Adenosine monophosphate, one metabolite from lower respiratory tract, promotes the progression of pulmonary arterial hypertension through the PI3K/AKT signaling pathway in pediatric with congenital heart disease

doi:10.21203/rs.3.rs-3343789/v1

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Adenosine monophosphate, one metabolite from lower respiratory tract, promotes the progression of pulmonary arterial hypertension through the PI3K/AKT signaling pathway in pediatric with congenital heart disease

https://doi.org/10.21203/rs.3.rs-3343789/v1

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Background The PI3K/AKT pathway is a crucial signaling pathway linked to cell growth and death in pulmonary arterial hypertension (PAH). In this study, we profiled the metabolic changes associated with PAH and investigated the role of adenosine monophosphate (AMP) in regulating PI3K/AKT signaling during PAH progression.

Methods The metabolites in the lung tissue lavage fluids from PAH patients and healthy controls were subjected to metabolite profiling. Human pulmonary artery endothelial cells (HPAECs) were subjected to AMP treatment, and the cell phenotype changes were assessed by CCK-8 proliferation assay, migration assay and in vitro tube formation assay. The impacts of AMP on PI3K/AKT pathway and the epithelial-mesenchymal transition (EMT) in HPAECs were examined by Western blot. A rat model of PAH was established to evaluate the roles of AMP and PI3K/AKT signaling in PAH progression.

Results We identified AMP as an upregulated metabolite in the lung tissue lavage fluids of PAH patients. AMP treatment inhibited the cell growth and angiogenic potential of HPAECs, while the AMP enhanced the EMT and migration in HPAECs. AMP treatment increased the phosphorylation levels of PI3K, AKT and mTOR in HPAECs. PI3K knockdown could reverse the effects of AMP on HPAECs. In the rat model of PAH, AMP administration aggravated the fibrosis of pulmonary artery by augmenting PI3K/AKT signaling, which could be alleviated by PI3K inhibitor.

Conclusion Our study showed that AMP promotes PAH progression by regulating the PI3K/AKT signaling pathway and inducing EMT in pulmonary artery endothelial cells. Targeting AMP/PI3K/AKT signaling could be employed as strategy to alleviate PAH.

Pulmonary arterial hypertension

Adenosine monophosphate

PI3K/AKT signaling pathway

Due to right ventricular (RV) enlargement and mortality, PAH, a potentially fatal condition, causes a gradual rise in pulmonary vascular resistance and PAH[1–2]. Researchers have found that this deadly injury is caused by increased inflammation, DNA damage, and epigenetic changes[3–5]. Vascular thickening is one of the primary causes of right ventricular failure and cardiac dysfunctions in PAH, a multifactorial cardiopulmonary illness. The prevalence of PAH is approximately 25 cases per population of 1 million, with an incidence of about five cases per population of 1 million annually [6]. Toxin- and drug-evoked PAH, idiopathic PAH, congenital heart disease (CHD), connective disease, heritable PAH, portal hypertension, and other types of PAH may be classified based on clinical criteria. Epidemiological studies have shown that patients with PAH have a less than 60% 5-year overall survival rate [7, 8]. Although our understanding of disease progression and mechanisms of PAH has improved in recent years, the etiology of PAH still needs to be fully understood. Pulmonary hypertension is assumed to have a shared etiology, including endothelial dysfunction, endothelial and smooth muscle cell proliferation, and enhanced vasoconstriction.Because of this, modern treatments focus on using drugs that improve endothelial function, like those that target endothelin-1, nitric oxide (NO), or prostacyclin [9, 10].

However, these treatments didn't help people live longer, and patients often stop taking drugs because they have harmful side effects or are hard to take. Inhibition of normal apoptosis, excessive angiogenesis, and plexiform intima damage is recognized to contribute to the etiology of PAH [11, 12]. Endothelial cells are the primary impediment to molecules passing through the arterial wall. The endothelium mediates numerous processes in hypoxia-induced hypertension. In adult angiogenesis, endothelial cell migration must be strictly regulated. [13, 14]. Despite decades of research into hypoxia's role in endothelial dysfunction, the cellular and molecular processes behind how hypoxia affects PAECs still need to be understood entirely. Finding molecules that regulate angiogenesis and PAEC survival is crucial for the development of PAH. The balance between cellular proliferation and apoptosis controls the continuous turnover of cell populations as a physiological force, but when it is disrupted, abnormal growth and death underlie several illnesses. Several essential intracellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/AKT signaling, control proliferation and apoptosis [15, 16].

The phosphorylation of membrane lipid phosphoinositides by PI3K, a lipid kinase family member, initiates the following signals. The primary downstream signal is AKT, which affects the activity of multiple cell fate-regulating proteins and is activated in a PI3K-dependent way. In PAH, it has been shown that the PI3K/AKT pathway is a crucial signaling pathway linked to cell growth and death [17].

We collected alveolar lavage fluid from patients with PAH for metabolome detection in the early stage. We found that differential metabolites (diethyl pyridine-2,4-dicarboxylate, AMP, fumaric acid, and hexamethylenetetramine) were highly enriched in patients with pulmonary arterial hypertension. At the same time, through KEGG signaling pathway enrichment of differential metabolites, it was found that there were significant changes in the PI3K signaling pathway [18]. This research sought to determine if AMP promoted PAH by modulating the PI3K/AKT signaling pathway.

Patients who underwent clinical bronchoscopy at the Fuwai Yunnan Cardiovascular Hospital (Kunming, China) were recruited in this research. The pulmonary microbial community was easily affected by multi-factors, and subjects were excluded from our study if they (1) have other cardiopulmonary diseases, lung comorbidities, pulmonary infection, metabolic diseases, and other systemic diseases; (2) take any particular drugs, such as antibiotics, in the 3 months before enrollment; (3) have special diets such as feeding with breast or formula; and (4) participate in other clinical studies or were unable to provide consent for care or BALF collection. Finally, a total of 68 patients were enrolled in the current research. Bronchoalveolar lavage fluid (BALF) was collected into the sterile tube according to a standardized protocol with minimum oral contamination. Two-milliliter BALF was collected from each participant, flash-frozen in liquid nitrogen, and transferred to − 80◦C later until further processing.

The metabolomic assay required BALF samples that were thawed on ice after being taken from liquid nitrogen. Methanol and L-2-chlorobenzalanine were used to extract the metabolites. The UHPLC -Q Exactive HF-X system (Thermo) was utilized to conduct metabolomic analysis on all samples. The instrument was outfitted with an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm.i.d., 1.8µm; Waters, Milford, USA), and the column temperature was kept at 40°C. The gradient elution of the Analytes was performed as previously described. Quality control samples are prepared by mixing equal volumes of all assay samples and are utilized to assess the stability of the assay system. The metabolism data that was obtained was further advanced based on previously reported findings. A principal component analysis (PCA) was conducted to analyze global similarities, which included quality control samples (QC). The student's t-test was combined with multivariate analysis. The method of orthogonal partial least-squares discriminate analysis (OPLSDA) was employed to identify differential metabolites (DMs) between groups (VIP > 1, p-value < 0.05). DMs were selected based on Fisher's exact test and clustering, and the Pearson coefficient was used to organize them. Differentially enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) was performed to investigate the metabolomic pathways potentially involved in the pathogenesis of CHD-PAH. This was achieved by integrating databases of HMDB, KEGG compounds, and LIPID MAPS.

Cell culture and grouping in vitro

Life Technologies provided Dulbecco's modified eagle medium and FBS, while the HPAECs were supplied by the ATCC in Manassas, Virginia. All cells were cultured in high glucose DMEM with 10% FBS, penicillin (100 U/mL), and streptomycin (100 g/mL) in an incubator at 37°C and 5% CO2. After cells reached 80–90% confluence (ethylenediaminetetraacetic acid), they were digested with 1x trypsin + EDTA.

To experiment regulatory effects of diethyl pyridine-2,4-dicarboxylate, AMP, fumaric acid and hexamethylenetetramine on pulmonary artery endothelial cells, five groups designed including; HPAECs, HPAECs + of diethyl pyridine-2,4-dicarboxylate (0, 1, 2, 4, and 8 mg/L), HPAECs + AMP (0, 1, 2, 4, and 8 mg/L), HPAECs + fumaric acid (0, 1, 2, 4, and 8 mg/L) and HPAECs + hexamethylenetetramine (0, 1, 2, 4, and 8 mg/L).

Based on the above results, we selected AMP for an in-depth study of the signaling pathway and for validation of signaling pathways that regulate angiogenesis, new grouping designed as HPAECs, HPAECs + AMP, HPAECs + AMP + PI3K knockdown-NC, HPAECs + AMP + PI3K knockdown.

Animal grouping and in vivo

Animal studies were approved by the Thical Committee of Laboratory Animals of Fuwai Yunnan Cardiovascular Hospital. Anesthesia with sodium pentobarbital was utilized for all surgical procedures.

Fifty male SD rats (180–200 g) entered this study (5 rats in each sub-group, a total of ten groups in 2 different parts). To induce PAH, male SD rats (180–200 g) were injected with a monocrotaline (MCT, Sigma) solution, and the model was formed within 28 days. This modeling was induced for all groups except the control groups. The animal experiment consists of two different parts. First, evaluation of the association of AMP with the development of PAH. For this reason, rats were divided into five groups, including the control group (SD rats), mock surgical group, pulmonary hypertension group, Pulmonary hypertension group + AMP (6 mg/kg/d), and Pulmonary hypertension group + AMP (6 mg/kg/d) + PI3K knockdown. The second part of the animal study evaluated the relationship between injection of AMP and inducing pulmonary hypertension. In this way, we divided rats into five groups, including the Control group (SD rats), mock surgical group, SD rats + AMP (12 mg/kg/d), SD rat + AMP + PI3K knockdown NC, and SD rat + AMP + PI3K knockdown.

Cell Proliferation

To plate the transfected cells into 96-well plates, 2000 cells per well were employed. Cell counting kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) solution was added to each well after 24, 48, 72, and 96 hours of culture, respectively. After 2 hours of incubation, each well's optical density (OD) was measured in the microplate reader at an absorption wavelength of 490 nm.

Real-time PCR analysis

Using an RNA Extraction Kit (QIAGEN, Netherlands), total RNA was extracted from growing cells, and target messenger RNAs were amplified using a reverse transcriptase-polymerase chain reaction. The 7500 Fast Real-Time PCR System was the equipment of choice for real-time PCR. After an initial denature at 95 degrees Celsius for twenty seconds, fifty cycles of denature at 95 degrees Celsius for five seconds and annealing/extension at 60 degrees Celsius for thirty seconds were performed. This study utilized TaqMan Gene Expression Assays from Life Technologies.

Western blot

Total protein was extracted from rat tissues using cell lysis buffer and the TissueLyser LT to homogenise the samples. Protein lysates from HPAECs were extracted using RIPA buffer. Both buffers included phosphatase and protease inhibitors. To quantify the protein content, the mixture was centrifuged at 12,000 rpm for 15 minutes at a low temperature after being immersed in an ice bath for 5 minutes. The experiment's procedures performed WB. Using SDS-PAGE, equal quantities of protein were separated and transferred to membranes, decoloured at room temperature (using TBST buffer solution) and blocked for one hour at room temperature. The membrane was exposed after adding the colour-developing solution and diluting the primary and secondary antibodies. The antibodies were diluted to corresponding concentrations of 1:200 and 1:5,000, and exposure was timed correctly. Identification was made of the protein expression of P-AKT, AKT, P-PI3K, PI3K, FOXO1, mTOR, VEGF, α-SMA, Tropomyosin, P16, P53, N-cadherin, and Vimentin. Using Image J, the grey values of protein bands were statistically assessed.

Angiogenesis assay

14 103 HPAECs were seeded in growth factor-free media containing 0.5% FBS on 40 L of growth factor-reduced Matrigel (CorningTM, # 354230) in a 96-well plate.Following 18 hours of incubation, the cells were secured in 4% formaldehyde solution in PBS and photographed utilizing an Olympus IX 70-phase contrast microscope with a Peltier CCD camera.The software known as Image J was utilized to determine the overall length of the tube.

Immunofluorescence

Before the cells were fixed in a 4% paraformaldehyde solution for 20 minutes, they were subjected to PBS washing to eliminate debris. Bovine serum albumin was utilised to block the tissue slices and cell cultures overnight at 4 degrees Celsius before applying primary antibodies(α-SMA Monoclonal Antibody,Abbkine Scientific Co., Ltd.,China). The following day, we observed an overnight stay in the dark to facilitate the incubation of the secondary fluorescent antibodies(Abbkine Scientific Co., Ltd.,China). To serve as a counterstain, all slices were stained with nuclear 4,6-diamino-2-phenylindole (DAPI; Zhongshan Jinqiao Biotechnology Co., Ltd., China). Leica manufactured the confocal laser scanning microscope for immunofluorescence imaging in Wetzlar, Germany. This allowed for the comparison and observation of various things.

Migration Assays

Five horizontal mark lines were placed at the bottom of a six-well plate before cells were added, and after they had grown to 90% confluence, cells were starved for 12 hours. A 1 ml pipette tip formed three straight scratches vertically in the middle of the well, photographed before the cells were incubated. The scratch was shot 24 hours later for comparison with the earlier pictures. ImageJ software computed The scratch area, and the cell migration ratio was calculated.

Staining with hematoxylin and eosin

Reverse gradient alcohol rehydration was used to prepare tissue samples for HE staining, followed by a 5-minute haematoxylin staining period. The tissues were differentiated with dilute hydrochloric acid, washed with running water, and then submerged in 0.6% ammonia until they turned blue. The tissues were then rinsed once more with running water. The samples were then sealed using gradient alcohol dehydration after being stained with eosin for 1–3 minutes.

Elastic fiber staining

After undergoing reverse gradient alcohol rehydration, tissue samples were immersed in Wiegert's stain for five minutes. The tissues were cleaned with Wiegert bleach for one to two minutes, separated with an acidic differentiation solution for two to three seconds, washed with running water for ten minutes, and then dyed again with Van Gieson's (VG) staining solution(Sigma-Aldrich,USA) for thirty seconds. The samples were then sealed using a gradient alcohol dehydration process.

Statistical analysis

The findings of three or more determinations were combined to represent the data as averages and standard deviations (SD). Data were examined using the GraphPad Prism program (Verse 8.0, San Diego, CA, USA). A two-tailed t-test was employed to establish statistical significance when only two groups were compared. A one-way or two-way analysis of variance was used when comparing more than two groups. Statistics were deemed significant for P values below 0.05.

AMP inhibited the cell viability of HPAECs

The results of the CCK8 test showed that AMP significantly inhibited the proliferation of pulmonary artery endothelial cells after the cells were treated at 8 mg/L, respectively, with diethylpyridine-2, 4-dicarboxylic acid, AMP, fumaric acid and hexamethylenetetramine(Fig. 1A).

AMP decreases the expression level of VEGF in HPAECs

qRT-PCR used to detect the expression of VEGF and α-SMA in HPAECs after treatment with diethyl pyridine-2,4-dicarboxylate, AMP, fumaric acid and hexamethylenetetramine. Our results showed that the expression level of VFGF and α-SMA decreased most significantly after AMP treatment, and VEGF was a key factor in maintaining vascular homeostasis(Fig. 1B).

AMP promoted the expression of P-PI3K, P-mTOR and P-AKT

WB results showed that diethyl pyridine-2,4-dicarboxylate, AMP, fumaric acid and hexamethylenetetramine all promoted the expression of P-PI3K, P-mTOR and P-AKT, while the expression levels of mTOR, PI3K and AKT were unchanged. Among them, the activation effect of AMP is the most significant. WB results also showed that the expression level of p53 and p16 was upregulated due to treatment by diethyl pyridine-2,4-dicarboxylate and fumaric acid. However, the expression of p53 and p16 in cells treated with AMP and Hexamethylenetetramine did not change(Fig. 1C).

AMP cannot alter apoptosis in HPAECs

Flow cytometry was used to examine the apoptosis rate in each group. Our results showed after treatment with diethyl pyridine-2,4-dicarboxylate and fumaric acid, the Apoptosis rate increased, but apoptosis did not change significantly after treatment with AMP and hexamethylenetetramine(Fig. 2A). The immunofluorescence results showed that after AMP treatment, the cells changed from pebble-like to Spindle apparatus-like, and the Cytoskeleton changed(Fig. 2B)

AMP could promote EMT in vascular endothelial cells

The expression levels of epithelial-mesenchymal transition-related proteins were up-regulated, and the results showed a significant increase in expressions of VEGF, α-SMA, N-cadherin and Vimentin. So, our findings suggest that AMP could promote EMT in vascular endothelial cells(Fig. 3A).

AMP increase the cell migration of HPAECs

Cell Scratch was used to Detect Cell Migration, and the results showed that the AMP group had the most significant increase in the cell migration of HPAECs(Fig. 3B).

AMP reduced the ability of angiogenesis in HPAECs

The results of angiogenesis experiments showed that diethyl pyridine-2,4-dicarboxylate, AMP, fumaric acid and hexamethylenetetramine all reduced the ability of angiogenesis, among which the inhibition of AMP was the most significant(Fig. 3C).

PI3K knockdown could reverse the effects of AMP on HPAECs

WB results showed that PI3K knockdown could reverse the effect of AMP inhibiting the expression of VEGF and the effect of AMP on the expression of P-PI3K, P-mTOR and P-AKT. PI3K knockdown reversed the up-regulation of epithelial-mesenchymal transition-related proteins caused by AMP (Fig. 4A, Fig. 4B). Cell Scratch showed PI3K knockdown reversed the effect of the AMP group on promoting cell migration of HPAECs (Fig. 5A). Finally, the results of angiogenesis experiments showed that PI3K knockdown reversed the angiogenesis ability inhibited by AMP (Fig. 5B). The results showed that PI3K knockdown restored the cell Spindle apparatus-like changes induced by AMP treatment (Fig. 5C).

Results of animal study

PI3K knockdown could reverse the inhibitory effect of AMP in vivo

WB results showed that AMP inhibited VEGF expression, but PI3K knockdown could reverse the inhibition of AMP on the expression of VEGF. Also, PI3K knockdown can reverse the promoting effect of AMP on EMT-related molecules. So, this finding suggests that PI3K knockdown could reverse the activation effect of AMP on the PI3K signaling pathway (Fig. 6A and Fig. 6B).

AMP can cause pulmonary artery remodeling in healthy SD rats

They showed that 6 mg/kg/d began to cause pulmonary artery remodeling, 12 mg/kg/d was more serious, and 24 mg/kg/d SD rats had a higher mortality rate. Choose 12 mg/kg/d as the follow-up experiment concentration. This finding suggests AMP can cause pulmonary artery remodeling in healthy SD rats, which shows that AMP may be one of the reasons for the induction of PAH(Fig. 7A and Fig. 7B).Double staining of Hematoxylin and eosin staining and Elastic fibre staining were used to evaluate the direct effect of AMP on healthy SD rats to induce PAH(Fig. 7C).

Pulmonary arterial hypertension, often known as PAH, is characterized by a sustained rise in pulmonary vascular resistance and pulmonary arterial pressure. This chronic and progressive disease of the pulmonary vasculature may ultimately lead to right heart failure and mortality. Multiple variables contribute to the genesis of PAH. These include genetic susceptibility, inflammation, an increase in vascular tone, an increase in pulmonary artery cell proliferation and resistance to apoptosis [19-21].There is an urgent need to study novel molecular pathways and create treatment techniques since the effectiveness of the presently available therapy for PAH could be better. There are few medications available to enhance the long-term prognosis [22,23]. In recent years, PI3K/AKT signaling has garnered much interest as a possible therapeutic target for treating PAH. The PI3K/AKT signaling is involved in many biological functions throughout an individual's lifetime. The roles played by the PI3K/AKT signaling have been the subject of continuous research. Researchers revealed that PI3K/AKT may mediate cell proliferation and death [24,25]. In addition, PI3K/AKT affects survival and inhibits unnatural death, which is accomplished mainly via the suppression of specific pro-apoptotic proteins like Caspase-9 [26].

In addition, research has been done to determine the function of the PI3K/AKT pathway in the aetiology of PAH. According to the findings of several studies, the PI3K/AKT pathway may be implicated in the growth and death of PAH cells. It is still unclear what function PI3K/AKT might play in the anti-apoptotic effects of PAH medicines. Because the growing body of data suggests that it is involved in vasoconstriction and remodeling, the PI3K/AKT signaling pathway is a primary focus of vasofunctional medicine research[25]. PI3K and AKT are two proteins that can regulate the bioactivity of the pulmonary vascular system. According to the findings of previous studies, regulating the PI3K/AKT signaling is an essential component of PAH treatment [27]. In the current study, we controlled the PI3K/AKT signaling pathway to investigate the potential effect that AMP could have on PAH. According to the results of our study, AMP was able to decrease the viability of HPAEC cells. In addition, AMP could stimulate EndMT in the endothelial cells of blood vessels, a significant funding source for our research. It is hypothesized that phenotypically altered, dedifferentiated, and partially dysfunctional pulmonary endothelial cells contribute directly, by transforming into smooth muscle-like cells and indirectly, through the action of paracrine effects, to the occlusive vascular remodeling that occurs in PAH [28,29]. Because EndMT is a critical developmental phase in which mature endothelial cells lose their unique protein expression, polarity, and shape to take on the mesenchymal property, it has received interest as a putative source of these highly proliferative, smooth muscle-like cells mesenchymal cells in PAH. One of the reasons EndMT may be a source of these cells in PAH is because of this [30].

Additionally, our findings indicated that AMP decreased HPAECs' capacity for angiogenesis and decreased their VEGF and -SMA expression levels. The malfunctioning of PAECs, which includes increased adhesion capacity and unchecked angiogenesis, is one of PAH's primary clinical and pathologic characteristics. Consequently, the pulmonary distal arteries are more susceptible to vascular remodeling [31]. Endothelial dysfunction is the primary initiating component of PAH pathogenesis, and endothelial proliferation and migration are crucial for angiogenesis[32].

Our results indicated that AMP increases the cell migration of HPAECs but couldn’t alter apoptosis in HPAECs. To evaluate the role of PI3K/AKT in AMP effects on PAH, we used PI3K knockdown groups in both in vivo and in vitro experiments. Our funding showed that PI3K knockdown could reverse the effect of AMP inhibiting the expression of VEGF and reverse the expression of P-PI3K, P-mTOR and -AKT. PI3K knockdown also reversed the up-regulation of EndMT proteins. The results of angiogenesis experiments showed that PI3K knockdown reversed the angiogenesis ability inhibited by AMP. Cell Scratch assay also showed PI3K knockdown reversed the effect of the AMP group on promoting cell migration of HPAECs. Animal experiments assessed this finding, and we delivered PI3K knockdown can alleviate pulmonary artery remodeling induced by AMP in vivo and also reverse the inhibitory effect of AMP on VEGF expression in vivo—this finding ability of AMP, which causes pulmonary artery remodeling in healthy SD rats. Controlling the proliferation, migration, apoptosis, and dedifferentiation of PASMCs, all of which may be mediated via the PI3K/AKT signaling, TRIM32 has been implicated in the development of PAH. Hu and others showed this[33]. As shown by Yang et al.[34], ghrelin can reverse the hypoxia-induced dysfunction of HPAEC by blocking the PI3K/AKT pathway. In recent research, Zhang et al. showed the relevance of 12-LO/12-HETE and the PI3K/AKT pathway in the etiology of PAH[35]. In addition, they provided unique insights into the origins of hypoxic PAH and underscored the importance of PI3K/AKT signaling for 12-LO/12-HETE's actions.

PAH is a very harmful cardiovascular disease, especially for children. Understanding its mechanism is of great significance for future medical monitoring of the occurrence and development of PAH. Our previous metabolome test on alveolar lavage fluid in children with PAH showed that AMP was one of the risk factors for PAH[18]. To understand its role in the development of PAH, we provided treatment of pulmonary endothelial cells and SD rats with AMP, which can reduce cell viability by promoting the occurrence of EMT. Our study is the first to discover the role and effect of metabolites in PAH, which will provide a marker for diagnosing PAH. At the same time, it may be used as a drug target for treating PAH. Of course, further in vitro and in vivo studies are needed. Although this study confirmed the role of AMP in the development of PAH at the cellular and animal levels, the source of metabolites in the development of PAH has yet to be clearly explained, which will be the focus of our subsequent study.

In conclusion, our study showed that AMP could activate the PI3K/AKT signaling pathway, pro, mote cell migration, and promote the EMT of vascular endothelial cells. The results of animal experiments showed that AMP could promote arterial remodeling in rats with PAH and induce arterial remodeling in healthy SD rats by promoting EMT in vascular endothelial cells.

Author Contributions Statement

Run-Wei Ma, Li-Ming Cheng and Xiao-Yu Zhang conceived and designed the experiments, wrote, revised and finalized the manuscript. Xiang Li, Yao Deng, Min-Zhang Zhao and Hao-Bo Ren conducted experiments. Chun-Jie Mu and Wen-Ting Gui performed analyzed the data. All authors reviewed the content and approved the final version for publication.

Funding

This work was supported by the Foundation of Medical Specialists (YNWR-MY-2020-044), Yunnan Provincial Clinical Research Center for Cardiovascular Diseases - New Technology Research and Development Project for Diagnosis and Treatment of Major Cardiovascular Diseases (202102AA310002) and Foundation Program of Yunnan Provincial Cardiovascular Clinical Medical Center (FZX2019-06-01).

Conflict of Interest

The authors declare no competing interests.

Acknowledgments

The authors thank all the subjects who participated in this study..

Ethical statement

Informed consent was obtained from all participants in this study. The institutional review board of Fuwai Yunnan Cardiovascular Hospital approved all experimental procedures and protocols.

Data availability statements

The data supporting this study's findings are available from the corresponding author when it's a reasonable request.

Human subjects/informed consent statement

The authors carried out no human studies for this article.

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No competing interests reported.

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Adenosine monophosphate, one metabolite from lower respiratory tract, promotes the progression of pulmonary arterial hypertension through the PI3K/AKT signaling pathway in pediatric with congenital heart disease

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Abstract

Figures

Introduction

Material and Methods

Cell culture and grouping in vitro

Animal grouping and in vivo

Cell Proliferation

Real-time PCR analysis

Western blot

Angiogenesis assay

Immunofluorescence

Migration Assays

Staining with hematoxylin and eosin

Elastic fiber staining

Statistical analysis

Results

AMP inhibited the cell viability of HPAECs

AMP decreases the expression level of VEGF in HPAECs

AMP promoted the expression of P-PI3K, P-mTOR and P-AKT

AMP cannot alter apoptosis in HPAECs

AMP could promote EMT in vascular endothelial cells

AMP increase the cell migration of HPAECs

AMP reduced the ability of angiogenesis in HPAECs

PI3K knockdown could reverse the effects of AMP on HPAECs

Results of animal study

PI3K knockdown could reverse the inhibitory effect of AMP in vivo

AMP can cause pulmonary artery remodeling in healthy SD rats

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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