The Protective of hUC- MSCs Derived Exosomes on Pulmonary Vascular Remodeling in Experimental Pulmonary Hypertension

Pulmonary hypertension (PH) is a life-threatening disease, so far no effective method for it. Therefore, the main aim of the present study was to investigate the protective effect of exosomes derived from human umbilical cord msenchymal stem cells (hUC-MSCs) on PH vascular remodeling and reveal the underlying mechanism. We established monocrotaline-induced PH (MCT-PH) rats and hypoxia induced pulmonary artery smooth muscle cells (H-PASMC) model in vivo and vitro . Exosomes derived from hUCMSCs (hUC-MSC-EXO) were administration, respectively. Post-operation, our results showed that hUC-MSC-EXO signicantly reduced the right ventricular systolic pressure (RVSP) and the right ventricular hypertrophy index (RVHI), inhibited pulmonary vascular remodeling. Furthermore, the hypoxia-induced PASMC proliferation was obviously inhibited in vitro . More-importantly, the results showed that the protein expression levels of BMPR2, BMP-4 and BMP-9 were signicantly increased, but the levels of nuclear factor-κB (NF-κB) p65 and bone morphogenetic protein (BMP) antagonists Gremlin-1 were signicantly decreased in hUC-MSC-EXO than that in MCT-PH and hypoxia-induced PASMC groups in vivo and vitro , respectively. Taken together, this present study provided the rm information for the use of this new method in treatment of PH, and the mechanism was associated with regulation of BMP signaling pathway.


Introduction
Pulmonary hypertension (PH) is a life-threatening disease, characterized by pulmonary vascular remodeling and right ventricular failure [1,2]. So far, there is no effective treatment for it and the mortality rate is still high. Our previous study showed that intravenous delivery of mesenchymal stem/stromal cell (MSC) could improve monocrotaline (MCT)-induced experimental PH vascular remodeling and right ventricular impairments [3,4]. However, the e cacy of MSC-based therapies has been increasingly attributed to the paracrine secretion, particularly exosome. Exosome, a subset of extracellular vesicles, are thought to function primarily as intercellular communication vehicles to transfer bioactive lipids, proteins and nucleic acids between cells to elicit biological responses in recipient cells.
process of pulmonary arterial remodeling in PH [15]. BMP9 not only protected pulmonary arterial endothelial cells from apoptosis and promotes vascular stability, but also increased BMPR2 gene expression [16]. Nuclear factor-κB, an in ammation-related transcription factor, upgulation of NF-κB signaling can promote vascular intima formation, smooth muscle cells proliferation and remodeling [17].
Report shows that pulmonary arterial endothelial cell apoptotic and endothelial to mesenchymal transition (Endo-MT) events caused by NF-κB signaling pathways in the lungs has signi cant correlation with BMP pathway [18], however, the basic mechanism has not been fully understood.
Here, the purpose of this experiment was to observe the protective effect of hUC-MSC-EXO against PH and explore the mechanism was related to the up-regulation the BMP signal pathway.

Animal
All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Also, all experiments were approved by the Institutional Animal Care and Use Committee of Shandong University. The monocrotaline-induced PH (MCT-PH) rat model has been well-established extensively. All animals were anaesthetized by iso urane inhalation (1.5-2%) and then euthanized by cervical dislocation.

Preparation of exosomes
Healthy pregnant women umbilical cord collection from obstetrics of our hospital. We stated that the experiment process conform to the principles outlined in the declaration of Helsinki and investigators have obtained the informed written consent before enrolling participants in clinical trials. Isolation of MSCs from human umbilical cord (hUCMSCs) Wharton's Jelly with some modi cation [10].The immunotyping characterization of hUCMSCs occurred at passage 3-4 using human speci c antibodies CD34, CD45, CD73, CD90, CD105, HLA-DR (BD Biosciences Pharmingen, San Diego, CA) by a uorescence-activated cell sorter (FACS, BD FACSAria II). Adipogenic, osteogenic and chondrogenic differentiation capacity ofT of hUCMSCs was performed by special medium (Cyagen US Inc), and then stained with Alizarin Red-S,Toluidine blue and Oil Red-O, respectively. The plates were seen using microscope, and images were taken from them.
When reached 90% con uences, the adherent cells were incubated in DMEM with 5% exosome-depleted FBS for 24 h, and 5-8 passages hUC-MSC were used for experiments. The conditioned medium was centrifuged at 4°C at 300 g for 10 min at 2000 g for 10 min and nally at 10 000 g for 30 min to remove the cells and debris, followed by centrifugation of the supernatant at 100 000 g at 4°C for 1 h. hUC-MSC-EXO was resuspended in PBS and ltered with a 0.22 µm micro ltration membrane, centrifuged again in PBS at 100,000 g for 1 h to collect the exosomes. The protein concentration of hUC-MSC-EXO was determined using a bicinchoninic acid (BCA) assay kit.
The morphology of MSC-EXO was examined using a transmission electron microscope (TEM) according to the manufacturer's instructions. Brie y, the prepared exosomes were stained with phosphotungstic acid solution and then performed under a Hitachi-9000 TEM system. Western blotting was used to detect the protein markers of hUC-MSC-EXO, such as CD63, CD81, TS101 and ALIX.

Animals experimental design
Sprague-Dawley rats PH model was established by a single subcutaneous injection of MCT(60 mg/kg; Sigma, St. Louis, MO, USA) [15]. Three weeks later, the animals received were given at a dose of 50 µg/day hUC-MSC-EXO or an equal volume of culture medium (hUC-MSC-CM) via tail vein injection once daily for 3 days, starting 1 weeks after the last series MCT injections [5]. The protein concentration of hUC-MSC-EXO was determined by a BCA assay kit, and 50 μg protein in 100 μl PBS was used. The rats were randomly divided into 4 groups (n=10): Control, MCT-PH, hUC-MSC-EXO, and hUC-MSC-CM groups. The animals were evaluated at 4 weeks after MCT injection.
Hemodynamic and the right ventricular hypertrophy assessment Post-operation, all animals were anaesthetized by iso urane inhalation (1.5-2%) and then euthanized by cervical dislocation. Hemodynamic data were recorded eight weeks after operation as previously described with some modi cations. Via femoral vein access, a 5F Swan-Ganz catheter (Edwards Lifesciences Corp, Irvine, CA) was advanced into the pulmonary artery for determination of heart rate (HR), systemic blood pressure (SBP) and right ventricular systolic pressure (RVSP). For assessment of right ventricular hypertrophy, the left ventricle (LV) plus the septum (LV+S) were harvested, and the weight ratio of the RV to LV+S weight calculated to quantify the right ventricular hypertrophy. The index by the formula: RV/(LV+S)×100.

Histology and immunology analysis
Post-operation, the lung and heart were quickly harvested and xed in 4% paraformaldehyde and embedded in para n, the serially sectioned at a thickness of 4-5 µm were stained with hematoxylin-eosin (H&E). To evaluate pulmonary artery structural remodeling, the vascular wall thickness (WT), vascular external diameter (ED), vascular wall area (WA) and total vascular area (TA) to calculate WT% (WT/ED) and WA% (WA/TA) were measured as our previously [6,7]. Fibrosis area of heart and lung were analyzed by Masson's trichrome staining, and then the sections were captured as digital images. Images were taken with an Eclipse 90i microscope (Nikon, Tokyo, Japan).
Immuno uorescence were used to analysis the expression of CD31 and a-SMA. Brie y, after blocking with 5% bovine serum albumin for 30 min at room temperature, the lung sections were incubated overnight at 4°C with anti-CD31 (AF3628) and α-SMA (ab21027) antibodies. Then, sections were further incubated with a antibody for 2 h at room temperature. Subsequently, the sections were followed by 1-h incubation in the dark with orescence isothiocyanate-conjugated secondary antibody. Images were taken with ZEISS LSM800 confocal microscope (Tokyo, Japan). All experiments were performed by two examiners blinded to treatment assignment.

Cell proliferation assays
Cell proliferation was monitored using a cell counting kit (CCK)-8 assay, Brie y, PASMCs were seeded into 96-well plates at about at a density of 5×10 3 cells/well. The cells were subjected to hypoxia with or without hUC-MSC-EXO at different time points, and then CCK-8 reagent (10 µl) was added to each each well and further incubated for 3 h. The absorbance was measured at 450 nm in a spectrophotometer. Analysis of cellular deoxyribonucleic acid (DNA) content discloses frequencies of cells in G0/1, S and G2/M phase by Flow Cytometry used Cell Cycle and Apoptosis Analysis Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions.

Real-time PCR analysis
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to detect the relative expression of CD31 and a-SMA using genespeci c primers as described previously. Brie y, total RNA in lung tissues or PAEC was extracted using RNeasy kit (Qiagen, Valencia, CA). ABI Prism 7900 sequence detection system software (version 2.2) was used to analysis the data were, and β-actin was used as an internal control for input RNA. The primers were designed by the Primer Express software package.

Statistical analysis
All data are expressed as mean±SD. Comparisons of parameters between 2 groups were made with unpaired Student t test. Comparisons of parameters among 3 groups were made with one-way analysis of variance (ANOVA), followed by the Scheffe post hoc test. Statistical analysis was carried out by using the SPSS 19.0 software. P<0.05 was regarded as signi cant statistical difference.

Characterization of hUC-MSCs and hUC-MSC-EXO
Flow cytometric analysis demonstrated that the majority of hUC-MSCs expressed high levels of the CD73, CD90, and CD105 markers, whereas CD34, CD45 and HLA-DR markers were relatively absent ( Figure 1A).
According to the Alizarin Red-S, Oil Red-O and Toluidine blue staining, it was recognized that hUC-MSCs have the ability to differentiate into osteocytes, adipocytes and cartilage ( Figure 1B). Characterization of the shape and size of MSC-EXO was carried out by transmission electron microscope, the range of hUC-MSC was between 50-150 nm in size ( Figure 1C). The protein expression of hUC-MSC-EXO markers was detected by western blot, the results showed that an enrichment of CD9, CD63, CD81, ALIX and TSG101 levels in hUC-MSC-EXO ( Figure 1D).

hUC-MSC-EXO attenuated MCT-induced PH pulmonary artery remodeling
In the present study, no animals in control and hUC-MSC-CM groups were dead of acute pulmonary edema within the rst week of the MCT administration, however, two animals in MCT-PH group and one in hUC-MSC-EXO group were dead, three replacement animals was utilized following the same procedure and treatment. Post-operation, we evaluate MCT-induced lung and heart injury by detecting RVSP and right ventricular hypertrophy index RV/(LV+S), as shown in Figure 2A, a signi cant increased of RVSP and RV/(LV+S) in MCT-PH and hUC-MSC-CM groups as compared with control (P<0.05), these indicated that we successfully established PH model in rats. However, the increased of RVSP and RV/(LV+S) were signi cantly inhibited by hUC-MSC-EXO (P<0.05). There was no signi cantly different in HR and SBP between groups (P>0.05).
Lung sections were stained with H&E and Masson's trichrome was used to analysis the medal thickness of pulmonary arterial walls and the degree of brosis. As shown in Figure 2B, WT% and WA% of muscular arteries with an external diameter of 15 to 50 µm were signi cantly increased in MCT-PH and hUC-MSC-CM groups than that in control, but notably decreased in hUC-MSC-EXO group (P<0.05). The same results also showed in Masson's stained, Figure 2C and 2D showed that hUC-MSC-EXO treatment reduced heart and lung brosis than that in MCT-PH or hUC-MSC-CM group (P<0.05).

Effect of hUC-MSC-EXO on MCT-Induced vascular proliferation
To determine whether MCT-Induced vascular proliferation, the smooth muscularization cells marker α-SMA and endothelial cell marker protein marker CD31 were detected by RT-PCR, western blot and double immuno uorescence staining for. Our results showed that the protein and mRNA expression of α-SMA was signi cantly increased, but CD31 was signi cantly decreased in MCT-PH and hUC-MSC-CM groups than that in control, however, which were signi cantly repaired in hUC-MSC-EXO group as compared with MCT-PH and hUC-MSC-CM groups (P<0.05, Figure3A-3C). On the other hand, our results also shwed that hUC-MSC-EXO could inhibition of MCT-induced pulmonary vascular endothelial-to-mesenchymal transition (Endo-MT). Furthermore, western blot results showed that the protein expression of proliferating cell nuclear antigen (PCNA) and cell-cycle regulatory protein D1(Cyclin D1) was signi cantly decreased, but the protein expression of cell cycle inhibitor P27 kip1 was signi cantly increased in hUC-MSC-EXO group than that in MCT-PH and CM groups ( Figure 4D, P<0.05).

Effect of hUC-MSC-EXO on PASMC proliferation
The PASMC proliferation were measured by CCK8 when cells treatment with a hypoxia or normoxia condition in the presence or absence of hUC-MSC-EXO fraction for 24 h, 48 h and 72h. The results indicated that the proliferation was signi cantly reduced in hUC-MSC-EXO group as compared with hypoxic exposure groups( Figure 4A, P<0.05). Cells were harvested and analyzed by ow cytometry at hypoxic exposure 48 h to measured the change of cell cycle. Our results showed that the percentage of G0/G1 phase was signi cantly increased in hUC-MSC-EXO group as compared with the hypoxic group, while the parentage of G2/M+S phase was signi cantly decreased, makes cells stay at rest and promotes the transition from S phase to mitosis, the G2 phase (P<0.05, Figure 4B).
The cells proliferation was also detected by immuno uorescence in vitro. The results showed that the protein expression of proliferating cell nuclear antigen Ki67 was obviously decreased in hUC-MSC-EXO treatment group as compared with hypoxia group ( Figure 4C, P < 0.05). Furthermore, western blot results showed that the protein expression of Cyclin D1 was signi cantly decreased, but the protein expression of P27 kip1 was signi cantly increased in H-PASMCs group than that in control. However, hUC-MSC-EXO administration could signi cantly restore these results than that in H-PASMCs group ( Figure 4D, P<0.05).

Effect of hUC-MSC-EXO on BMP signaling pathway
Our results of western blot showed that BMPR2 signaling, characterized by rising of BMPR2, BMP4, BMP9 and ID1 expression were signi cantly lower, BMP antagonists Gremlin-1 was higher in MCT-PH and hypoxia groups as compared with control, but an obviously up-regulation of BMPR2, BMP4, BMP9 and ID1, down-regulation of Gremlin-1 in hUC-MSC-EXO group than those in MCT-PH and hypoxia groups respectively in vivo and vitro(P<0.05, Figure 5A and 5B).
NF-κB is a key transcriptional regulator factor, plays a key role in the process of vascular remodeling in a variety of physiological and pathophysiological states. Reports have previously showed a direct association of NF-κB with BMP signaling in the lungs of MCT-PH, inhibition of NF-κB attenuated PH by regulating BMPR2-ID axis gene. Here, our western blot and immuno uorescence results showed that the protein expression expression of NF-κB p65, p-NF-κB p65 and the ratio between phospho and total NF-kB p65 (p-p65/p65) were down-regulation in hUC-MSC-EXO group than in PH and hypoxia groups (P<0.05, Figure 5C and 5D).

Discussion
Pulmonary hypertension (PH) is a kind of refractory rare lung diseases, distal pulmonary arterial remodeling is the characteristic of it [19,20]. The pathogenesis of PH is not clear yet, and no effective therapy is available for it. Previous studies [21,22,18] suggested the exosomes isolated from mesenchymal stem cells has the potential to inhibition of vascular remodeling in PH. In the present study, our data con rmed that hUC-MSC-EXO could signi cantly reduce RVSP and RV/(LV +S) as compared with the MCT-PH rats in vivo and inhibition of PASMSs proliferation induced by hypoxia in vitro, which is considered as a novel potential therapeutic approach for PH.
Bone morphogenetic proteins (BMPs) and their receptors were required for PH-induced right ventricular hypertrophy, which playing an important role in the remodeling of pulmonary resistance vessels in the process of PH occurs[8, 23,24]. BMPR2 mutations have been reported in more than 70% of heritable cases of PH and approximately 20% of apparently sporadic cases of idiopathic PH [25,26]. Loss of BMPR 2 or dysfunction of BMP signaling were associated with the occurrence of PH [16]. BMPs involved in a wide range of cell function including proliferation, migration, differentiation, and apoptosis. Mounting evidence indicates that several BMPs, including BMPR2, BMP4 and BMP9 play as an important role during endocrine regulator of pulmonary arterial remodeling, cardiovascular, metabolic, and haematopoietic function [13][14][15][16]. BMPR2 signaling as a cause of increased proliferation of PASMCs playing an important role in the remodeling of pulmonary resistance vessels in PH [25], BMP9 could protect pulmonary arterial endothelial cells from apoptosis and promotes vascular stability, increase BMPR2 gene expression, and further enhancement of BMPR2 signaling.
The induction of inhibitor of DNA binding protein (ID) expression by BMP contributes to its proangiogenic response. Regulation of Id proteins by BMPs, with relevance to PH, play an main effect in the smooth muscle cell function . Id family of transcription factors, especially ID1 and ID3 as important functional targets of BMP signaling, are potently regulated by BMP signalling in PAMSCs and might play a complementary and partially redundant role in regulating cell cycling in vascular and other tissues [27].
ID family of transcription factors as important functional targets of BMP signaling, with relevance to PH, both ID1 and ID3 were downregulated in the lungs along with BMPR2 in MCT rats [28,29]. In the present study, our data corroborated with the association of BMPs with PH, the results demonstrate the expression levels of BMP signaling related protein molecules BMP2, BMP4, BMP9, BMPR2 and ID1 were restored when the PH rats were treatment with hUC-MSC-EXO compared with the MCT-PH rats. To further explore the underlying mechanism of hUC-MSC-EXO against PH, here, PAMSC were induced by hypoxia in vitro in the presence or absence of hUC-MSC-EXO. The effect of hUC-MSC-EXO on proliferation and apoptosis abilities of PAMSC were also analyzed, the results showed the protein expression of CyclinD1 was signi cantly higher, but the the expression of P27 were signi cantly lower in hUC-MSC-EXO group than that in H-PASMCs group. Therefore, our data provide a strong evidence for the inhibition of hUC-MSC-EXO on PH vascular remodeling.
In PAMSCs, BMPR2 mutation, leads to a proproliferative, apoptosis-resistant cell phenoype, that may contribute to the process of vascular obliteration observed in PH. BMP signaling is tightly regulated by in ammatory mediators/in ammation [30,31]. In ammatory cytokines are associated with the pathogenesis of PH, in ammatory molecules were signi cant in ltration of macrophages and accumulation in the mice treated with MCT [32,33]. In ammatory cytokines stimulation reduced the expression of BMP2 and BMPR2, promote excessive PASMC proliferation and pulmonary vascular remodeling in the setting of BMPR2 de ciency in PASMCs [34]. Reports have previously showed a direct association of NF-κB with BMP signaling in the lungs of MCT-PH, inhibition of NF-κB attenuated PH and RVH by regulating BMPR2-ID axis gene in heart [12,35]. Mover over, we further con rmed that NF-κB signaling were downregulated in the hUC-MSC-EXO group in vivo and vitro. The present study at least part suggested that mechanism of hUC-MSC-EXO against PH was through inhibition of NF-κB signaling and activation BMP signaling on experimental PH and pulmonary vascular remodeling. Collectively, these data suggested that the mechanism of hUC-MSC-EXO promote apoptosis and inhibit proliferation in vitro is likely to be relevant with BMP signaling and maybe associated with anti-in ammatory response .

Conclusions
The present study demonstrated that hUC-MSC-EXO attenuated PH pulmonary vascular remodeling through increased the vascular remodeling associated BMPR2 signaling pathway.