Pulmonary microvascular endothelial cells from irreversible PAH showed apoptosis resistance

Three weeks after monocrotaline injection following left pneumectomy, the rats have developed irreversible PAH, characterizing -with significantly increased mean pulmonary arterial pressure (mPAP) (Fig. 1A). In addition, the immunohistochemistry staining in small pulmonary arteries showed the existence of the vWF-expressing endothelial cells lining vascular channels in PAH rats but not control ones (Fig. 1B, C), indicating that a severe pulmonary proliferative lesion, plexiform-like lesions were formed in the PAH rats. Cultured cells obtained from PAH rats exhibited polygon or fusiform morphologies (Fig Suppl A) and were strongly positive for CD31 and CD 309 as detected by flow cytometry, respectively (Fig Suppl B, C). Decreased cell apoptosis was confirmed by the presence of lower caspase-3/caspase-7 activity among irreversible PAH pulmonary arterial endothelial cells (PAH) cultured for 1 to 3 days compared with that from control rats (Con, Fig. 1H). Furthermore, apoptosis was quantitated by Annexin V-FITC/PI staining based on flow cytometry. Consistently, the rate of apoptosis in PAH group was significantly lower than that of Con group (Fig. 1D, E, G). PACEs treated by hypoxia (5%O2) for 24 h induced apoptosis-resistance (AR), with the feature of decreased caspase-3/caspase-7 activity and lower apoptosis rate (Fig. 1F, G, H). The surviving apoptosis resistant cells were harvested and cultured for further use in subsequent experiments.

Fig. 1

Endothelial cells isolated from pulmonary arteries developing plexiform lesions or treated by hypoxia showed apoptotic resistance. Figure 1A. The mean pulmonary arterial pressure increased significantly higher in Pneu + MCT treated rats than that in Sham + veh treated rats (50.4 ± 8.8 mmHg vs.15 ± 2.4 mmHg, n = 10 in each group, unpaired two-tailed t-test, p < 0.0001) 21 days after treatment. Figure 1B-C, Immunostaining with von Willebrand factor (vWF) showed normal small artery in rats received sham procedure + vehicle treatment (B) and abnormal channels formed by a diffuse population of endothelial cells in rats received Pneu + MCT treatment(C). Figure 1D-G, The PAECs isolated from pulmonary arteries of PAH rats (PAH group) or received hypoxia treatment (AR group) were less apoptotic compared with control rats [Con group (41.00 ± 3.39)%, PAH group(11.00 ± 1.87)%, AR group (14.2 ± 2.39)%, n = 5 in each group, One-way ANOVA Tukey’s multiple comparisons test, PAH vs. Con, p < 0.0001, AR vs. Con, p = 0.0002]. Figure 1H, The caspase-3/caspase-7 activity showed that the reduction in apoptosis occurred over days of culture in PAECs either isolated from PAH rats or caused by hypoxia treatment (n = 5 in each group, Two-way ANOVA Tukey’s multiple comparisons test, relative fluorescence light units: Day 1, PAH vs. Con, p = 0.0192, AR vs. Con, p = 0.0311; Day 2, PAH vs. Con, p = 0.0005, AR vs. Con, p = 0.0024; Day 3, PAH vs. Con, p = 0.0003, AR vs. Con, p = 0.0021). Veh: vehicle, Pneu: pneumectomy, MCT: monocrotaline. #: p < 0.05, vs. sham + veh group, *p < 0.05, vs. con group

Apoptotic-resistance PAECs displayed fragmented mitochondrial morphology

As shown in Fig. 2B, the mitochondria in PAH group showed shorter, rounded structures in transmission electron microscopic images, while the ones in control group displayed elongated, tubular structures (Fig. 2B), and the length of mitochondria in PAECs were significantly longer than that in PAH group (Fig. 2D). In addition, the mitochondrial morphology was further analyzed using a mitochondrial marker (Mito Tracker Green) by confocal microscopy. Representative images of the mitochondria are shown in Fig. 2F, and the marked mitochondrial fragmentation was observed in the PAH group compared to the Con group (Fig. 2E). The PAECs in PAH group have a significantly higher percent of fragmented mitochondria than that in Con group (Fig. 2H). In hypoxia induced apoptotic-resistance PAECs (AR group, Fig. 2C, G), mitochondria displayed short isolated dot-like spheres in both transmission electron microscopic and confocal microscopic images, with increased fragmented mitochondria indicating as shorter mitochondria and higher percent of fragmented mitochondria when compared to Con group (Fig. 2D, H).

Fig. 2

Apoptosis resistant PAECs display fragmented mitochondrial morphology. A-C, Representative images from transmission electron microscopy showed shorter, rounded mitochondria in PAECs from PAH and AR group rats and elongated, tubular structures in PAECs from Con group rats. D, Mitochondria showed significantly shorter in structure in PAECs from both PAH (0.97 ± 0.29 μm, vs. Con group, n = 41 in each group, One-way ANOVA Tukey’s multiple comparisons test, p < 0.0001) and AR group (1.16 ± 0.24 μm, vs. Con group, n = 41 in each group, One-way ANOVA Tukey’s multiple comparisons test, p < 0.0001) rats than that in Con group (3.06 ± 0.56 μm) when analyzed at least 40 mitochondria per experiment. E–G, Representative images of mitochondria with more fragmentation by MitoTracker Green-labeling were presented in PAECs from PAH and AR group, while more tubular mitochondria were displayed in Con group. H, The percentage of cells with fragmented mitochondria in PAECs from PAH [(55.2 ± 7.3)%, vs. Con group, n = 45 in each group, Two-way ANOVA Tukey’s multiple comparisons test, p < 0.0001] and AR group[(44.4 ± 4.2)%, vs. Con group, Two-way ANOVA Tukey’s multiple comparisons test, p < 0.0001] were higher than from Con group [(7.3 ± 5.5)%] when assessed 150 mitochondria in each experimental group. *p < 0.05, vs. con group

Drp1 played a key role in the development of apoptotic-resistance PAECs

The role of a key protein Drp1 in regulation of mitochondrial fission was investigated. Immunofluorescence showed that Drp1 was overexpressed in both PAH (Fig. 3B) and AR (Fig. 3C) group. The endogenous protein expression of Drp1 detected by western blotting analysis were significantly higher in both PAH and AR group than that in Con group as well (Fig. 3D).

Fig. 3

DRP1 was involved in development of apoptosis resistant PAECs. A-C, Immunofluorescence for Drp1 protein expression showed enhanced intensity in PAECs both from PAH and AR group. D, DRP1 protein expression analyzed by western blotting were increased significantly in PAH (vs. Con group, n = 4 in each group, One-way ANOVA Tukey’s multiple comparisons test, p = 0.0115) and AR group (vs. Con group, n = 4 in each group, One-way ANOVA Tukey’s multiple comparisons test, p = 0.0278) when compared to Con group. E, Immunoblot of DRP1 following knockdown via siRNAs compared to nontargeting control siRNA sequence (NTRNA). F–H, The transfection of Drp1-SiRNA blocked the development of apoptosis resistant induced by hypoxia (5% O2) in PAECs as assessed by flow cytometry with PE Annexin V/7-AAD Apoptosis Detection Kit (apoptosis rate: NTRNA + AR, 12.60 ± 1.52 vs. SiDrp1 + AR, 33.20 ± 2.87, n = 5 in each group, unpaired two-tailed t-test, p < 0.0022). *p < 0.05, vs. con group, #p < 0.05, vs. NTRNA group, **p < 0.05, vs. NTRNA + AR group

As the up-regulated expression of Drp1 protein in both PAH and AR group, this suggests a potential function for Drp1 in the formation of apoptosis resistant PAECs. Drp1 was silenced in PAECs using SiRNA method, and the expression in PAECs that detected by western blotting was reduced significantly after transfection of Drp1-SiRNA (Fig. 3E). Drp1 silence prevented mitochondrial fission in hypoxia induced apoptosis resistant PAECs which showing as less fragmented and more elongated mitochondria in Drp1 silenced PAECs under hypoxia (5% O2) circumstance for 24 h (SiDrp1 + AR, Fig. 4). Consistently, quantitative analysis by flow cytometry indicated that Drp1 silence significantly increased the percentage of apoptosis cells in PAECs after hypoxia treatment (Fig. 3F-H). Similarly, Mdivi-1, a Drp1 inhibitor, rescued the mitochondrial fission and apoptosis resistance in PAECs isolated from PAH rats showing pulmonary plexiform-like lesions, which assessed via the quantification of the mean mitochondrial length (Fig. 5A-C), percent of fragmented mitochondria in cells (Fig. 5D-F) and apoptosis rate (Fig. 5G-I).

Fig. 4

Drp1 knockdown affected the mitochondrial morphology. A-C Transmission electron microscopy images of mitochondria showed that the elongated, tubular mitochondria increased instead of shorter, rounded mitochondria after siDrp1 transfection, indicating as increased length of mitochondria in siDrp1 + AR group than in NTRNA + AR (1.94 ± 0.45 vs. 1.27 ± 0.29, n = 45 in each group, unpaired two-tailed t-test, p < 0.0001). D-F, In hypoxia environment (5% O2), decreased fragmented mitochondria were presented in PAECs with siRNA transfection for Drp1 when compared that received nontargeting control siRNA transfection [Percent of Fragment mitochondria: (50 ± 5)% vs. (38 ± 8.3)%, n = 150 in each group, unpaired two-tailed t-test, p < 0.0001]. *p < 0.05, vs. NTRNA + AR group

Fig. 5

Pharmacologic blockade of DRP1 improved mitochondria fission and apoptosis in PAH PAECs. A-C, Mdivi-1 treatment on PAECs from PAH rats increased the length of mitochondria significantly compared to vehicle treatment as analyzing by Transmission electron microscopy (0.99 ± 0.26 vs. 1.8 ± 0.34, n = 45 in each group, unpaired two-tailed t-test, p < 0.0001). D-F. Reduced mitochondrial fragmentation were displayed in mdivi-1treated-PAH PAECs labeled by MitoTracker Green compared to vehicle treated [percent of fragmentation mitochondria: (52.5 ± 5.86)% vs. (42.8 ± 4.36)%, n = 150 in each group, unpaired two-tailed t-test, p < 0.0001]. G-I, The apoptosis rate were elevated in PAECs from PAH rats after midivi-1 treatment when analyzed using flow cytometry for apoptosis [apoptosis rate: (12.00 ± 2.60)% vs.(33.00 ± 5.10)%, n = 5 in each group, unpaired two-tailed t-test, p = 0.0004].* p < 0.05, vs. DMSO + PAH group

STAT3 activation mediated the expression of Drp1 during the process of apoptotic-resistance PAECs formation

Phosphorylation of STAT3 plays a major role in preserving mitochondrial function in endothelial cells (Banerjee et al. 2017), here, the effects of phosphorylated STAT3 on mitochondrial fission were investigated in apoptotic-resistance PAECs. The expression of STAT3 in Con group, PAH group and AR group were similar without significant difference among them (Fig.6A-B), while apoptosis resistant PAECs (both PAH group and AR group) have significantly higher phosphorylated STAT3 on Tyr 705 than Con group (Fig. 6A-C). An inhibitor of STAT3 phosphorylation, AG490, prevented the overexpression of Drp1 in PAECs received hypoxia treatment for 24 h (Fig. 6D-F), in addition, the apoptosis in AG-490 treated PAECs under hypoxia environment increased significantly when compared to AR group (Fig. 6G-I).

Fig. 6

STAT3 phosphorylation at Tyr 705 affected Drp1 protein expression and apoptosis in PAECs under hypoxia. A, Immunoblot of STAT3 and STAT3Tyr705 phosphorylation in PAECs from Con, PAH and AR group. B. The protein expression of STAT3 among the three groups were similar with statistical difference (n = 3 in each group, One-way ANOVA, p = 0.729), while the levels of STAT3 Tyr705 phosphorylation were higher in both PAH and AR group than that in Con group (n = 3 in each group, One-way ANOVA Tukey’s multiple comparisons test, PAH vs. Con, p = 0.0004, and AR vs. Con, p = 0.0008). C. PAECs from both PAH and AR group had increased percent of phosphorylated STAT3 Tyr705 compared with controls (n = 3 in each group, One-way ANOVA Tukey’s multiple comparisons test, PAH vs. Con, p = 0.0014, and AR vs. Con, p = 0.0043). D-E, Representative immunofluorescence images showed weakened Drp1 protein expression in PAECs with AG-490 (an inhibitor of STAT3Tyr705 phosphorylation) treatment under hypoxia (5% O2). F, The decreased Drp1 expression in PAECs received AG-490 treatment under hypoxia compared to receive vehicle treatment under hypoxia (n = 4 in each group, unpaired two-tailed t-test, p = 0.0401). G-I, The AG-490 treated-PAECs under hypoxia showed increased apoptosis than vehicle-treated PAECs as assessing by flow cytometry [(13.60 ± 3.85)% vs.(36.0 ± 4.47)%, n = 4 in each group, unpaired two-tailed t-test, p = 0.0406]. *p < 0.05, vs. Con group, #p < 0.05, vs. saline + AR group

Discussion

Recently, mitochondrial dysfunction has become the focus of intensive investigation in PAH, and enhanced mitochondrial fragmentation has also been found in pulmonary arterial smooth muscle cells and right ventricle from animal models of PAH (Culley and Chan 2018). However, there are needsto characterize mitochondria involving in progressive increase in pulmonary vascular resistance due to pathologic remodeling of the pulmonary vasculature, and the effects on endothelial cells remain one of uncharted territory for future discovery (Marshall et al. 2018). We reported that apoptosis-resistant PAECs either from PAH rats developing plexiform-like lesions or induced by hypoxia showed increased mitochondrial fragmentation. The fragmented phenotype in PAECs was associated with up-regulation of the mitochondrial fission regulator Drp1, and Drp1 silence using siRNA or pharmacological blocked by Mdivi-1 prevented the development of mitochondrial fission. Furtherly, activation of STAT3 via phosphorylation at Tyr705 has been proved to play a crucial role in Drp1-mediated mitochondrial fission in the apoptosis-resistant PAECs, providing a basic but novel mechanism underlying the development of PAH.

Irreversible PAH is characterized by plexiform vascular lesions in pulmonary arterioles, which are hypothesized to arise from apoptosis-resistant PAECs (Masri et al. 2007). The young rats received left pneumectomy and monocrotaline injection, following White RJ et al.’s method (White et al. 2007) developed plexiform lesions as identified by immunohistochemical staining with vWF. Primary cultures of PAECs from the animal model showed decreased apoptosis, which confirmed again that apoptosis-resistant PAECs were the pathological hallmark of irreversible PAH. To explore the mechanisms, apoptosis-resistant PAECs were also induced by hypoxia treatment according to Yongmei Cao et al.’s method (Cao et al. 2016).

Although there are several molecules involved in the process of mitochondrial fission, Drp1 is indispensable during mammalian mitochondrial fission. Drp1-mediated mitochondrial fission is intricately related to many distinct pathological conditions, such as cancer (Altieri 2019), neurodegenerative diseases (Alexiou et al. 2019) and cardiovascular diseases. It is believed that Drp1 contributes to the process of apoptosis suppression of pulmonary arterial smooth muscle cells (Zhang et al. 2016) and angiogenesis of PAECs (Shen et al. 2015) and plays multiple roles in pulmonary vascular remodeling. Here we found that impaired mitochondrial dynamics and excessive mitochondrial fission induced by Drp1 overexpression could be an important mechanism in apoptosis-resistant PAECs. First, the percent of fragmented mitochondria increased in PAECs either from irreversible PAH rats or after hypoxia treatment, meanwhile, the length of mitochondria in these two types of apoptosis-resistant PAECs decreased obviously. Second, the increased mitochondrial fission was associated with overexpression of Drp1 by western blotting analysis, and targeting Drp1 using siRNA and pharmacological blocker Midivi-1 inhibited the mitochondrial fission and following apoptosis-resistance.

As a key fission protein in mitochondrial dynamics, Drp1 was involved in pathological injury of endothelial cells in many cardiovascular diseases through influencing cellular energy, ROS generation, intracellular calcium levels, apoptogenic protein production, and so on. In Kawasaki disease murine model, mitochondrial dysregulated fission caused by Drp-1 overexpression precipitated the arterial endothelial cells injury, which could be reversed by Masitinib targeting Drp1(An et al. 2023). Similarly, Drp1 was vital in mediating the overexpression of miR-199b-5p regulated oxidized low-density lipoprotein–induced mitochondrial dysfunction and apoptotic effects in human umbilical vein endothelial cells by targeting AKAP1-mediated mitochondrial fission (Cui et al. 2022). The endothelial cell apoptosis associated with the pathophysiology of atherosclerosis could be alleviated by Mdivi-1, an inhibitor of Drp1, through inhibiting mitochondrial fission. Interestingly, a classical anti-atherosclerotic drug, atorvastatin, prominently alleviated the mitochondrial dynamics disorder and vascular endothelial cell injury both in vitro and in vivo via inhibiting the expression of Drp1 (Liu et al. 2023). The role of Drp1 both in endothelial cell-related pathological process and in pulmonary artery muscle smooth cells hyperproliferation in PAH were reported in previous studies, whereas this work advances the mechanisms of Drp1-mediated mitochondrial fission in the development of apoptotic resistance in PAECs in PAH.

STAT3 plays a critical role in the development of apoptosis abnormality in tumor cell lines, which promotes an apoptosis-resistant environment via the activation of survivin, NFAF and Bcl-2 (Marshall et al. 2018). Increased and sustained STAT3 phosphorylation has been reported in endothelial cells localizing in plexiform lesions of idiopathic human lungs as well as in PAECs from idiopathic PAH human lungs. We reported that apoptosis-resistant PAECs isolated from monocrotaline injection following left pneumonectomy-induced PAH rats or induced by hypoxia treatment demonstrated increased phosphorylation of STAT3 at Tyr 705, and when STAT3 phosphorylation inhibited by AG-490, the apoptosis increased in PAECs cultured in a 5% O2 environment. These findings confirmed the role of Tyr 705 STAT3 phosphorylation in the regulation of apoptosis in a hypoxia-induced apoptosis resistant PAECs.

Recently, several studies have demonstrated that STAT3 is involved in regulating mitochondrial function, either via its transcriptional activity or independent of its transcriptional activity, but the report of its regulation in mitochondrial dynamics is rare. This year, Zhou K et al. firstly showed that JAK2/STAT3 pathway played a key role in regulating mitochondrial dynamics in microglia through regulation of Drp1 (Zhou et al. 2019). Consistently, we found that, in the development of apoptosis resistant PAECs, Tyr 705 STAT3 phosphorylation played a crucial role in mediating mitochondrial fission through affecting Drp1 expression. STAT3 activation was associated with overexpression of Drp1 in both PAECs isolated from PAH rat lung and apoptosis resistant PAECs induced by hypoxia treatment, while AG490 prevented the phosphorylation of STAT3 at tyr705 in hypoxia-induced apoptosis resistant PAECs, and those were associated with the decreased expression of Drp1 protein. Furtherly, Drp1 silencing or blocked by Midivi-1 in hypoxia-treated PAECs showed similar results of apoptosis as AG490 treatment, validating Drp1 regulation by the STAT3 phosphorylation.

Limitations

Most studies indicated that phosphorylation is the major modification for enhancing the effects of Drp1, but in present study, we showed that the overexpression of Drp1 is one of the mechanisms in the regulation of mitochondrial fission after activation of STAT3 during the development of apoptosis resistance in PAECs, therefore, the phosphorylation or other type of post-translation modifications for Drp1 may need to further investigate. Similar result has been reported by Marsboom et al., in which the upregulation of total Drp1 was involved in driving mitochondrial fission (Marsboom et al. 2012).