Pulmonary Arterial Hypertension In Mice Research Articles

Abstract 4119280: The role of colony-stimulating factor 1 receptor and perivascular macrophages in pulmonary arterial hypertension

Background: Pulmonary arterial hypertension (PAH) is a life-threatening disease caused by pulmonary artery remodeling, inflammation, and altered immune responses. The proliferation of pulmonary arterial smooth muscle cells and the accumulation of perivascular macrophages are crucial factors for the development of pulmonary artery remodeling. However, the mechanism for the progression of pulmonary artery remodeling through the perivascular macrophages has not been fully investigated. In this study, we examined the significance of colony-stimulating factor 1 receptor (CSF1R), which regulates the differentiation and proliferation of macrophages in PAH. Methods and Results: The numbers of pulmonary arterial perivascular CSF1R-positive cells significantly increased in lungs from PAH patients, compared to controls. The CSF1R phosphorylation on whole lungs was significantly increased in monocrotaline (MCT)-induced PAH rats and SU5416/Hypoxia (SuHx)-induced PAH mice. Next, we evaluated the therapeutic potential of a CSF1R inhibitor or anti-CSF1R antibody in vivo . Blocking CSF1R with a CSF1R inhibitor or anti-CSF1R antibody significantly decreased right ventricular systolic pressure, right ventricular fibrosis, perivascular CD68-positive macrophages and Arg1-positive M2 macrophages in PAH rats and mice. CSF1R knockdown using antisense oligonucleotide in the lung significantly decreased right ventricular systolic pressure in SuHx-induced PAH mice. Next, we focused on C-C motif chemokine ligand 2 (CCL2) whose gene expression is elevated in macrophages of PAH model. The culture with a conditioned medium from lung-derived M2 macrophages accelerated the proliferation of pulmonary arterial smooth muscle cells, while adding a CCL2 neutralizing antibody into the conditioned medium inhibited their proliferation. Conclusion: CSF1R has a critical role in the proliferation of pulmonary arterial perivascular macrophages and the progression of PAH, and M2-derived CCL2 contributed to the proliferation of pulmonary arterial smooth muscle cells. CSF1R is a novel therapeutic target in PAH.

Ginsenoside Rg1 improves hypoxia-induced pulmonary vascular endothelial dysfunction through TXNIP/NLRP3 pathway-modulated mitophagy

BackgroundVascular endothelial dysfunction (VED) is one of the main pathogenic events in pulmonary arterial hypertension (PAH). Previous studies have demonstrated that the ginsenoside Rg1 (Rg1) can ameliorate PAH, but the mechanism by which Rg1 affects pulmonary VED in hypoxia-induced PAH remains unclear. MethodsNetwork pharmacology, molecular docking and other experiments were used to explore the mechanisms by which Rg1 affects PAH. A PAH mouse model was established via hypoxia combined with the vascular endothelial growth factor (VEGFR) inhibitor su5416 (SuHx), and a cell model was established via hypoxia. The functions of Rg1 in VED, oxidative stress, inflammation, mitophagy, and TXNIP and NLRP3 expression were examined. ResultsIn hypoxia-induced VED, progressive exacerbation of oxidative stress, inflammation, and mitophagy were observed, and were associated with elevated TXNIP and NLRP3 expression in vivo and in vitro. Rg1 improved hypoxia-induced impaired endothelium-dependent vasodilation and increased nitric oxide (NO) and endothelial NO synthase (eNOS) expression. Rg1, SRI37330 (a TXNIP inhibitor), MCC950 (an NLRP3 inhibitor), and Liensinine (a mitophagy inhibitor) attenuated oxidative stress, inflammation, and mitophagy by reducing the expression of TXNIP and NLRP3 in mice and cells. Furthermore, the combination of SB203580 (a mitophagy agonist) with Rg1 disrupted the protective effect of Rg1 on hypoxia-induced pulmonary artery and human pulmonary artery endothelial cells (HPAECs). ConclusionRg1 improves hypoxia-induced pulmonary vascular endothelial dysfunction through TXNIP/NLRP3 pathway-modulated oxidative stress, inflammation and mitophagy.

ScRNA‐seq reveals NAMPT‐mediated macrophage polarization shapes smooth muscle cell plasticity in pulmonary arterial hypertension

AbstractPhenotypic switching of smooth muscle cells (SMC) is a crucial process in the pathogenesis of pulmonary arterial hypertension (PAH). However, the underlying mechanism is unclear. Here, we performed single‐cell RNA sequencing on pulmonary arteries obtained from lung transplantation to explore the cellular heterogeneity and gene expression profile of the main cell types. We identified three distinct SMC phenotypes, namely contractile, fibroblast‐like, and chondroid‐like, and observed an enhanced transition from contractile to fibroblast‐like phenotype in PAH by pseudo‐time analysis and in vitro. We also revealed a classically activated (M1) polarization of macrophages and an increased pro‐inflammatory macrophage‐SMC crosstalk in PAH via intercellular communication. Notably, Nicotinamide phosphoribosyltransferase (NAMPT) emerges as a key player in macrophage polarization. The macrophages overexpress Nampt in Sugen/hypoxia (Su/Hx) ‐induced PAH mice and significantly downregulate the pro‐inflammation secretion pattern with Nampt interference. In a cellular coculture system, Nampt knockdown in macrophages significantly inhibits the fibroblast‐like phenotypic switching of SMCs. Finally, we identified Ccl2/5 as a key cytokine for SMC phenotypic modulation. Collectively, these findings provide a cell atlas of normal human pulmonary arteries and demonstrate that NAMPT‐driven M1 macrophage polarization promotes the fibroblast‐like phenotypic switching of SMCs through CCR2/CCR5 cellular crosstalk in PAH.

Empagliflozin Attenuates Pulmonary Arterial Remodeling Through Peroxisome Proliferator-Activated Receptor Gamma Activation.

The loss of peroxisome proliferator-activated receptor gamma (PPARγ) exacerbates pulmonary arterial hypertension (PAH), while its upregulation reduces cell proliferation and vascular remodeling, thereby decreasing PAH severity. SGLT2 inhibitors, developed for type 2 diabetes, might also affect signal transduction in addition to modulating sodium-glucose cotransporters. Pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) were treated with three SGLT2 inhibitors, canagliflozin (Cana), dapagliflozin (Dapa), and empagliflozin (Empa), to investigate their antiproliferative effects. To assess the impact of Empa on PPARγ, luciferase reporter assays and siRNA-mediated PPARγ knockdown were employed to examine regulation of the γ-secretase complex and its downstream target Notch3. Therapy involving daily administration of Empa was initiated 21 days after inducing hypoxia-induced PAH in mice. Empa exhibited significant antiproliferative effects on fast-growing IPAH PASMCs. Empa activated PPARγ to prevent formation of the γ-secretase complex, with specific impacts on presenilin enhancer 2 (PEN2), which plays a crucial role in maintaining γ-secretase complex stability, thereby inhibiting Notch3. Similar results were obtained in lung tissue of chronically hypoxic mice. Empa attenuated pulmonary arterial remodeling and right ventricle hypertrophy in a hypoxic PAH mouse model. Moreover, PPARγ expression was significantly decreased and PEN2, and Notch3 levels were increased in lung tissue from PAH patients compared with non-PAH lung tissue. Empa reverses vascular remodeling by activating PPARγ to suppress the γ-secretase-Notch3 axis. We propose Empa as a PPARγ activator and potential therapeutic for PAH.

High relative humidity environment alleviates hypoxia-induced pulmonary arterial hypertension in mice

The environment has long been considered a crucial factor influencing the onset and progression of pulmonary diseases. Environmental therapy is also a practical treatment approach for many conditions. While research has explored the effects of factors like air pressure and oxygen concentration on pulmonary arterial hypertension (PAH), the impact of air humidity on PAH has not been investigated. In this study, we examined the role of different air humidity levels in a mouse model of PAH by controlling relative humidity. We induced PAH in mice using 10 % hypoxia, which led to significant thickening of the pulmonary vasculature, elevated right ventricular systolic pressure, and an increased right ventricular hypertrophy index (RVHI). However, when exposed to an environment with 80–95 % relative humidity, there was a marked reduction in the extent of pulmonary vascular remodeling, decreased vascular thickening, and lower RVHI, effectively preserving right heart function. Notably, changes in the Bmpr2/Tgf-β signaling pathway were significant and may play a pivotal role in this protective effect. In summary, our findings indicate that high relative humidity confers a protective effect on hypoxia-induced PAH in mice, providing novel insights into potential treatments for PAH.

Erythrophagocytosis-induced ferroptosis contributes to pulmonary microvascular thrombosis and thrombotic vascular remodeling in pulmonary arterial hypertension

BackgroundWhether primary or just as a complication from the progression of pulmonary arterial hypertension (PAH), thrombosis seems to be an important player in this condition. The crosstalk between red blood cells (RBCs) and pulmonary microvascular endothelial cells (PMVECs) and their role in PAH remain undefined. ObjectivesThe goals of this study were to assess the role of RBC–PMVEC interaction in microvascular thrombosis and thrombotic vascular remodeling under hypoxic conditions. MethodsWe established an in vitro hypoxic coincubation model of RBC and PMVEC as well as a hypoxic mouse model. We investigated erythrophagocytosis (EP), ferroptosis, thrombosis tendency, and pulmonary hemodynamics in experimental PAH. ResultsIncreased EP in PMVEC triggered ferroptosis, enhanced procoagulant activity, and exacerbated vessel remodeling under hypoxic conditions. In the PAH mouse model induced by chronic hypoxia, EP-induced ferroptosis followed by upregulated TMEM16F led to a high tendency of thrombus formation and thrombotic vascular remodeling. Inhibition of ferroptosis or silencing of TMEM16F could alleviate hypercoagulable phenotype, reverse right ventricular systolic pressure, right ventricular hypertrophy index, and remodeling of pulmonary vessels. ConclusionThese results illustrate the pathogenic RBC–PMVEC interactions in PAH. Inhibition EP, ferroptosis, or TMEM16F could be a novel therapeutic target to prevent PAH development and thrombotic complications.

5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase promotes pulmonary arterial smooth muscle cell proliferation via the Ras signaling pathway.

Pulmonary arterial hypertension (PAH) is a debilitating vascular disorder characterized by abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and collagen synthesis, contributing to vascular remodeling and elevated pulmonary vascular resistance. This study investigated the critical role of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) in cell proliferation and collagen synthesis in PASMCs in PAH. Here we show that ATIC levels are significantly increased in the lungs of monocrotaline (MCT)-induced PAH rat model, hypoxia-induced PAH mouse model, and platelet-derived growth factor (PDGF)-stimulated PASMCs. Inhibition of ATIC attenuated PDGF-induced cell proliferation and collagen I synthesis in PASMCs. Conversely, overexpression or knockdown of ATIC causes a significant promotion or inhibition of Ras and ERK activation, cell proliferation, and collagen synthesis in PASMCs. Moreover, ATIC deficiency attenuated Ras activation in the lungs of hypoxia-induced PAH mice. Furthermore, Ras inhibition attenuates ATIC overexpression- and PDGF-induced collagen synthesis and PASMC proliferation. Notably, we identified that transcription factors MYC, early growth response protein 1 (EGR1), and specificity protein 1 (SP1) directly binds to promoters of Atic gene and regulate ATIC expression. These results provide the first evidence that ATIC promotes PASMC proliferation in pulmonary vascular remodeling through the Ras signaling pathway.NEW & NOTEWORTHY Our findings highlight the important role of ATIC in the PASMC proliferation of pulmonary vascular remodeling through its modulation of the Ras signaling pathway and its regulation by transcription factors MYC, EGR1, and SP1. ATIC's modulation of Ras signal pathway represents a novel mechanism contributing to PAH development.

CircST6GAL1 knockdown alleviates pulmonary arterial hypertension by regulating miR-509-5p/multiple C2 and transmembrane domain containing 2 axis.

Hypertension is a main contributing factor of cardiovascular diseases; deregulated circular RNAs are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Herein, we evaluated the function and mechanism of circST6GAL1 in PAH process. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in hypoxic environment for functional analysis. The cell counting kit-8, 5-ethynyl-2'-deoxyuridine, wound healing, and flow cytometry assays were used to investigate cell proliferation, migration, and apoptosis. qRT-PCR and Western blotting analyses were used for level measurement of genes and proteins. The binding between miR-509-5p and circST6GAL1 or multiple C2 and transmembrane domain containing 2 (MCTP2) was analyzed by dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays. The monocrotaline (MCT)-induced PAH mouse models were established for in vivo assay. CircST6GAL1 was highly expressed in PAH patients and hypoxia-induced HPASMCs. Functionally, circST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs. Mechanistically, circST6GAL1 directly targeted miR-509-5p, and MCTP2 was a target of miR-509-5p. Rescue assays showed that the regulatory effects of circST6GAL1 deficiency on hypoxia-induced HPASMCs were abolished. Moreover, forced expression of miR-509-5p suppressed HPASMC proliferation and migration and induced cell apoptosis under hypoxia stimulation, while these effects were abolished by MCTP2 overexpression. Moreover, circST6GAL1 silencing improved MCT-induced pulmonary vascular remodeling and PAH. CircST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs, and alleviated pulmonary vascular remodeling in MCT-induced PAH mouse models through the miR-509-5p/MCTP2 axis, indicating a potential therapeutic target for PAH.

Hypusine Signaling Promotes Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both invitro and invivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.

Allele-specific control of rodent and human lncRNA KMT2E-AS1 promotes hypoxic endothelial pathology in pulmonary hypertension.

Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)-dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N-methyltransferase 2E-antisense 1 (KMT2E-AS1) and histone lysine N-methyltransferase 2E (KMT2E). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α-dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)-specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1/KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

Human Umbilical Cord Mesenchymal Stromal Cell-Derived Exosomes Alleviate Hypoxia-Induced Pulmonary Arterial Hypertension in Mice Via Macrophages.

Pulmonary hypertension (PH) is an intractable, severe, and progressive cardiopulmonary disease. Recent findings suggest that human umbilical cord mesenchymal stromal cells (HUCMSCs) and HUCMSC-derived exosomes (HUCMSC-Exos) possess potential therapeutic value for PH. However, whether they have beneficial effects on hypoxic pulmonary hypertension (HPH) is unclear. Exos are released into the extracellular environment by the fusion of intracellular multivesicular bodies with the cell membrane, and they play an important role in cellular communication. Exos ameliorate immune inflammation levels, alter macrophage phenotypes, regulate mitochondrial metabolic function, and inhibit pulmonary vascular remodeling, thereby improving PH. Macrophages are important sources of cytokines and other transmitters and can promote the release of cytokines, vasoactive molecules, and reactive oxygen species, all of which are associated with pulmonary vascular remodeling. Therefore, the aim of this study was to investigate whether HUCMSC-Exos could improve the lung inflammatory microenvironment and inhibit pulmonary vascular remodeling by targeting macrophages and identifying the underlying mechanisms. The results showed that HUCMSC-Exos promoted M2 macrophage polarization, decreased pro-inflammatory factors, increased IL-10 levels, and inhibited IL-33/ST2 axis expression, thereby inhibiting hypoxia-induced proliferation of pulmonary artery smooth muscle cells and ameliorating HPH.

Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells: Role of cystathionine γ-lyase/hydrogen sulfide pathway.

Ferroptosis can induce low retention and engraftment after mesenchymal stem cell (MSC) delivery, which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension (PAH) therapy. Interestingly, the cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway may contribute to mediating ferroptosis. However, the influence of the CSE/H2S pathway on ferroptosis in human umbilical cord MSCs (HUCMSCs) remains unclear. To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H2S pathway-mediated ferroptosis, and to investigate the functions of the CSE/H2S pathway in ferroptosis in HUCMSCs and the underlying mechanisms. Erastin and ferrostatin-1 (Fer-1) were used to induce and inhibit ferroptosis, respectively. HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE. A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions, and pulmonary pressure and vascular remodelling were measured. The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging. Cell viability, iron accumulation, reactive oxygen species production, cystine uptake, and lipid peroxidation in HUCMSCs were tested. Ferroptosis-related proteins and S-sulfhydrated Kelch-like ECH-associating protein 1 (Keap1) were detected by western blot analysis. In vivo, CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxia-induced PAH. In vitro, CSE overexpression improved H2S production and ferroptosis-related indexes, such as cell viability, iron level, reactive oxygen species production, cystine uptake, lipid peroxidation, mitochondrial membrane density, and ferroptosis-related protein expression, in erastin-treated HUCMSCs. In contrast, in vivo, CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice. In vitro, CSE inhibition decreased H2S levels and restored ferroptosis in Fer-1-treated HUCMSCs. Interestingly, upregulation of the CSE/H2S pathway induced Keap1 S-sulfhydration, which contributed to the inhibition of ferroptosis. Regulation of the CSE/H2S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH. Moreover, the protective effect of the CSE/H2S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling. The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Canagliflozin ameliorates hypobaric hypoxia-induced pulmonary arterial hypertension by inhibiting pulmonary arterial smooth muscle cell proliferation

ABSTRACT Pulmonary arterial hypertension (PAH) is a disease with a high mortality and few treatment options to prevent the development of pulmonary vessel remodeling, pulmonary vascular resistance, and right ventricular failure. Canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is originally used in diabetes patients which could assist the glucose excretion and decrease blood glucose. Recently, a few studies have reported the protective effect of SGLT2 inhibitor on monocrotaline-induced PAH. However, the effects of canagliflozin on hypobaric hypoxia-induced PAH as well as its mechanism still unclear. In this study, we used hypobaric hypoxia-induced PAH mice model to demonstrate if canagliflozin could alleviate PAH and prevent pulmonary vessel remodeling. We found that daily canagliflozin administration significantly improved survival in mice with hypobaric hypoxia-induced PAH compared to vehicle control. Canagliflozin treatment significantly reduced right ventricular systolic pressure and increased pulmonary acceleration time determined by hemodynamic assessments. Canagliflozin significantly reduced medial wall thickening and decreased muscularization of pulmonary arterioles compared to vehicle treated mice. In addition, canagliflozin inhibited the proliferation and migration of pulmonary arterial smooth muscle cells through suppressing glycolysis and reactivating AMP-activated protein kinase signaling pathway under hypoxia condition. In summary, our findings suggest that canagliflozin is sufficient to inhibit pulmonary arterial remodeling which is a potential therapeutic strategy for PAH treatment.

Abstract 18413: Therapeutic Genome Editing of Endothelial FABP4 by Endothelium-Targeted Nanoparticle Delivery of the CRISPR/Cas9 System Inhibits Pulmonary Arterial Hypertension in Mice and Rats

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling. The molecular mechanisms of obliterative pulmonary vascular remodeling remain elusive. Hypothesis: Therapeutic genome editing of endothelial Fabp4 will knockout FABP4 expression selectively in endothelial cells and inhibit PAH in mice and rats. Methods: Employing the newly developed endothelial cell (EC)-targeted nanoparticle delivery technology, plasmid DNA expressing CRISPR/Cas9 controlled by Cdh5 promoter and Fabp4 -specific guide RNAs driven by U6 promoter was administered i.v. to adult SD rats and Egln1 Tie2Cre mice which exhibit severe PAH and obliterative pulmonary vascular remodeling. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, right ventricular systolic pressure (RVSP), ratio of right ventricular versus left ventricular plus septum (RV/LV+S) and pulmonary vascular remodeling were determined. Pharmacological FABP4 inhibitor was also employed to treat MCT rats as a comparison. Similarly, the pulmonary hypertensive phenotype in genome-edited Egln1 Tie2Cre mice was also characterized. Results: Nanoparticle delivery of the all-in-one CRISPR rCdh5 plasmid DNA knockout Fabp4 selectively in endothelial cells in adult rats and Egln1 Tie2Cre mice leading to reduced RVSP, RV hypertrophy and pulmonary vascular remodeling in MCT rats and also in Egln1 Tie2Cre mice. Obliterative pulmonary vascular remodeling seen in Egln1 Tie2Cre mice was inhibited and the mortality rate in endothelial Fabp4-deficient Egln1 Tie2Cre mice was reduced. Treatment of FABP4 inhibitor also inhibits PAH in MCT rats. We also observed a marked increase of FABP4 expression in endothelial cells of lung samples of IPAH patients. Overexpression of FABP4 in primary cultures of human lung microvascular endothelial cells resulted in a 70% increase of cell proliferation. Conclusions: These findings demonstrate endothelial FABP4 is a promising target for PAH therapy. EC-targeted nanoparticle delivery of the genome editing system may represent an effective and safer gene therapy approach for PAH treatment.

Abstract 17836: ATP Citrate Lyase Coordinates Lipid Synthesis and Expression Cell Cycle Regulating Genes to Promote Vascular Remodeling in Pulmonary Arterial Hypertension

INTRODUCTION: Pulmonary arterial hypertension (PAH) is characterized by progressive obliteration of distal pulmonary arteries (PAs) due to enhanced proliferation, suppressed apoptosis and increased migration of PA smooth muscle cells (PASMCs). Like cancer cells, this abnormal phenotype of PASMCs is driven by epigenetic reprogramming and fueled by a metabolic shift towards glycolysis. ATP-Citrate Lyase (ACLY) is a nuclear-cytosolic enzyme that converts citrate to acetyl-CoA, which serves as substrate for histone acetyltransferases regulating gene expression and as a building block for lipid synthesis (required for proliferating cells to generate membrane). ACLY has recently emerged as a key player and therapeutic target in cancer. However, its role in PAH is unknown. METHODS/RESULTS: ACLY, p-ACLY (active form) expression and its nuclear localization were increased in distal PAs and PASMCs from PAH patients (WB and IF, p<0.01). Similar results were observed in the Sugen/Hypoxia (Su/Hx) animal models. In vitro , ACLY inhibition (BMS, siRNA) decreases PAH-PASMCs proliferation (PLK1; Ki67; p<0,01) and survival (Survivin; Annexin-V; p<0.01). These effects were accompanied with a decreased glycolysis (PFKBP3, p-PDH) and increased OCR/ECAR (Seahorse). Moreover, ACLY inhibition decreases the PAH-PASMCs migratory potential. Using RNA-Seq, we demonstrated that inhibition of ACLY downregulates genes associated with cell division and lipid synthesis. Further experiments revealed that ACLY promotes nuclear acetyl-CoA production favoring acetylation of H3K27, H3K9, H4, and GCN5-mediated transcriptional activation of genes responsible for cell cycle progression. Accordingly, we demonstrated that GCN5 inhibition decreases proliferation and survival of PAH-PASMCs (Ki67, AnnexinV). In vivo , Acly loss-of-function targeted to SMCs conferred protection against Su/HX-induced PAH in mice (echo and RHC). Accordingly, pharmacological inhibition of ACLY using BMS-303141 or Bempedoic Acid improved hemodynamics (RVSP, mPAP, SV, TPR, p<0.05) and vascular remodeling (EVG, p<0.05) in Su/Hx rats with established PAH. CONCLUSION: We demonstrated that ACLY inhibition may represent a novel therapeutic avenue to improve vascular remodeling in PAH.

Abstract 15967: Unravelling the Role of Deoxyhypusine Synthase-Mediated Hypusination of eIF5A in Pulmonary Arterial Hypertension

Introduction: Pulmonary Arterial Hypertension (PAH) is characterized by progressive distal pulmonary arteries (PAs) obstruction leading to heart failure and death. PA smooth muscle cells (PASMCs) from PAH patients display a “cancer-like” phenotype that contributes to PA remodeling. Appreciation of the pivotal role of translational control in hyperproliferating diseases is steadily increasing. In this regard, eukaryotic translation initiation factor 5A (eIF5A) was shown to provide cancer cells with a competitive advantage by increasing translation of mRNAs with oncogenic proprieties. Strikingly, eIF5A is the only protein containing the unique spermidine-derived amino acid hypusine required for its function. Hypusine formation is catalyzed by the sequential actions of deoxyhypusine synthase (DHPS) and deoxyhypusine hydrolase (DOHH). We hypothesized that increased Hyp eIF5A in PAH-PASMCs is required to promote translational efficiency of a set of factors conferring a higher survival and proliferative capacity, leading to pulmonary vascular remodeling. Methods/Results: As assessed by LC-MSMS and WB, expression levels of DHPS, DOHH and both total and hypusinated forms of eIF5A were found increased in PAs and PASMCs from PAH patients and animal models. In vitro , both molecular and pharmacological inhibition of DHPS and DOHH significantly attenuated PAH-PASMCs survival (WB Survivin; Annexin V labeling) and proliferation (WB MCM2, PLK1; Ki67 labeling and EdU incorporation). Hypusine signaling promoted the expression of a broad array of proteins involved in oxidative phosphorylation, supporting the bioenergetic requirements of cell survival and proliferation (LC-MSMS, WB, Seahorse). In vivo , smooth muscle cells-targeted inactivation of one allele of Dhps conferred partial protection against the development of Sugen/Hypoxia (Su/Hx)-induced PAH in mice. Accordingly, pharmacological inhibition of DHPS using GC7 improved hemodynamics (RVSP, mPAP, CO) and vascular remodeling (EVG) in monocrotaline and Su/Hx rats with established PAH. Conclusion: We showed for the first time that hypusine signaling is implicated in PAH development and represents a new promising therapeutic target.

Abstract 14829: Inflammatory Cells and Cytokines Are Increased in Mice With Pulmonary Arterial Hypertension Associated With Hematopoietic Dnmt3a Depletion

Background: Pulmonary Arterial Hypertension (PAH) is characterized by pulmonary vascular remodeling of the precapillary pulmonary arteries. Increasing evidence suggests that inflammation plays an essential role in PAH development. Understanding the involvement of immunity in this context can provide valuable insights into the pathogenesis of PAH. DNA Methyltransferase 3A ( DNMT3A ) mutations can lead to altered DNA methylation patterns in immune cells, potentially affecting the expression of immune-regulatory genes. Here, we investigate the role of inflammation in PAH driven by hematopoietic Dnmt3a depletion in mice. Methods: We used male and female conditional, Vav-Cre-driven hematopoietic Dnmt3a knockout mice ( Dnmt3a –/– ) and compared them to controls (n=3/group). Lung tissue and plasma were collected. Single-cell suspensions of digested lungs were stained to detect total leukocytes (CD45+), T cells (CD3+), B cells (CD19+), macrophages (Ly6G-, F4/80+), and neutrophils (Ly6G+, F4/80-) via flow cytometry. Plasma samples were screened for 32 distinct cytokines using a cytokine array. Results: Dnmt3a –/– mice spontaneously developed PAH at 4.5 months. PAH was associated with a 19% increase in total CD45+ leukocytes in the lungs compared to controls. T cells, and B cells were increased in Dnmt3a –/– mice compared to controls (7.9% and 3.1%, respectively). In contrast, there was no difference in lung neutrophil count. Two subsets of macrophages were observed based on granularity. Compared to controls, Dnmt3a –/– mice had a 29% increase in agranular macrophages and a 19.7% increase in granular macrophages. IL-13, known for its role in alternative monocyte/macrophage activation in PAH patients associated with systemic sclerosis (SSc-PAH), was significantly increased in the plasma of Dnmt3a –/– mice compared to controls (1.8 times higher, p=0.02). IL-12p40, a chemoattractant for macrophages, showed a trend towards being increased in Dnmt3a –/– mice compared to controls (5.7 times higher, p=0.14). Conclusion: Deletion of DNMT3A in the hemopoietic system triggers an inflammatory form of PAH in mice, associated with increased macrophages, B- and T-cells in the lung. IL-13 may be a potential diagnostic marker in PAH.

RGS5 as a Biomarker of Pericytes, Involvement in Vascular Remodeling and Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by a sustained rise in mean pulmonary artery pressure. Pulmonary vascular remodeling serves an important role in PAH. Identifying a key driver gene to regulate vascular remodeling of the pulmonary microvasculature is critical for PAH management. Differentially expressed genes were identified using the Gene Expression Omnibus (GEO) GSE117261, GSE48149, GSE113439, GSE53408 and GSE16947 datasets. A co-expression network was constructed using weighted gene co-expression network analysis. Novel and key signatures of PAH were screened using four algorithms, including weighted gene co-expression network analysis, GEO2R analysis, support vector machines recursive feature elimination and robust rank aggregation rank analysis. Regulator of G-protein signaling 5 (RGS5), a pro-apoptotic/anti-proliferative protein, which regulate arterial tone and blood pressure in vascular smooth muscle cells. The expression of RGS5 was determined using reverse transcription-quantitative PCR (RT-qPCR) in PAH and normal mice. The location of RGS5 and pericytes was detected using immunofluorescence. Compared with that in the normal group, RGS5 expression was upregulated in the PAH group based on GEO and RT-qPCR analyses. RGS5 expression in single cells was enriched in pericytes in single-cell RNA sequencing analysis. RGS5 co-localization with pericytes was detected in the pulmonary microvasculature of PAH. RGS5 regulates vascular remodeling of the pulmonary microvasculature and the occurrence of PAH through pericytes, which has provided novel ideas and strategies regarding the occurrence and innovative treatment of PAH.

Therapeutic potential and protective role of GRK6 overexpression in pulmonary arterial hypertension

Abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key mechanism in the development of pulmonary arterial hypertension (PAH). Signal transducer and activator of transcription 3 (STAT3) signalling plays a critical role in modulating PASMC proliferation, and G-protein-coupled receptor kinase 6 (GRK6) regulates the STAT3 pathway. However, the mechanism underlying the relationship between GRK6 and PAH remains unclear. In this study, we aimed to investigate the role of GRK6 in PAH and determine its potential as a therapeutic target. We utilised hypoxia- and SU5416-induced PAH mouse models and a monocrotaline-induced PAH rat model to analyse the involvement of GRK6. We conducted gain- and loss-of-function experiments using mouse PASMCs. Modulation of GRK6 expression was achieved via a lentiviral vector in vitro and an adeno-associated virus serotype 1 encoding GRK6 in vivo. GRK6 was significantly downregulated in the lung tissues of PAH mice and rats, predominantly in PASMCs. Knockout of GRK6 exacerbated PAH, while both therapeutic and prophylactic overexpression of GRK6 alleviated PAH, as evidenced by a reduction in right ventricular systolic pressure, right ventricular wall to left ventricular wall plus ventricular septum ratio, pulmonary vascular media thickness, and pulmonary vascular muscularisation. Mechanistically, GRK6 overexpression attenuated hypoxia-induced PASMC proliferation and STAT3 phosphorylation. Conversely, knockdown of GRK6 promoted hypoxia-induced proliferation, which was mitigated by a STAT3 inhibitor. Our findings highlight the potential protective and beneficial roles of GRK6 in PAH; we propose a lung-targeted GRK6 gene therapy utilizing adeno-associated virus serotype 1 as a potential treatment approach for patients with PAH.

HIV-derived proteins contribute to the development of pulmonary arterial hypertension in mice

Pulmonary arterial hypertension (PAH) is a life-threatening lung disease caused by remodeling of pulmonary vascular walls which is associated with a sustained elevation in pulmonary arterial pressure leading to RV failure and ultimately death. HIV infection increases the risk of developing severe PAH and HIV-associated PAH has a poor prognosis with elevated mortality. Despite significant effort to understand its pathophysiology, the identification of signaling pathways involved in HIV-mediated onset and progression of PAH remains scarce. The aim of the present study is to unravel the mechanistic links between HIV and PAH and to investigate how HIV-derived proteins contribute to the pathological processes of pulmonary vascular remodeling (PVR) of PAH and right ventricle (RV) hypertrophy. Three different HIV mouse models have been used to answer these questions. Our results showed that transgenic HIV-1 Tg26 mice, which mimics people living with HIV (PLWH) with low viremia and without progression to AIDS, exhibit increased right ventricular systolic pressure (RVSP), Fulton-index and thickness of pulmonary arterial wall indicating PVR and RV hypertrophy in these mice. More importantly, transfer of bone marrow (BMT) from Tg26 to wild-type (WT) mice and chronic treatment with HIV-encoded Tat protein induce development of PAH and RV hypertrophy in WT mice indicating that HIV-derived proteins contribute to the pathogenesis of PAH in mouse models of HIV. In order to explore signaling pathways involved in the HIV-related PAH, RNA sequencing and qPCR were performed using lung samples from control/Tg26 mice and BMT mice. We found increased expression of the proliferative marker Ki67 in Tg26 lung endothelial cells (EC). In Tg26 lung vascular smooth muscle cells (VSMC), markers of contractile SMC phenotype (SM22, SM MHC) were decreased while markers for synthetic SMC phenotype (Vimentin, Rbp-1) were upregulated suggesting increased EC proliferation and SMC de-differentiation in Tg26 mice. Moreover, we identified ten genes which expression levels were up/ downregulated in the lungs of Tg26 mice which can potentially contribute to the development of HIV-associated PAH. In conclusion, we have established a novel model of HIV, the BMT Tg26 to WT mice, to study the contribution of immune cell derived viral proteins to PAH and demonstrated that at least one viral protein, the HIV-Tat, is involved in the development of PAH and RV hypertrophy associated with HIV. Additional studies are needed to further elucidate the role of these signaling mechanisms in HIV-induced PAH in order to develop effective therapy for the prevention and treatment of this lung disorder. Supported by NIH NHLBI 1R01HL147639 (EBdC) and Augusta University IGP IGPP00030 (LK). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.