpression under PDGF treatment, but in contrast, pyruvate production was inhibited in either PDGF-treated or no-PDGF-treated PASMCs with YULINK knockdown.

Since glucose transporter 1 (GLUT1), the major member of the Glut family, was reported to be expressed in vascular smooth muscle cells and facilitate glucose uptake for vascular contractility and reactivity [22], we next examined whether the alteration of YULINK expression further affected GLUT1 expression. Consistently, while YULINK knockdown decreased GLUT1 expression in PASMCs, the level of GLUT1 was increased with YULINK overexpression regardless of the presence or absence of PDGF treatment (Fig. 3C). Additionally, the level of hexokinase II (HK-2), the key catalytic kinase that converts glucose to glucose-6-phosphate in the process of glycolysis, was affected in the same pattern as GLUT1 by western blotting analysis (Fig. 3C).

To investigate whether YULINK and GLUT1 had protein interactions under PDGF treatment, immunofluorescent analysis was performed for PASMCs with or without PDGF treatment. As shown in Fig. 3D, GLUT1 and YULINK were not only increased but also colocalized in PASMCs treated with PDGF. Taken together, YULINK expression could regulate glucose uptake and glycolysis through GLUT1 protein interaction under PDGF treatment, thereby implying a possible role of YULINK in PAH-related pathogenesis.

In addition to PDGF-treated PASMCs, PASMCs derived from monocrotaline-induced PAH rat models were also examined. As shown in Additional file 1: Fig. S2, medial hypertrophy was noted by hematoxylin and eosin (H & E) staining of the pulmonary artery in the MCT-induced PAH group, confirming the efficacy of the MCT treatment. Since GLUT1 is known to localize on the cell membrane and functions as a glucose transporter, we conducted further investigations to determine whether YULINK also exhibits an increase in cell membrane localization in PASMCs during PAH pathogenesis. As shown in Fig. 3E, F, increased YULINK expression was noted in both whole-cell lysates and membrane fractions in PASMCs under PDGF treatment and in PAH-PASMCs. A co-immunoprecipitation analysis provided further evidence of the colocalization of YULINK and GLUT1 on the cell membrane in PASMCs. Under the conditions related to PAH pathogenesis, it was evident that GLUT1 was notably present in the protein extracts that were pulled down along with YULINK (Fig. 3G). These findings suggested that YULINK could be increased in the cell membrane of PASMCs for protein interaction with GLUT1 during PAH pathological development.

Enhanced YULINK expression in MCT-induced PAH rats and a clinical PAH patient

How YULINK is expressed in MCT-induced rats in vivo was next examined. Immunohistochemical (IHC) staining of the pulmonary artery in the MCT-induced PAH group revealed increased brown staining in the thickening vasculature of the pulmonary artery compared to the normal control. In normal animals, YULINK expression is minimal within the pulmonary artery. However, following PAH induction, there is a significant increase in the expression of YULINK, and it is distributed more extensively throughout the tissue including in media muscular layer (Fig. 4A), which indicated increased YULINK expression in the pulmonary artery of the MCT-induced PAH rat model.

Fig. 4

Enhanced YULINK expression in MCT-induced PAH rats and human PAH specimen. A Pulmonary artery tissues were derived from normal and MCT-induced PAH rats for IHC staining. The brown color in the photomicrograph indicates the expression of YULINK expression. B Tissue sample from the right pulmonary artery of a clinical patient with severe PAH was subjected to IHC staining to assess YULINK expression. A normal pulmonary artery tissue was used as a control for comparison. Scale bar in 1–3: 100 µM, and 4: 200 µM

In addition to the animal model, we obtained a human right pulmonary artery specimen of an individual with severe PAH and performed immunohistochemistry (IHC) staining to analyze YULINK expression in an actual clinical case. Consistent with the results observed in rat tissue, our findings in the human specimen also demonstrate a significant increase in YULINK expression particularly in media muscular layer in the human PAH specimen compared to the control from a normal pulmonary artery specimen (Fig. 4B).

YULINK suppression inhibited the migration and proliferation of PASMCs derived from an MCT-induced PAH rat model (PAH-PASMCs)

To investigate whether cell migration and proliferation could be correlated with YULINK expression in PAH-PASMCs, cells with or without YULINK knockdown were analyzed by Transwell migration and proliferation assays. In comparison with PASMCs derived from normal rats, PAH-PASMCs exhibited a four- to fivefold increase in cell migration (Fig. 5A). However, when YULINK was knocked down, the cell migration was dramatically reduced by half in PAH-PASMCs (Fig. 5A); furthermore, as shown in Fig. 5B, increased expression of YULINK and FAK phosphorylation were noted in PAH-PASMCs compared with PASMCs and were further enhanced by PDGF treatment in both PASMCs and PAH-PASMCs, although both were inhibited in PAH-PASMCs with YULINK knockdown.

Fig. 5

YULINK knockdown reversed the enhancement of cell migration and proliferation in PAH-PASMCs. PASMCs and cells derived from MCT-induced PAH rats with or without YULINK KD were prepared for Transwell cell migration analysis. A Microscopy images presented with bar graphs indicate cell migration in PASMCs. Magnification ×200. B The cells were incubated with PDGF (20 ng/ml) for 24 h before harvest for protein extractions. Western blot analysis indicates the activation and expression of the indicated proteins. β-actin served as an internal control. The numbers labeled below the respective blot lanes indicate the relative fold normalized with the internal control. C MTT analysis of the 5-day proliferation of the cells. D The production of pyruvate in the cells. The values represent the mean of three independent experiments ± standard deviation. *p < 0.05 compared to normal PASMCs. #p < 0.05 between compared groups

Consistently, cell proliferation was enhanced by 50% in a 5-day proliferation analysis in PAH-PASMCs compared to PASMCs; however, cell proliferation was inhibited in PAH-PASMCs with YULINK knockdown (Fig. 5C). As shown in Fig. 5D, pyruvate production increased in PAH-PASMCs, which indicated increased glycolysis compared to that in PASMCs, whereas it decreased in PAH-PASMCs with YULINK knockdown compared to that in PASMCs. Taken together, PAH-PASMCs revealed increased cell migration, proliferation and glycolysis compared to PASMCs, which could be associated with YULINK expression.

YULINK participated in cell migration and proliferation of PASMCs via the PI3K-AKT signaling pathway

The phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway is one of the major downstream effects of the PDGF signaling pathway [23] and has been demonstrated to play an essential role in PDGF-induced PASMC proliferation, migration, and PAH progression [8, 24]. To explore the role of YULINK and its potential mechanism in PAH-related cell migration and proliferation, PASMCs derived from normal or MCT-induced PAH rats with or without YULINK knockdown were treated with PDGF for further experiments. As shown in Fig. 6A, the expression of YULINK and PDGFR was enhanced in PASMCs and PAH-PASMCs under PDGF treatment. Increased PI3K and AKT phosphorylation in response to PDGF treatment was found in the western blotting analysis, which indicated that PI3K-AKT signaling was activated in PASMCs and PAH-PASMCs under PDGF treatment. When YULINK was suppressed in PAH-PASMCs, the expression of PDGFR and the phosphorylation of PI3K and AKT were both inhibited with or without PDGF treatment.

Fig. 6

YULINK regulated cell migration and proliferation in PASMCs via PI3K-AKT signaling. A PASMCs and PAH-PASMCs with or without YULINK KD were treated with or without 20 ng/ml PDGF for 24 h for protein extraction. Cell lysates were subjected to western blot analysis for the indicated proteins. β-Actin served as an internal control. The numbers labeled below the respective blot lanes indicate the relative fold normalized with the internal control. B Representative images (magnification 200×) together with the bar graphs indicate the cell migration analysis of PASMCs, PASMCs with or without YULINK OE treated with LY294002 (10 µM), and PAH-PASMCs treated with LY294002. C A 5-day cell proliferation assay in the cells under the indicated treatments. *p < 0.05 compared to control PASMCs. #p < 0.05 between compared groups

To further examine whether YULINK could be involved in PASMC migration and proliferation via PI3K-AKT signaling, a PI3K inhibitor, LY294002, was administered to PASMCs with or without YULINK overexpression and PAH-PASMCs for further analysis. As shown in Fig. 6B, while treatment with LY294002 alone significantly reduced cell migration in PASMCs, the cell migration ability was rescued in PASMCs with YULINK overexpression under LY294002 treatment. In Fig. 6C, similar results were found in the cell proliferation experiment, with a 5-day proliferation assay showing significant inhibition of PASMC proliferation under LY294002 treatment. Furthermore, the downregulated cell proliferation induced by LY294002 treatment was restored in PASMCs overexpressing YULINK.

Taken together, the cell migration and proliferation of PASMCs under PDGF treatment or PAH-PASMCs might be regulated by YULINK through the PI3K-AKT signaling pathway.