Remodeling In Hypoxic Pulmonary Hypertension Research Articles

Hypoxic Preconditioned Tibetan Mesenchymal Stem Cell-derived Exosomes Ameliorate Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension Rats

BackgroundPulmonary hypertension associated with hypoxia frequently manifests in a range of long-standing lung disorders. A strategy to augment the therapeutic efficacy of mesenchymal stem cells (MSCs) involves subjecting them to hypoxic preconditioning, which bolsters the emission of exosomes exhibiting enhanced bioactivities through paracrine mechanisms. The objective of this research is to explore the healing impact of preconditioning with hypoxia on MSC-extracted exosomes in relation to vascular structural changes within the lungs of rodents afflicted by hypoxia-induced pulmonary arterial hypertension. Methods and ResultsThis research entailed the successful isolation, cultivation of mesenchymal stem cells from Tibetan umbilical cords, alongside the extraction of their exosomes. Our results highlight that exosomes derived from hypoxia-conditioned Tibetan cord mesenchymal stem cells (H-Tibetan-exo) demonstrated superior efficacy in suppressing pulmonary vascular structural changes compared to those from normoxic conditions (N-Tibetan-exo) in a rat model of hypoxia-induced pulmonary hypertension (HPH).In an effort to replicate pulmonary hypertension conditions within a laboratory setting, we subjected rat pulmonary arterial smooth muscle cells (rPASMCs) to a low oxygen environment. Our findings revealed that exosomes derived from high-altitude Tibetans (H-Tibetan-exo) showed a superior capacity in suppressing the growth of rPASMCs compared to those obtained from normoxic Tibetans (N-Tibetan-exo). Additionally, the Transwell assay revealed a more significant inhibition of rPASMCs migration by H-Tibetan-exo. The investigation of PI3K-AKT-mTOR pathway protein expression through western blot analysis revealed that under hypoxic conditions, there was an elevation in the phosphorylation status of these proteins in both cellular and tissue settings. Furthermore, the H-Tibetan-exo exhibited more effective inhibition of rPASMCs proliferation and a more significant downregulation of PI3K-AKT-mTOR pathway protein phosphorylation levels compared to the N-Tibetan-exo. ConclusionsH-Tibetan-exo significantly ameliorates pulmonary vascular remodeling in hypoxic pulmonary hypertension more effectively than N-Tibetan-exo. The enhancement observed could be linked to reduced activity of the PI3K-AKT-mTOR pathway. This discovery sheds fresh light on managing hypoxic pulmonary hypertension, highlighting the potential therapeutic benefits of H-Tibetan-exo in mitigating its development.

CircLMBR1 inhibits phenotypic transformation of hypoxia-induced pulmonary artery smooth muscle via the splicing factor PUF60

Phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) contributes to vascular remodeling in hypoxic pulmonary hypertension (PH). Recent studies have suggested that circular RNAs (circRNAs) may play important roles in the vascular remodeling of hypoxia-induced PH. However, whether circRNAs cause pulmonary vascular remodeling by regulating the phenotypic transformation in PH has not been investigated. Microarray and RT-qPCR analysis identified that circLMBR1, a novel circRNA, decreased in mouse lung tissues of the hypoxia-SU5416 PH model, as well as in human PASMCs and mouse PASMCs exposed to hypoxia. Overexpression of circLMBR1 in the Semaxinib (SU5416) mouse model ameliorated hypoxia-induced PH and vascular remodeling in the lungs. Notably, circLMBR1 was mainly distributed in the nucleus and bound to the splicing factor PUF60. CircLMBR1 suppressed the phenotypic transformation of human PASMCs and vascular remodeling by inhibiting PUF60 expression. Furthermore, we identified U2AF65 as the downstream regulatory factor of PUF60. U2AF65 directly interacted with the pre-mRNA of the contractile phenotype marker smooth muscle protein 22-α (SM22α) and inhibited its splicing. Meanwhile, hypoxia exposure increased the formation of the PUF60–U2AF65 complex, thereby inhibiting SM22α production and inducing the transition of human PASMCs from a contractile phenotype to a synthetic phenotype. Overall, our results verified the important role of circLMBR1 in the pathological process of PH. We also proposed a new circLMBR1/PUF60–U2AF65/pre-SM22α pathway that could regulate the phenotypic transformation and proliferation of human PASMCs. This study may provide new perspectives for the diagnosis and treatment of PH.

Nur77 induced by HIF-1α mediates vascular remodeling in hypoxic pulmonary hypertension.

Nur77 is a member of the NR4A subfamily of orphan nuclear receptors that is expressed and has a function within the immune system. This study aimed to investigate the role of Nur77 in hypoxic pulmonary hypertension. SPF male SD rats were exposed in hypobaric chamber simulating 5000 m high altitude for 0, 3, 7, 14, 21 or 28 days. Rat pulmonary artery smooth muscle cells (RPASMCs) were cultured under normoxic conditions (5% CO2-95% ambient air) or hypoxic conditions (5% O2 for 6 h, 12 h, 24 h, 48 h). Hypoxic rats developed pulmonary arterial remodeling and right ventricular hypertrophy with significantly increased pulmonary arterial pressure. The levels of Nur77, HIF-1α and PNCA were upregulated in pulmonary arterial smooth muscle from hypoxic rats. Silencing of either Nur77 or HIF-1α attenuated hypoxia-induced proliferation. Silencing of HIF-1α down-regulated Nur77 protein level, but Nur77 silence did not reduce HIF-1α. Nur77 was not con-immunoprecipitated with HIF-1α. This study demonstrated that Nur77 acted as a downstream regulator of HIF-1α under hypoxia, and plays a critical role in the hypoxia-induced pulmonary vascular remodeling, which is regulated by HIF-1α. Nur77 maybe a novel target of HPH therapy.

ADAR1 regulates vascular remodeling in hypoxic pulmonary hypertension through N1-methyladenosine modification of circCDK17

Pulmonary hypertension (PH) is an extremely malignant pulmonary vascular disease of unknown etiology. ADAR1 is an RNA editing enzyme that converts adenosine in RNA to inosine, thereby affecting RNA expression. However, the role of ADAR1 in PH development remains unclear. In the present study, we investigated the biological role and molecular mechanism of ADAR1 in PH pulmonary vascular remodeling. Overexpression of ADAR1 aggravated PH progression and promoted the proliferation of pulmonary artery smooth muscle cells (PASMCs). Conversely, inhibition of ADAR1 produced opposite effects. High-throughput whole transcriptome sequencing showed that ADAR1 was an important regulator of circRNAs in PH. CircCDK17 level was significantly lowered in the serum of PH patients. The effects of ADAR1 on cell cycle progression and proliferation were mediated by circCDK17. ADAR1 affects the stability of circCDK17 by mediating A-to-I modification at the A5 and A293 sites of circCDK17 to prevent it from m1A modification. We demonstrate for the first time that ADAR1 contributes to the PH development, at least partially, through m1A modification of circCDK17 and the subsequent PASMCs proliferation. Our study provides a novel therapeutic strategy for treatment of PH and the evidence for circCDK17 as a potential novel marker for the diagnosis of this disease.

基于伏邪理论探讨低氧性肺动脉高压肺血管重塑

基于伏邪理论探讨低氧性肺动脉高压肺血管重塑

Sulforaphane alleviated vascular remodeling in hypoxic pulmonary hypertension via inhibiting inflammation and oxidative stress

Hypoxic pulmonary hypertension (HPH) is a cardiopulmonary disease featured by pulmonary vascular remodeling, which is due to abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) and dysfunction of endothelial cells (ECs). Sulforaphane (SFN) is a natural isothiocyanate extracted from cruciferous vegetables with promising anti-inflammatory and anti-oxidative activities. This study aimed to explore the effect and mechanism of SFN on HPH. Male mice were exposed to persistent chronic hypoxia for 4 weeks to induce HPH. The results demonstrated that SFN repressed the increased right ventricular systolic pressure (RVSP) and attenuated the right ventricular hypertrophy and pulmonary arteries remodeling in HPH mice. In particular, after SFN treatment, the CD68 positive cells in lung sections were reduced; TNF-α and IL-6 levels in lungs and serum declined; activation of NF-κB in PASMCs was inhibited in response to hypoxia. Besides, SFN enhanced the superoxide dismutase (SOD) activity in serum, SOD2 expression, total glutathione levels, and GSH/GSSG ratio in PASMCs, along with a decrease in malondialdehyde (MDA) contents in serum and ROS production in PASMCs after hypoxia exposure. Notably, SFN, as an Nrf2 activator, reversed the reduction in Nrf2 expression in hypoxic PASMCs. In vitro, SFN treatment inhibited hyperproliferation and promoted apoptosis of PASMCs under hypoxia conditions. SFN also prevented the apoptosis of pulmonary microvascular ECs caused by hypoxia. Therefore, these data suggested that SFN could significantly restrain the inflammation and oxidative stress, thereby inhibiting PASMCs proliferation, promoting PASMCs apoptosis, and reversing hypoxia injury in ECs to improve pulmonary vascular remodeling.

M6A Modification-mediated GRAP Regulates Vascular Remodeling in Hypoxic Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling induced by human pulmonary arterial smooth muscle cell (HPASMC) proliferation, migration, and apoptosis resistance. m6A (N6-methyladenosine) is the most prevalent RNA posttranscriptional modification in eukaryotic cells. However, its role in PAH remains elusive. We designed this study to investigate whether m6A modification and its effector proteins play a role in pulmonary vascular resistance. Lung samples were used to profile m6A concentrations in control subjects and patients with PAH. Bioinformatics analysis, real-time PCR, immunohistochemistry, and Western blotting were used to determine the role of m6A effectors in PAH. The biological effects of GRAP modified by m6A were investigated using invitro and invivo models. Furthermore, RIP-PCR was used to assess the writers and readers of GRAP. In this study, we revealed that m6A-modified GRAP mRNA was upregulated in PAH lung samples, cHx/Su-induced mouse models, and hypoxia-stimulated HPASMCs; however, GRAP mRNA and protein were abnormally downregulated. Functionally, overexpression of GRAP drastically alleviated the proliferative and invasive ability of PAH HPASMCs through inhibition of the Ras/ERK signaling pathway invitro and invivo. In addition, METTL14 (methyltransferase-like 14) and the m6A binding protein YTHDF2 were significantly increased in PAH. Moreover, we found that m6A-modified GRAP mRNA was recognized by YTHDF2 to mediate the degradation. GRAP expression was consistently negatively correlated with METTL14 and YTHDF2 invivo and invitro. Taken together, for the first time, our findings highlight the function and therapeutic target value of GRAP and extend our understanding of the importance of RNA epigenetics in PAH.

Piperlongumine attenuates vascular remodeling in hypoxic pulmonary hypertension by regulating autophagy

ObjectiveThe aim of this study was to determine the therapeutic effect of piperlongumine on hypoxic pulmonary hypertension. MethodsA hypoxic pulmonary hypertension rat model was constructed, primary rat pulmonary artery smooth muscle cells (PASMCs) were isolated, and the proliferation of PASMCs was measured by Cell Counting Kit‑8 assay. The expression of autophagic proteins microtubule-associated protein 1 light chain 3B (LC3B) and P62 were examined by western blot. Autophagic flux in PASMCs was detected by tandem mRFP-GFP-LC3 fluorescence analysis. ResultsHypoxia-induced proliferation of PASMCs was significantly inhibited by piperlongumine exposure. Treatment with piperlongumine elevated LC3B II/LC3B I protein ratio and decreased the expression of P62 protein in both PASMCs and rat lung tissues. Tandem mRFP-GFP-LC3 fluorescence analysis showed that piperlongumine increased autophagic flux in PASMCs. Inhibition of autophagy using 3-methyladenine (3-MA) attenuated the inhibitory effect of piperlongumine on proliferation of PASMCs. Chronic hypoxia exposure led to a significant increase in rat right ventricle systolic pressure, right ventricular hypertrophy, wall thickness and area of pulmonary artery, and muscularization of pulmonary arterioles, which was obviously suppressed by administration of piperlongumine. 3-MA attenuated the alleviating effects of piperlongumine on pulmonary vascular remodeling. ConclusionsPiperlongumine attenuates vascular remodeling in hypoxic pulmonary hypertension by regulating autophagy. Piperlongumine treatment may serve as a promising therapy for hypoxic pulmonary hypertension.

NPS2143 Modulates the Phenotypic Switching of PASMCs by Inhibiting Autophagy in Hypoxia-Induced Pulmonary Hypertension

Background and Objectives: Pulmonary Artery Hypertension (PAH) is considered as a malignant tumor in cardiovascular disease. Our previous study found that Calcium-Sensing Receptor (CaSR) is involved in pulmonary vascular remodeling in hypoxic pulmonary hypertension (HPH). However, the relationship of Pulmonary Artery Smooth Muscle Cell (PASMC) phenotypic switching, proliferation, and autophagy in CaSR-related HPH remain unclear. The purpose of this study was to detect the role of a CaSR antagonist, NPS2143, on the vascular remodeling by autophagy modulation under hypoxia. Methods: Hypoxic rat PAH model were simulated in vivo. Meanwhile, mean Pulmonary Artery Pressure (mPAP) was measured while RVI, WT%, and WA% indices were calculated. Immunohistochemistry and Western blot were used to detect phenotypic switching and cell proliferation in pulmonary arteriole. Cell viability was determined in vitro by CCK8 and cell cycle. Cell proliferation, phenotypic switching, autophagy level and PI3K/Akt/mTOR pathways were investigated in human PASMCs through mRNA or Western blot methods. Results: Rats with hypoxic-induced PAH had an increased mPAP, RVI, WT% and WA%. Moreover, expression of CaSR was significantly increased, followed by activation of autophagy (increased LC3b and decreased p62), phenotypic switching of PASMCs (reduced calponin, SMA-a and increased OPN) and pulmonary vascular remodeling. However, NPS2143 weakened these hypoxic effects. The results using hypoxic-induced human PASMCs confirmed that NPS2143 suppressed autophagy and reversed phenotypic switching in vitro by inhibiting PI3K/Akt/mTOR pathways. Conclusions: Our study demonstrates that NPS2143 was conducive to inhibit the proliferation and reverse phenotypic switching of PASMCs by regulating autophagy levels in HPH and vascular remodeling.

Overexpressed lncRNA AC068039.4 Contributes to Proliferation and Cell Cycle Progression of Pulmonary Artery Smooth Muscle Cells Via Sponging miR-26a-5p/TRPC6 in Hypoxic Pulmonary Arterial Hypertension.

Hypoxic pulmonary hypertension (HPH) is a devastating and incurable disease characterized by pulmonary vascular remodeling, resulting in right heart failure and even death. Accumulated evidence has confirmed long coding RNAs (lncRNAs) are involved in hypoxia-induced pulmonary vascular remodeling in HPH. The exact mechanism of lncRNA in hypoxic pulmonary hypertension remains unclear. Microarray analysis was applied to investigate the profiles of lncRNA expression in pulmonary artery smooth muscle cells (PASMCs) cultured under hypoxia and normoxia condition. qRT-PCR was performed for the expression of lncRNAs, miRNA, and mRNAs, western blot analysis was employed for the detection of the expression of proteins. CCK-8 and transwell chamber assay were applied for the assessment of PASMC proliferation and migration, respectively. Besides, flow cytometry was performed for assessments of cell cycle progression. The binding between AC068039.4 and miR-26a-5p, miR-26a-5p, and TRPC6 3'UTR was detected by dual luciferase reporter assay. A total of 1,211 lncRNAs (698 up-regulated and 513 down-regulated) were differently expressed in hypoxia-induced PASMCs. Consistent with microarray analysis, quantitative PCR verified that AC068039.4 was obviously up-regulated in hypoxia-induced PASMCs. Knocking down AC068039.4 alleviated proliferation and migration of PASMCs and regulated cell cycle progression through inhibiting cells entering the G0/G1 cell cycle phase. Further experiment indicated AC068039.4 promoted hypoxic PASMCs proliferation via sponging miR-26-5p. In addition, transient receptor potential canonical 6 (TRPC6) was confirmed to be a target gene of miR-26a-5p. In conclusion, downregulation of lncRNA AC068039.4 inhibited pulmonary vascular remodeling through AC068039.4/miR-26a-5p/TRPC6 axis, providing new therapeutic insights for the treatment of HPH.

Circ-calm4 Serves as an miR-337-3p Sponge to Regulate Myo10 (Myosin 10) and Promote Pulmonary Artery Smooth Muscle Proliferation.

Pulmonary artery smooth muscle cell proliferation is the pathological basis of pulmonary vascular remodeling in hypoxic pulmonary hypertension. Recent studies suggest that circular RNA (circRNA) can regulate various biological processes, including cell proliferation. Therefore, it is possible that circRNA may have important roles in pulmonary artery smooth muscle cell proliferation in hypoxic pulmonary hypertension. In the present study, we aimed to identify functional circRNAs and clarify their roles and mechanisms in pulmonary artery smooth muscle cell proliferation in pulmonary hypertension. RNA sequencing identified 67 circRNAs that were differentially expressed in hypoxic lung tissues of mice. Screening by bioinformatics and quantitative polymerase chain reaction revealed significant elevation of a circRNA derived from alternative splicing of the calmodulin 4 gene (designated circ-calm4). Notably, this circRNA absorbed miR-337-3p. We further identified Myo10 (myosin 10) as a target protein of miR-337-3p. miR-337-3p bound to the 3'-untranslated region of Myo10 mRNA, thereby attenuating the translation of Myo10. Using loss-of-function and gain-of-function approaches, we found that circ-calm4 regulated cell proliferation by regulating the cell cycle. Additionally, we verified the functions of miR-337-3p and Myo10 in hypoxic pulmonary artery smooth muscle. Our results suggested that the circ-calm4/miR-337-3p/Myo10 signal transduction axis modulated the proliferation of pulmonary artery smooth muscle cells at the molecular level, thus establishing potential targets for the early diagnosis and treatment of pulmonary hypertension.

Extracellular Superoxide Dismutase Regulates Early Vascular Hyaluronan Remodeling in Hypoxic Pulmonary Hypertension

Chronic hypoxia leads to pathologic remodeling of the pulmonary vasculature and pulmonary hypertension (PH). The antioxidant enzyme extracellular superoxide dismutase (SOD3) protects against hypoxia-induced PH. Hyaluronan (HA), a ubiquitous glycosaminoglycan of the lung extracellular matrix, is rapidly recycled at sites of vessel injury and repair. We investigated the hypothesis that SOD3 preserves HA homeostasis by inhibiting oxidative and enzymatic hyaluronidase-mediated HA breakdown. In SOD3-deficient mice, hypoxia increased lung hyaluronidase expression and activity, hyaluronan fragmentation, and effacement of HA from the vessel wall of small pulmonary arteries. Hyaluronan fragmentation corresponded to hypoxic induction of the cell surface hyaluronidase-2 (Hyal2), which was localized in the vascular media. Human pulmonary artery smooth muscle cells (HPASMCs) demonstrated hypoxic induction of Hyal2 and SOD-suppressible hyaluronidase activity, congruent to our observations in vivo. Fragmentation of homeostatic high molecular weight HA promoted HPASMC proliferation in vitro, whereas pharmacologic inhibition of hyaluronidase activity prevented hypoxia- and oxidant-induced proliferation. Hypoxia initiates SOD3-dependent alterations in the structure and regulation of hyaluronan in the pulmonary vascular extracellular matrix. These changes occurred soon after hypoxia exposure, prior to appearance of PH, and may contribute to the early pathogenesis of this disease.

LncRNA Tug1 involves in the pulmonary vascular remodeling in mice with hypoxic pulmonary hypertension via the microRNA-374c-mediated Foxc1

Hypoxic pulmonary hypertension (HPH) is a serious and potentially devastating disorder of the pulmonary circulation with complicated mechanisms. Long non-coding RNA (lncRNA) has been revealed to participate in HPH development. This study aimed to explore how lncRNA Tug1 affected the pulmonary vascular remodeling in HPH. A mouse model of HPH and a pulmonary artery smooth muscle cell (PASMC) model of HPH (HPH-PASMCs) were established, where the expression of lncRNA Tug1 was determined. Then, the interaction among lncRNA Tug1, miR-374c, and Foxc1 was assessed. Finally, in order to determine the effects of lncRNA Tug1 on PASMC activities and pulmonary vascular remodeling after HPH, the expression of lncRNA Tug1 was silenced in HPH-PASMCs and HPH mice, with the proliferation, apoptosis, and migration of PASMCs as well as blood pressure in mice measured, respectively. The obtained data revealed that lncRNA Tug1 was highly expressed in HPH mice and HPH-PASMCs. In addition, lncRNA Tug1 up-regulated the expression of Foxc1 by binding to miR-374c. Notably, silencing of lncRNA Tug1 inhibited the proliferation and migration, but promoted the apoptosis of PASMCs. Moreover, lncRNA Tug1 silencing was observed to attenuate the pulmonary vascular remodeling in HPH mice through the Foxc1-mediated NOTCH signaling pathway. Taken conjointly, silencing of lncRNA Tug1 down-regulated the Foxc1 expression by binding to miR-374c, thereby inhibiting the proliferation and migration, while promoting apoptosis of PASMCs to impede pulmonary vascular remodeling in HPH via the Notch signaling pathway. This study provided novel therapeutic insights for treating HPH.

MiRNA let-7b promotes the development of hypoxic pulmonary hypertension by targeting ACE2.

Angiotensin-converting enzyme 2 (ACE2) protects against hypoxic pulmonary hypertension (HPH) by inhibiting the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). Under hypoxia, the hypoxia-inducible factor 1α (HIF-1α) inhibits ACE2 indirectly; however, the underlying mechanism is unclear. In the present study, we found that exposure to chronic hypoxia stimulated microRNA (miRNA) let-7b expression in rat lung via a HIF-1α-dependent pathway. Let-7b downregulated ACE2 expression by directly targeting the coding sequence of ACE2. Our in vitro and in vivo results revealed that let-7b contributed to the pathogenesis of HPH by inducing PASMCs proliferation and migration. Let-7b knockout mitigated right ventricle hypertrophy and pulmonary vessel remodeling in HPH by restoring ACE2 expression. Overall, we demonstrated that HIF-1α inhibited ACE2 expression via the HIF-1α-let-7b-ACE2 axis, which contributed to the pathogenesis of HPH by stimulating PASMCs proliferation and migration. Since let-7b knockout alleviated the development of HPH, let-7b may serve as a potential clinical target for the treatment of HPH.

A Time- and Compartment-Specific Activation of Lung Macrophages in Hypoxic Pulmonary Hypertension.

Studies in various animal models suggest an important role for pulmonary macrophages in the pathogenesis of pulmonary hypertension (PH). Yet, the molecular mechanisms characterizing the functional macrophage phenotype relative to time and pulmonary localization and compartmentalization remain largely unknown. In this study, we used a hypoxic murine model of PH in combination with FACS to quantify and isolate lung macrophages from two compartments over time and characterize their programing via RNA sequencing approaches. In response to hypoxia, we found an early increase in macrophage number that was restricted to the interstitial/perivascular compartment, without recruitment of macrophages to the alveolar compartment or changes in the number of resident alveolar macrophages. Principal component analysis demonstrated significant differences in overall gene expression between alveolar and interstitial macrophages (IMs) at baseline and after 4 and 14 d hypoxic exposure. Alveolar macrophages at both day 4 and 14 and IMs at day 4 shared a conserved hypoxia program characterized by mitochondrial dysfunction, proinflammatory gene activation, and mTORC1 signaling, whereas IMs at day 14 demonstrated a unique anti-inflammatory/proreparative programming state. We conclude that the pathogenesis of vascular remodeling in hypoxic PH involves an early compartment-independent activation of lung macrophages toward a conserved hypoxia program, with the development of compartment-specific programs later in the course of the disease. Thus, harnessing time- and compartment-specific differences in lung macrophage polarization needs to be considered in the therapeutic targeting of macrophages in hypoxic PH and potentially other inflammatory lung diseases.

MicroRNA let-7g inhibited hypoxia-induced proliferation of PASMCs via G0/G1 cell cycle arrest by targeting c-myc

AimsPulmonary hypertension (PH) is a proliferative disorder characterized by enhanced proliferation and suppressed apoptosis of intrapulmonary vascular smooth muscle cells. Recently, network-based bioinformatics have identified let-7 family, a tumor suppressive microRNA, regulate multiple interacting targets relevant to PH. However, the role of let-7 in vascular homeostasis in PH remains unknown. Thus, we wanted to investigate the role of let-7 in hypoxia-induced PASMCs proliferation and the underlying mechanism in hypoxic pulmonary hypertension (HPH). Main methodsThe male Sprague–Dawley (SD) rats were exposed to hypoxia (10% O2) for 21days to induce HPH. The expression of let-7 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Primary rat PASMCs were exposed to hypoxia (3% O2). MTS and EDU were performed to evaluate PASMCs proliferation. The mRNA and protein expression of c-myc, Bmi-1 and p16 were determined by qRT-PCR and Western blotting, respectively. The functions of let-7g on PASMCs proliferation, c-myc, Bmi-1 and p16 expression were assessed by let-7g mimic and inhibitor transfection. Key findingsAmong let-7 family members, only let-7b and let-7g were significantly down-regulated in remodeled pulmonary artery in HPH rats. Furthermore, only let-7g level was decreased in hypoxic PASMCs. Either hypoxia or let-7g inhibitor stimulated proliferation of PASMCs, let-7g mimic inhibited hypoxia-induced PASMCs proliferation. C-myc was the target of let-7g in PASMCs. Transfect of let-7g mimic inhibited hypoxia-induced c-myc, Bmi-1 up-regulation and p16 down-regulation, which ultimately controls cell cycle progression. SignificanceLoss of inhibition on c-myc-Bmi-1-p16 signaling pathway by let-7g may lead to PASMCs proliferation and vascular remodeling in HPH.

MicroRNA-103/107 is involved in hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by targeting HIF-1β

AimsActivation of hypoxia inducible factor-1 (HIF) is a hallmark in hypoxia-induced pulmonary hypertension (HPH). microRNAs play a significant role in regulating proliferation of pulmonary arterial smooth muscle cells (PASMCs) in pulmonary hypertension. Previous studies have shown that HIF-1β is a target of miR-103/107. In this present study, we aimed to investigate whether miR-103/107 regulate vascular remodeling in HPH via HIF-1β. Main methodsThe HPH model was built by hypoxia exposure in rats. Real-time PCR and Western blotting were used to determine the expression of miR-103/107 and HIF-1β. Proliferation of PASMCs was examined by 5-bromo-2′-deoxyuridine (BrdU) incorporation method. The functions of miR-103/107 on PASMCs proliferation, HIF-1α and HIF-1β expression were assessed by transfecting miR-103/107 mimics and inhibitors. Key findingsSignificant down-regulation of miR-103/107 was observed in remodeled intrapulmonary vascular in HPH rats and hypoxia-exposured PASMCs, whereas HIF-1α and HIF-1β expression were up-regulated. Hypoxia exposure induced significant proliferation of PASMCs, overexpression of miR-103/107 inhibited but miR-103/107 inhibitors exacerbated PASMCs proliferation. Gain-of-function and loss-of-function experiments showed that miR-103/107 expression was inversely correlated with HIF-1β level. No significant changes of HIF-1α expression were observed under miR-103/107 mimic treatment. SignificanceLoss of suppression on HIF-1β by miR-103/107 may contribute to excess proliferation of PASMCs and vascular remodeling in hypoxic pulmonary hypertension.

Metabolic reprogramming and inflammation act in concert to control vascular remodeling in hypoxic pulmonary hypertension

Pulmonary hypertension (PH) is a complex, multifactorial syndrome that remains poorly understood despite decades of research. PH is characterized by profound pulmonary artery (PA) remodeling that includes significant fibro-proliferative and inflammatory changes of the PA adventitia. In line with the emerging concept that PH shares key features with cancer, recent work centers on the idea that PH results from a multistep process driven by reprogramming of gene-expression patterns that govern changes in cell metabolism, inflammation, and proliferation. Data demonstrate that in addition to PA endothelial cells and smooth muscle cells, adventitial fibroblasts from animals with experimental hypoxic PH and from humans with PH (hereafter, termed PH-Fibs) exhibit proinflammatory activation, increased proliferation, and apoptosis resistance, all in the context of metabolic reprogramming to aerobic glycolysis. PH-Fibs can also recruit, retain, and activate naïve macrophages (Mϕ) toward a proinflammatory/proremodeling phenotype through secretion of chemokines, cytokines, and glycolytic metabolites, among which IL-6 and lactate play key roles. Furthermore, these fibroblast-activated Mϕ (hereafter, termed FAMϕ) exhibit aerobic glycolysis together with high expression of arginase 1, Vegfa, and I1lb, all of which require hypoxia-inducible factor 1α and STAT3 signaling. Strikingly, in situ, the adventitial Mϕ phenotype in the remodeled PA closely resembles the Mϕ phenotype induced by fibroblasts in vitro (FAMϕ), suggesting that FAMϕ crosstalk involving metabolic and inflammatory signals is a critical, pathogenetic component of vascular remodeling. This review discusses metabolic and inflammatory changes in fibroblasts and Mϕ in PH with the goal of raising ideas about new interventions to abrogate remodeling in hypoxic forms of PH.

MiR-29a-3p attenuates hypoxic pulmonary hypertension by inhibiting pulmonary adventitial fibroblast activation.

Activation of pulmonary adventitial fibroblasts plays a key role in the pulmonary vascular remodeling in hypoxic pulmonary hypertension. Previous studies showed that miRNAs participated in the regulation of fibroblast activation. This study explored the role of miR-29 in the activation of pulmonary adventitial fibroblasts and the therapeutic potential in hypoxic pulmonary hypertension. We found that hypoxia-induced pulmonary adventitial fibroblasts activation was accompanied with a drastic decrease of miR-29a-3p expression. Knockdown of hypoxia-inducible factor-1 α or Smad3 reversed the hypoxia-induced decrease of miR-29-3p in cultured pulmonary adventitial fibroblasts. In vitro, miR-29a-3p mimic inhibited the hypoxia-induced proliferation, migration, and secretion of pulmonary adventitial fibroblasts, suppressed the hypoxia-induced expression of α-smooth muscle actin and extracellular matrix collagen in pulmonary adventitial fibroblasts; however, miR-29a-3p inhibitor mimicked the effect of hypoxia on the activation of pulmonary adventitial fibroblasts. Further studies revealed that preventative or therapeutic administration of miR-29a-3p significantly decreased pulmonary artery pressure and right ventricle hypertrophy index and ameliorated pulmonary vascular remodeling in hypoxic pulmonary hypertension rats. These findings suggest that miR-29a-3p regulates the activation and phenotype of pulmonary adventitial fibroblasts in hypoxia and has preventative and therapeutic potential in hypoxic pulmonary hypertension.

Disodium Cromoglycate Attenuates Hypoxia Induced Enlargement of End-Expiratory Lung Volume in Rats

Mechanism responsible for the enlargement of end-expiratory lung volume (EELV) induced by chronic hypoxia remains unclear. The fact that the increase in EELV persists after return to normoxia suggests involvement of morphological changes. Because hypoxia has been also shown to activate lung mast cells, we speculated that they could play in the mechanism increasing EELV similar role as in vessel remodeling in hypoxic pulmonary hypertension (HPH). We, therefore, tested an effect of mast cells degranulation blocker disodium cromoglycate (DSCG) on hypoxia induced EELV enlargement. Ventilatory parameters, EELV and right to left heart weight ratio (RV/LV+S) were measured in male Wistar rats. The experimental group (H+DSCG) was exposed to 3 weeks of normobaric hypoxia and treated with DSCG during the first four days of hypoxia, control group was exposed to hypoxia only (H), two others were kept in normoxia as non-treated (N) and treated (N+DSCG) groups. DSCG treatment significantly attenuated the EELV enlargement (H+DSCG = 6.1+/-0.8; H = 9.2+/-0.9; ml +/-SE) together with the increase in minute ventilation (H + DSCG = 190+/-8; H = 273 +/- 10; ml/min +/- SE) and RV/LV + S (H + DSCG = 0.39 +/- 0.03; H = 0.50 +/- 0.06).