Hypoxia-induced Pulmonary Hypertension Rat Model Research Articles

RAPID ENDOGENOUS PRODUCTION OF ENDOTHELIN-1 REVEALED BY AN ENDOTHELIN NEUTRALIZING ANTIBODY IN A SERIES OF EXPERIMENTAL AND MODELING STUDIES

Objective: The endothelin pathway is implicated in driving pathophysiological changes leading to pulmonary and systemic hypertension, chronic kidney disease and heart failure. Small molecule antagonists that selectively blocked endothelin A receptors (ETA) as well as non-selective ETA and endothelin B receptor (ETB) antagonists were successfully developed for the treatment of these vascular diseases. However, the treatment-related adverse events, such as fluid retention and edema, significantly limit the utility of this class of therapeutics, especially in the heart failure patients. Design and method: A monoclonal antibody that selectively neutralizes endothelin-1 (ET-1) may offer an attractive alternative therapeutic approach that remedies ETA-mediated vasoconstriction without impacting desirable ET-3/ETB mediated fluid regulation. A selective ET-1/2 neutralizing antibody, S69glC21 was discovered and optimized through protein engineering to 80 pM and 30 pM binding affinities to ET-1 and ET-2, respectively. Results: When administered into rats exogenously infused with ET-1, S69glC21 prevented blood pressure increase. In a hypoxia-induced pulmonary hypertension rat model, S69glC21 reduced pulmonary pressure. Compared to atrasentan, a selective small molecule ETA antagonist, S69glC21 had a distinct pharmacodynamic (PD) effect characterized by initial complete inhibition followed by a delayed rise of pulmonary pressure as the hypoxia condition persisted. To further dissect these findings, S69glC21 pharmacokinetics (PK) and circulating ET-1 target engagement (TE) analysis were performed. In the S69glC21 treated rats, antibody bound ET-1 was shown to accumulate relative to baseline plasma levels at all dose levels. In the 30 mpk group, the antibody bound ET-1 accumulated nearly 40,000-fold above the baseline plasma level of 2.25 ± 0.87 pg/mL indicating a rapid production rate of ET-1. Conclusions: Thus, PK/PD modeling reveals significant druggability challenges for the development of a highly effective, commercially viable ET-1 selective neutralizing antibody for the treatment of cardiovascular, pulmonary, and/or renal diseases.

Comparison of hypoxia-induced pulmonary hypertension rat models caused by different hypoxia protocols

Background and aim: Pulmonary hypertension (PH) is a serious and even fatal disorder with limited treatment strategies. The hypoxia-induced pulmonary hypertension (HPH) rat model is commonly used in this field. While the HPH rat model has strong predictability and repeatability, the model is a chronic model, making it time-consuming, costly, and complicated and limiting the progress of the experiments. Currently, there is no uniform international standard for the HPH model. Our study aimed to find a relatively effective and efficient HPH modeling protocol. Methods: We established HPH rat models with different total hypoxia periods and different daily hypoxia times, and assessed different hypoxia modeling modes in multiple dimensions, such as haemodynamics, right ventricular (RV) hypertrophy, pulmonary arterial remodeling, muscularization, inflammation, and collagen deposition. Results: Longer daily hypoxia time resulted in higher mean pulmonary arterial pressure (mPAP)/right ventricular systolic pressure (RVSP) and more obvious RV hypertrophy, as well as more severe pulmonary arterial remodeling and muscularization, regardless of the total period of hypoxia (3- or 4-week). Moreover, pulmonary perivascular macrophages and collagen deposition showed daily hypoxia time-dependent increases, both in 3- and 4-week hypoxia groups. Conclusion: Our findings showed that the 3-week continuous hypoxia mode was a relatively efficient way to reduce the time needed to induce significant disease phenotypes, which offered methodological evidence for future studies in building HPH models.

Tsantan Sumtang Restored Right Ventricular Function in Chronic Hypoxia-Induced Pulmonary Hypertension Rats.

Background: Tsantan Sumtang originated from Four Tantras, which consisted of Choerospondias axillaris (Roxb.) B. L. Burtt and A. W. Hill, Santalum album L., and Myristica fragrans Houtt. The three herbs are in ratio 1:1:1. This medication is widely used for cardiovascular diseases. Aims: The purpose of this study was to explore the effect of Tsantan Sumtang on right ventricular (RV) function in hypoxia-induced pulmonary hypertension (HPH) rats and investigate the underlying mechanism. Methods: Sixty male Sprague-Dawley (SD) rats were divided into control, hypoxia, and hypoxia + Tsantan Sumtang (1.0, 1.25, and 1.5 g•kg−1•d−1) groups. Chronic hypoxia was induced by putting the rats inside a hypobaric chamber for four weeks and adjusting the inner pressure and oxygen content to match an altitude of 4500 m. Echocardiography was used to assess RV function and right ventricular-pulmonary arterial (RV-PA) coupling. The physiological parameters of the animals were also evaluated. Morphological characteristics of RV were assessed by hematoxylin and eosin (H&E) staining and TEM. Masson’s trichrome staining, immunohistochemical staining, western blotting, and TUNEL assay were used to assess fibrosis and apoptosis levels. The antioxidant and anti-apoptosis properties of Tsantan Sumtang were also evaluated. The effect of Tsantan Sumtang on ROCK signaling pathway was evaluated using real-time quantitative PCR and western blotting. Results: We established an HPH rat model as indicated by the significant increases in the physiological parameters of the rats. Tsantan Sumtang showed a significant cardiac-protective function and an improved effect on RV-PA coupling. Moreover, Tsantan Sumtang treatment inhibited fibrosis and alleviated apoptosis and oxidative stress in RV. In terms of mechanism, Tsantan Sumtang reduced the expression of ROCK (ROCK1, ROCK2) in RV, inhibited cardiac remodeling-related transcription factors (NFATc3, P-STAT3), and regulated apoptosis-related proteins. Conclusion: Tsantan Sumtang was able to restore RV function, improve RV-PA coupling, recover hemodynamic and hematological indexes, and protect RV against structural maladaptive remodeling in the HPH rats. These findings demonstrated that Tsantan Sumtang protects the function of RV in HPH rats. The antioxidant and anti-apoptosis properties of Tsantan Sumtang may be responsible for inhibiting the ROCK signaling pathway.

Overexpression of ACE2 prevents hypoxia-induced pulmonary hypertension in rats by inhibiting proliferation and immigration of PASMCs.

Imbalance of the ratio of angiotensin converting enzyme (ACE) and ACE2 may lead to pathological conditions in lung. However, its effect on hypoxia-induced pulmonary hypertension (HPH) remains unclear. Therefore, the aim of this study was to investigate the effects of ACE2 overexpression on rat primary pulmonary arterial smooth muscle cells (PASMCs) and HPH rat model. ACE and ACE2 expression in rat PASMCs under hypoxia condition, as well as in HPH rat model, was detected. The overexpressed ACE2 gene was transfected into PASMCs by Lentiviral. Later, the proliferation and migration of PASMCs were evaluated. Meanwhile, the overexpressed ACE2 gene was transfected into rats and exposed to hypoxia for four weeks. Finally, the right ventricular systolic pressure, the right ventricular hypertrophy, and the percentage of the medial wall thickness were measured to evaluate the development of HPH. Imbalance of the expression of ACE/ACE2 was indicated in rat PASMCs under hypoxia condition and in the HPH rat model, respectively. The overexpression of ACE2 significantly inhibited PASMCs proliferation and migration. Moreover, the overexpressed ACE2 could significantly attenuate pulmonary hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy in HPH rat model. ACE2 is related to the formation of pulmonary vascular remodeling and pulmonary hypertension. Furthermore, it may prevent hypoxia-induced pulmonary hypertension by inhibiting the proliferation of PASMCs.

Drag-reducing polymers attenuates pulmonary vascular remodeling and right ventricular dysfunction in a rat model of chronic hypoxia-induced pulmonary hypertension.

Drag-reducing polymers (DRPs) was previously demonstrated to increase blood flow, tissue perfusion, and reduce vascular resistance. The purpose of this study was to investigate the effect of DRPs on pulmonary vascular remodeling and right ventricular dysfunction in a rat model of chronic hypoxia-induced pulmonary hypertension (HPH). A total of forty male Wistar rats were randomly and equally assigned into four experimental groups (Group I: normoxia + saline, Group II: normoxia + PEO, Group III: hypoxia + saline, Group IV: hypoxia + PEO) and maintained in normoxia (21% O2) or hypobaric hypoxia (10% O2). After four weeks, comparisons were made of the following aspects: the mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure (RVSP), right ventricular hypertrophy, wall thickness of pulmonary trunk and arteries, internal diameter of pulmonary arteries, cardiomyocyte cross-sectional area (CM CSA), and ultrastructure of right ventricular. Treatment with PEO in Group IV attenuated the increases in RVSP and mPAP (40.5±7.2 and 34.7±7.0 mmHg, respectively, both P < 0.05), compared with Group III. Distal vascular remodeling was visible as a significant increase in medial wall thickness (64.2±12.3% vs. 43.95±7.0%, P < 0.01) and a remarkable decrease in internal diameter of small pulmonary arteries (35.2±9.7μ m vs. 50.4±14.7μ m, P < 0.01) in Group III, to a greater extent than that detected in Group IV. Nevertheless, no significant histopathological differences in medial wall thickness was observed in pulmonary trunk between Group III and Group IV (P > 0.05), denoting that PEO chiefly attenuated the remodeling of small pulmonary arteries rather than main arteries in hypoxic environment. Infusion of DRPs (intravenous injection twice weekly) also attenuated the index of right ventricular hypertrophy, protected against the increase of cardiomyocyte cross-sectional area, and provided protection for cardiac ultrastructure. DRP treatment with intravenous injection elicited a protective effect against pulmonary vascular remodeling and right ventricular dysfunction in the rat model of HPH. DRPs may offer a new potential approach for the treatment of HPH, which may have theoretical significance and application value to society.

Structural and functional definition of the pulmonary vein system in a chronic hypoxia-induced pulmonary hypertension rat model.

Unlike the pulmonary artery (PA), the pathophysiological changes of the pulmonary vein (PV) in the development of pulmonary hypertension (PH) remain largely unknown. In this study, we comprehensively investigated the structural and functional changes in the PV isolated from the chronic hypoxia (CH; 10% O2, 21 days)-induced PH rat model (CHPH). Results showed that CH caused an increase in right ventricular pressure but did not affect the mean pulmonary venous pressure and the left atrial pressure. Similar to the PA, vascular lumen stenosis and medial thickening were also observed in the intrapulmonary veins isolated from the CHPH rats. Notably, CH induced more severe loss in the endothelium of intrapulmonary veins than the arteries. Then, the contractile response to 5-HT and U46619 was significantly greater in the intrapulmonary small veins (ISPV) and arteries (ISPA) isolated from CHPH rats than those from normoxic rats but not in the extrapulmonary and intrapulmonary large veins. Treatment with nifedipine (Nif), SKF96365 (SKF), or ryanodine and caffeine either partially attenuated (Nif) or dramatically abolished (SKF or ryanodine and caffeine) 5-HT-induced maximal contraction in ISPV from both normoxic and CHPH rats. Because of the severe loss of endothelium in the PV of CHPH rats, the decrease in acetylcholine (ACh)-induced endothelium-dependent relaxation was significantly larger in ISPV than ISPA, whereas the sodium nitroprusside-induced endothelium-independent relaxation was not altered in both ISPA and ISPV. In conclusion, our results provide fundamental data to comprehensively define the PV system in CHPH rat model.

Crosstalk between the Akt/mTORC1 and NF-κB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs.

Abnormal wound healing by pulmonary artery smooth muscle cells (PASMCs) promotes vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Increasing evidence shows that both the mammalian target of rapamycin complex 1 (mTORC1) and nuclear factor-kappa B (NF-κB) are involved in the development of HPH. In this study, we explored the crosstalk between mTORC1 and NF-κB in PASMCs cultured under hypoxic condition and in a rat model of hypoxia-induced pulmonary hypertension (HPH). We showed that hypoxia promoted wound healing of PASMCs, which was dose-dependently blocked by the mTORC1 inhibitor rapamycin (5-20 nM). In PASMCs, hypoxia activated mTORC1, which in turn promoted the phosphorylation of NF-κB. Molecular docking revealed that mTOR interacted with IκB kinases (IKKs) and that was validated by immunoprecipitation. In vitro kinase assays and mass spectrometry demonstrated that mTOR phosphorylated IKKα and IKKβ separately. Inhibition of mTORC1 decreased the level of phosphorylated IKKα/β, thus reducing the phosphorylation and transcriptional activity of NF-κB. Bioinformatics study revealed that dipeptidyl peptidase-4 (DPP4) was a target gene of NF-κB; DPP4 inhibitor, sitagliptin (10-500μM) effectively inhibited the abnormal wound healing of PASMCs under hypoxic condition. In the rat model of HPH, we showed that NF-κB activation (at 3 weeks) was preceded by mTOR signaling activation (after 1 or 2 weeks) in lungs, and administration of sitagliptin (1-5 mg/kg every day, ig) produced preventive effects against the development of HPH. In conclusion, hypoxia activates the crosstalk between mTORC1 and NF-κB, and increased DPP4 expression in PASMCs that leads to vascular remodeling. Sitagliptin, a DPP4 inhibitor, exerts preventive effect against HPH.

MiR-593-5p promotes the development of hypoxic-induced pulmonary hypertension via targeting PLK1.

The aim of this study was to investigate the role of microRNA-593-5p (miR-593-5p) in hypoxia-induced pulmonary hypertension (HPH), and to explore its underlying mechanism. Sprague-Dawley (SD) rats were housed in a hypoxia environment 8 hours per day for consecutive 4 weeks. After the establishment of the HPH rat model, we detected the right ventricular systolic pressure (RVSP) and right heart hypertrophy index (RVHI) in HPH rats and controls. The expression levels of miR-593-5p and polo-like kinase 1 (PLK1) in rat lungs were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Subsequently, miR-593-5p mimics and inhibitor were constructed and transfected into cells. The proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) were accessed by cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The protein level of PLK1 in PASMCs after transfection with miR-593-5p mimics or inhibitor was detected by Western blot. Dual-luciferase reporter gene assay was conducted to verify the binding condition of miR-593-5p and PLK1. Finally, rescue experiments were performed to explore whether the regulatory effect of miR-593-5p on HPH development was associated with PLK1. RVSP and RVHI in rats of the hypoxic group were significantly higher than those of controls. MiR-593-5p was significantly downregulated while PLK1 was remarkably upregulated in lung tissues of HPH rats than those of controls. Similarly, miR-593-5p expression in PASMCs decreased gradually with the prolongation of hypoxia induction. Overexpression of miR-593-5p remarkably inhibited the proliferation and migration of PASMCs. Subsequently, dual-luciferase reporter gene verified the binding condition of miR-593-5p and PLK1. Both the mRNA and protein levels of PLK1 were negatively regulated by miR-593-5p. Also, rescue experiments demonstrated that the inhibitory effects of miR-593-5p on the proliferation and migration of PASMCs could be reversed by PLK1 overexpression. MiR-593-5p is lowly expressed in lung tissues of HPH rats. Meanwhile, it stimulates the proliferation and migration of PASMCs via targeting PLK1, thereby promoting HPH development.

Involvement of CapG in proliferation and apoptosis of pulmonary arterial smooth muscle cells and in hypoxia-induced pulmonary hypertension rat model

ABSTRACTPurpose: Actin-binding protein capping protein gelsolin-like (CapG) was preferentially expressed in human pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, and reduced CapG expression was accompanied by impaired migration ability in vitro. We intended to investigate the effects of CapG on rat PASMCs and hypoxia-induced pulmonary hypertension (HPH) rat model. Materials and Methods: We investigated the effect of RNA interference-medicated down-regulation of CapG expression in rat PASMCs as well as in HPH rat model. The proliferation, apoptosis and cell cycle of PASMCs were evaluated. The HPH rat model was established by intratracheal instillation of lentiviral vector and subsequent hypoxia exposure for four weeks. Right ventricular systolic pressure, right ventricular hypertrophy and the percentage of medial wall thickness were measured to evaluate the development of HPH. Results: Knock-down CapG in PASMCs resulted in decreased proliferation, increased apoptosis and induced cell cycle inhibition. Down-regulation of CapG expression locally could attenuate pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy in HPH rat model. Conclusions: Our study indicated that CapG participated in the pathogenesis of pulmonary vascular remodeling in HPH rats, which was probably mediated by promoting the proliferation and inhibiting the apoptosis of PASMCs. We proposed CapG modulating protective effects of pulmonary hypertension.

Effects of tetrahydrobiopterin oral treatment in hypoxia-induced pulmonary hypertension in rat.

Endothelial nitric oxide synthase (eNOS) plays a major role in maintaining pulmonary vascular homeostasis. Tetrahydrobiopterin (BH4), an essential cofactor that stabilizes the dimerization of eNOS and balances nitric oxide (NO) and superoxide production, may have therapeutic potential in pulmonary hypertension. In the isolated perfused lung, we demonstrated a direct effect of exogenous administration of BH4 on pulmonary NO production, leading to acute vasorelaxation during the plateau phase of hypoxia-induced pulmonary vasoconstriction. In the chronic hypoxia-induced pulmonary hypertension rat model, chronic BH4 oral administration attenuated the pressor response to hypoxia (mean pulmonary artery pressure ± standard error of the mean, 31.8 ± 0.5 mmHg at 100 mg/kg/day; placebo group, 36.3 ± 0.6 mmHg; P < 0.05). During telemetric monitoring, right ventricular systolic pressure was reduced by approximately 50% after 1 week of BH4 treatment at 100 mg/kg/day. BH4 at 100 mg/kg/day reduced right ventricular hypertrophy (from 0.55 ± 0.01 to 0.50 ± 0.01; P < 0.05) and pulmonary vascular muscularization (from 79.2% ± 2% to 65.2% ± 3%; P < 0.01). BH4 treatment enhanced lung eNOS activity and reduced superoxide production, with a net increase in cyclic guanosine monophosphate levels. BH4 is effective in attenuating pulmonary hypertension in the hypoxic rat model when given as a rescue therapy.

Expression and role of factor inhibiting hypoxia-inducible factor-1 in pulmonary arteries of rat with hypoxia-induced hypertension

Hypoxia-inducible factor-1alpha subunit (HIF-1alpha) plays a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH) by transactivating it' target genes. As an oxygen-sensitive attenuator, factor inhibiting HIF-1 (FIH) hydroxylates a conserved asparagine residue within the C-terminal transactivation domain of HIF-1alpha under normoxia and moderate hypoxia. FIH protein is downregulated in response to hypoxia, but its dynamic expression and role during the development of HPH remains unclear. In this study, an HPH rat model was established. The mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. The pulmonary artery remodeling index became evident after 7 d of hypoxia, while the right ventricular hypertrophy index became significant after 14 d of hypoxia. The messenger RNA (mRNA) and protein expression of HIF-1alpha and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1alpha, were markedly upregulated after exposure to hypoxia in pulmonary arteries. FIH protein in lung tissues declined after 7 d of hypoxia and continued to decline through the duration of hypoxia. FIH mRNA had few changes after exposure to hypoxia compared with after exposure to normoxia. In hypoxic rats, FIH protein showed significant negative correlation with VEGF mRNA and VEGF protein. FIH protein was negatively correlated with mean pulmonary arterial pressure, pulmonary artery remodeling index and right ventricular hypertrophy index. Taken together, our results suggest that, in the pulmonary arteries of rat exposed to moderate hypoxia, a time-dependent decrease in FIH protein may contribute to the development of rat HPH by enhancing the transactivation of HIF-1alpha target genes such as VEGF.

Expression and role of factor inhibiting hypoxia-inducible factor-1 in pulmonary arteries of rat with hypoxia-induced hypertension

Hypoxia-inducible factor-1alpha subunit (HIF-1alpha) plays a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH) by transactivating it' target genes. As an oxygen-sensitive attenuator, factor inhibiting HIF-1 (FIH) hydroxylates a conserved asparagine residue within the C-terminal transactivation domain of HIF-1alpha under normoxia and moderate hypoxia. FIH protein is downregulated in response to hypoxia, but its dynamic expression and role during the development of HPH remains unclear. In this study, an HPH rat model was established. The mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. The pulmonary artery remodeling index became evident after 7 d of hypoxia, while the right ventricular hypertrophy index became significant after 14 d of hypoxia. The messenger RNA (mRNA) and protein expression of HIF-1alpha and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1alpha, were markedly upregulated after exposure to hypoxia in pulmonary arteries. FIH protein in lung tissues declined after 7 d of hypoxia and continued to decline through the duration of hypoxia. FIH mRNA had few changes after exposure to hypoxia compared with after exposure to normoxia. In hypoxic rats, FIH protein showed significant negative correlation with VEGF mRNA and VEGF protein. FIH protein was negatively correlated with mean pulmonary arterial pressure, pulmonary artery remodeling index and right ventricular hypertrophy index. Taken together, our results suggest that, in the pulmonary arteries of rat exposed to moderate hypoxia, a time-dependent decrease in FIH protein may contribute to the development of rat HPH by enhancing the transactivation of HIF-1alpha target genes such as VEGF.

Evidence for dysregulation of dimethylarginine dimethylaminohydrolase I in chronic hypoxia-induced pulmonary hypertension.

Chronic hypoxia-induced pulmonary hypertension is associated with increased pulmonary expression of nitric oxide synthase (NOS) enzymes. Nevertheless, some reports have indicated decreased pulmonary production of NO in the disease. To address this paradox, we determined pulmonary concentrations of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) in the hypoxia-induced pulmonary hypertension rat model. In addition, we determined whether dysregulation of the ADMA-metabolizing enzyme dimethylarginine dimethylaminohydrolase I (DDAH I) plays a role in this disease. Adult male rats were exposed for 1 week to either normoxia or hypoxia (10% oxygen). Lung tissues were used for Western blot analysis of endothelial NOS and DDAH I expression, measurement of lung NO and ADMA content, and in vitro assay of DDAH enzyme activity. Western blot analysis revealed a 1.9-fold increase in endothelial NOS protein and a 37% decrease in DDAH I protein in the lungs of hypoxia-exposed rats. Both pulmonary DDAH enzyme activity and NO content were significantly decreased in the hypoxic group (by 37% and 22%, respectively), but pulmonary ADMA concentrations were increased by 2.3-fold compared with the normoxic group. These data demonstrate that the rat chronic hypoxia-induced pulmonary hypertension model is associated with increased pulmonary concentrations of the NOS inhibitor ADMA. Moreover, pulmonary hypertensive rats exhibit reduced pulmonary expression and activity of the ADMA-metabolizing enzyme DDAH I. The decreased DDAH I and increased ADMA concentrations may therefore contribute to pulmonary hypertension via the competitive inhibition of pulmonary NOS enzymes.