Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.     

BMP4 Protects Rat Pulmonary Arterial Smooth Muscle Cells from Apoptosis by PI3K/AKT/Smad1/5/8 Signaling

Bone morphogenetic protein-4 (BMP4), a member of the transforming growth factor β (TGF-β) family of growth factors, is activated and increased under hypoxic conditions, which plays an important role in the progression of pulmonary arterial hypertension (PAH). Previous studies have shown that BMP4 is involved in the regulation of proliferation, differentiation, migration and apoptosis of various cell types. However, the precise mechanisms involved in the regulation of pulmonary artery smooth muscle cells (PASMCs) in PAH are still incompletely understood. It has been reported that AKT is a critical regulator of cell survival and vascular remodeling. Therefore, there may be crosstalk between BMP4 anti-apoptotic processes and PI3K/AKT survival effect in rat PASMCs. To test this hypothesis, we performed confocal, cell viability measurement, mitochondrial potential, real-time polymerase chain reaction (PCR), and Western blot analysis to determine the role of BMP4 on cell survival and apoptosis. We found that hypoxia up-regulated the expression of BMP4. BMP4 promoted cell survival, reduced mitochondrial depolarization, and increased the expression of Bcl-2 and procaspase-3 in PASMCs under serum-deprived condition. These effects were reversed by PI3K/AKT inhibitors ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin). Thus, these findings indicate that BMP4 protects PASMCs from apoptosis at least in part, mediated via the PI3K/AKT pathway.     

Regulation of Cancer Metabolism by Deubiquitinating Enzymes: The Warburg Effect

Cancer is a disorder of cell growth and proliferation, characterized by different metabolic pathways within normal cells. The Warburg effect is a major metabolic process in cancer cells that affects the cellular responses, such as proliferation and apoptosis. Various signaling factors down/upregulate factors of the glycolysis pathway in cancer cells, and these signaling factors are ubiquitinated/deubiquitinated via the ubiquitin–proteasome system (UPS). Depending on the target protein, DUBs act as both an oncoprotein and a tumor suppressor. Since the degradation of tumor suppressors and stabilization of oncoproteins by either negative regulation by E3 ligases or positive regulation of DUBs, respectively, promote tumorigenesis, it is necessary to suppress these DUBs by applying appropriate inhibitors or small molecules. Therefore, we propose that the DUBs and their inhibitors related to the Warburg effect are potential anticancer targets.     

Epigenetic Regulation of Pulmonary Arterial Hypertension-Induced Vascular and Right Ventricular Remodeling: New Opportunities?

Pulmonary artery hypertension (PAH) is a rare chronic disease with high impact on patients’ quality of life and currently no available cure. PAH is characterized by constant remodeling of the pulmonary artery by increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), fibroblasts (FBs) and endothelial cells (ECs). This remodeling eventually leads to increased pressure in the right ventricle (RV) and subsequent right ventricle hypertrophy (RVH) which, when left untreated, progresses into right ventricle failure (RVF). PAH can not only originate from heritable mutations, but also develop as a consequence of congenital heart disease, exposure to drugs or toxins, HIV, connective tissue disease or be idiopathic. While much attention was drawn into investigating and developing therapies related to the most well understood signaling pathways in PAH, in the last decade, a shift towards understanding the epigenetic mechanisms driving the disease occurred. In this review, we reflect on the different epigenetic regulatory factors that are associated with the pathology of RV remodeling, and on their relevance towards a better understanding of the disease and subsequently, the development of new and more efficient therapeutic strategies.     

Inhibition of KIF23 Alleviates IPAH by Targeting Pyroptosis and Proliferation of PASMCs

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive vascular disease with high mortality and heritability. Pyroptosis is a novel form of programmed cell death, and it is closely associated with IPAH. However, the roles of pyroptosis-related genes (PRGs) in IPAH are still largely unknown. In this study, we identified KIF23 as the most relevant gene for IPAH and pyroptosis, and its expression was significantly increased in pulmonary arterial smooth muscle cells (PASMCs) of IPAH. Besides, the pyroptosis level of PASMCs was also considerably upregulated in IPAH. Knockdown of KIF23 in PASMCs could significantly suppress the PASMCs’ pyroptosis and proliferation and then alleviate the increase in pulmonary arterial pressure, right ventricular hypertrophy, and pulmonary vascular resistance in IPAH. KIF23 regulated the expression of Caspase3, NLRP3, and HMGB1, and they were all involved in the PI3K/AKT and MAPK pathways, indicating that PI3K/AKT and MAPK pathways might participate in regulating PASMCs pyroptosis by KIF23. In conclusion, our study suggests that KIF23 may be a new therapeutic target for IPAH, which can alleviate the symptoms of IPAH by inhibiting the pyroptosis and proliferation of PASMCs.     

Signal Transduction during Metabolic and Inflammatory Reprogramming in Pulmonary Vascular Remodeling

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by (mal)adaptive remodeling of the pulmonary vasculature, which is associated with inflammation, fibrosis, thrombosis, and neovascularization. Vascular remodeling in PAH is associated with cellular metabolic and inflammatory reprogramming that induce profound endothelial and smooth muscle cell phenotypic changes. Multiple signaling pathways and regulatory loops act on metabolic and inflammatory mediators which influence cellular behavior and trigger pulmonary vascular remodeling in vivo. This review discusses the role of bioenergetic and inflammatory impairments in PAH development.     

E3 Ubiquitin Ligase APC/CCdh1 Regulation of Phenylalanine Hydroxylase Stability and Function

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by the dysfunction of the enzyme phenylalanine hydroxylase (PAH). Alterations in the level of PAH leads to the toxic accumulation of phenylalanine in the blood and brain. Protein degradation mediated by ubiquitination is a principal cellular process for maintaining protein homeostasis. Therefore, it is important to identify the E3 ligases responsible for PAH turnover and proteostasis. Here, we report that anaphase-promoting complex/cyclosome-Cdh1 (APC/C)^Cdh1 is an E3 ubiquitin ligase complex that interacts and promotes the polyubiquitination of PAH through the 26S proteasomal pathway. Cdh1 destabilizes and declines the half-life of PAH. In contrast, the CRISPR/Cas9-mediated knockout of Cdh1 stabilizes PAH expression and enhances phenylalanine metabolism. Additionally, our current study demonstrates the clinical relevance of PAH and Cdh1 correlation in hepatocellular carcinoma (HCC). Overall, we show that PAH is a prognostic marker for HCC and Cdh1 could be a potential therapeutic target to regulate PAH-mediated physiological and metabolic disorders.     

Baicalin Inhibits Hypoxia-Induced Pulmonary Artery Smooth Muscle Cell Proliferation via the AKT/HIF-1α/p27-Associated Pathway

Baicalin, a flavonoid compound purified from the dry roots of Scutellaria baicalensis Georgi, has been shown to possess various pharmacological actions. Previous studies have revealed that baicalin inhibits the growth of cancer cells through the induction of apoptosis. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by enhanced pulmonary artery smooth muscle cell (PASMCs) proliferation and suppressed apoptosis. However, the potential mechanism of baicalin in the regulation of PASMC proliferation and the prevention of cardiovascular diseases remains unexplored. To test the effects of baicalin on hypoxia, we used rats treated with or without baicalin (100 mg·kg⁻¹ each rat) at the beginning of the third week after hypoxia. Hemodynamic and pulmonary pathomorphology data showed that right ventricular systolic pressures (RVSP), the weight of the right ventricle/left ventricle plus septum (RV/LV + S) ratio and the medial width of pulmonary arterioles were much higher in chronic hypoxia. However, baicalin treatment repressed the elevation of RVSP, RV/LV + S and attenuated the pulmonary vascular structure remodeling (PVSR) of pulmonary arterioles induced by chronic hypoxia. Additionally, baicalin (10 and 20 μmol·L⁻¹) treatment suppressed the proliferation of PASMCs and attenuated the expression of hypoxia-inducible factor-α (HIF-α) under hypoxia exposure. Meanwhile, baicalin reversed the hypoxia-induced reduction of p27 and increased AKT/protein kinase B phosphorylation p-AKT both in vivo and in vitro. These results suggested that baicalin could effectively attenuate PVSR and hypoxic pulmonary hypertension.     

mTOR Signaling in Pulmonary Vascular Disease: Pathogenic Role and Therapeutic Target

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease without a cure. The exact pathogenic mechanisms of PAH are complex and poorly understood, yet a number of abnormally expressed genes and regulatory pathways contribute to sustained vasoconstriction and vascular remodeling of the distal pulmonary arteries. Mammalian target of rapamycin (mTOR) is one of the major signaling pathways implicated in regulating cell proliferation, migration, differentiation, and protein synthesis. Here we will describe the canonical mTOR pathway, structural and functional differences between mTOR complexes 1 and 2, as well as the crosstalk with other important signaling cascades in the development of PAH. The pathogenic role of mTOR in pulmonary vascular remodeling and sustained vasoconstriction due to its contribution to proliferation, migration, phenotypic transition, and gene regulation in pulmonary artery smooth muscle and endothelial cells will be discussed. Despite the progress in our elucidation of the etiology and pathogenesis of PAH over the two last decades, there is a lack of effective therapeutic agents to treat PAH patients representing a significant unmet clinical need. In this review, we will explore the possibility and therapeutic potential to use inhibitors of mTOR signaling cascade to treat PAH.     

Inhibition of Vascular Endothelial Cadherin Cleavage Prevents Elastic Fiber Alterations and Atherosclerosis Induced by Intermittent Hypoxia in the Mouse Aorta

Intermittent hypoxia (IH), the major feature of obstructive sleep apnea syndrome (OSAS), induces atherosclerosis and elastic fiber alterations. VE-cadherin cleavage is increased in OSAS patients and in an IH-cellular model. It is mediated by HIF-1 and Src-tyr-kinases pathways and results in endothelial hyperpermeability. Our aim was to determine whether blocking VE-cadherin cleavage in vivo could be an efficient strategy to inhibit deleterious IH-induced vascular remodeling, elastic fiber defects and atherogenesis. VE-cadherin regulation, aortic remodeling and atherosclerosis were studied in IH-exposed C57Bl/6J or ApoE-/-mice treated or not with Src-tyr-kinases inhibitors (Saracatinib/Pazopanib) or a HIF-1 inhibitor (Acriflavine). Human aortic endothelial cells were exposed to IH and treated with the same inhibitors. LDL and the monocytes transendothelium passage were measured. In vitro, IH increased transendothelium LDL and monocytes passage, and the tested inhibitors prevented these effects. In mice, IH decreased VE-cadherin expression and increased plasmatic sVE level, intima-media thickness, elastic fiber alterations and atherosclerosis, while the inhibitors prevented these in vivo effects. In vivo inhibition of HIF-1 and Src tyr kinase pathways were associated with the prevention of IH-induced elastic fiber/lamella degradation and atherogenesis, which suggests that VE-cadherin could be an important target to limit atherogenesis and progression of arterial stiffness in OSAS.     

Epigenetic Regulation of Endothelial Dysfunction and Inflammation in Pulmonary Arterial Hypertension

Once perceived as a disorder treated by vasodilation, pulmonary artery hypertension (PAH) has emerged as a pulmonary vascular disease with severe endothelial cell dysfunction. In the absence of a cure, many studies seek to understand the detailed mechanisms of EC regulation to potentially create more therapeutic options for PAH. Endothelial dysfunction is characterized by complex phenotypic changes including unchecked proliferation, apoptosis-resistance, enhanced inflammatory signaling and metabolic reprogramming. Recent studies have highlighted the role of epigenetic modifications leading to pro-inflammatory response pathways, endothelial dysfunction, and the progression of PAH. This review summarizes the existing literature on epigenetic mechanisms such as DNA methylation, histone modifications, and non-coding RNAs, which can lead to aberrant endothelial function. Our goal is to develop a conceptual framework for immune dysregulation and epigenetic changes in endothelial cells in the context of PAH. These studies as well as others may lead to advances in therapeutics to treat this devastating disease.     

Molecular Mechanisms of Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease that is precipitated by hypertrophic pulmonary vascular remodeling of distal arterioles to increase pulmonary artery pressure and pulmonary vascular resistance in the absence of left heart, lung parenchymal, or thromboembolic disease. Despite available medical therapy, pulmonary artery remodeling and its attendant hemodynamic consequences result in right ventricular dysfunction, failure, and early death. To limit morbidity and mortality, attention has focused on identifying the cellular and molecular mechanisms underlying aberrant pulmonary artery remodeling to identify pathways for intervention. While there is a well-recognized heritable genetic component to PAH, there is also evidence of other genetic perturbations, including pulmonary vascular cell DNA damage, activation of the DNA damage response, and variations in microRNA expression. These findings likely contribute, in part, to dysregulation of proliferation and apoptosis signaling pathways akin to what is observed in cancer; changes in cellular metabolism, metabolic flux, and mitochondrial function; and endothelial-to-mesenchymal transition as key signaling pathways that promote pulmonary vascular remodeling. This review will highlight recent advances in the field with an emphasis on the aforementioned molecular mechanisms as contributors to the pulmonary vascular disease pathophenotype.     

KCNK3 Mutation Causes Altered Immune Function in Pulmonary Arterial Hypertension Patients and Mouse Models

Loss of function KCNK3 mutation is one of the gene variants driving hereditary pulmonary arterial hypertension (PAH). KCNK3 is expressed in several cell and tissue types on both membrane and endoplasmic reticulum and potentially plays a role in multiple pathological process associated with PAH. However, the role of various stressors driving the susceptibility of KCNK3 mutation to PAH is unknown. Hence, we exposed kcnk3^fl/fl animals to hypoxia, metabolic diet and low dose lipopolysaccharide (LPS) and performed molecular characterization of their tissue. We also used tissue samples from KCNK3 patients (skin fibroblast derived inducible pluripotent stem cells, blood, lungs, peripheral blood mononuclear cells) and performed microarray, immunohistochemistry (IHC) and mass cytometry time of flight (CyTOF) experiments. Although a hypoxic insult did not alter vascular tone in kcnk3^fl/fl mice, RNASeq study of these lungs implied that inflammatory and metabolic factors were altered, and the follow-up diet study demonstrated a dysregulation of bone marrow cells in kcnk3^fl/fl mice. Finally, a low dose LPS study clearly showed that inflammation could be a possible second hit driving PAH in kcnk3^fl/fl mice. Multiplex, IHC and CyTOF immunophenotyping studies on human samples confirmed the mouse data and strongly indicated that cell mediated, and innate immune responses may drive PAH susceptibility in these patients. In conclusion, loss of function KCNK3 mutation alters various physiological processes from vascular tone to metabolic diet through inflammation. Our data suggests that altered circulating immune cells may drive PAH susceptibility in patients with KCNK3 mutation.     

Epigenetic Targets for Oligonucleotide Therapies of Pulmonary Arterial Hypertension

Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverse established arterial wall thickening, stiffening, and hypercontractility. Therefore, new approaches are needed to achieve long-acting prevention and reversal of occlusive pulmonary vascular remodeling. Several promising new drug classes are emerging from a better understanding of pulmonary vascular gene expression programs. In this review, potential epigenetic targets for small molecules and oligonucleotides will be described. Most are in preclinical studies aimed at modifying the growth of vascular wall cells in vitro or normalizing vascular remodeling in PAH animal models. Initial success with lung-directed delivery of oligonucleotides targeting microRNAs suggests other epigenetic mechanisms might also be suitable drug targets. Those targets include DNA methylation, proteins of the chromatin remodeling machinery, and long noncoding RNAs, all of which act as epigenetic regulators of vascular wall structure and function. The progress in testing small molecules and oligonucleotide-based drugs in PAH models is summarized.     

PDK1 Inhibitor BX795 Improves Cisplatin and Radio-Efficacy in Oral Squamous Cell Carcinoma by Downregulating the PDK1/CD47/Akt-Mediated Glycolysis Signaling Pathway

Background: Oral squamous cell carcinoma (OSCC) has a high prevalence and predicted global mortality rate of 67.1%, necessitating better therapeutic strategies. Moreover, the recurrence and resistance of OSCC after chemo/radioresistance remains a major bottleneck for its effective treatment. Molecular targeting is one of the new therapeutic approaches to target cancer. Among a plethora of targetable signaling molecules, PDK1 is currently rising as a potential target for cancer therapy. Its aberrant expression in many malignancies is observed associated with glycolytic re-programming and chemo/radioresistance. Methods: Furthermore, to better understand the role of PDK1 in OSCC, we analyzed tissue samples from 62 patients with OSCC for PDK1 expression. Combining in silico and in vitro analysis approaches, we determined the important association between PDK1/CD47/LDHA expression in OSCC. Next, we analyzed the effect of PDK1 expression and its connection with OSCC orosphere generation and maintenance, as well as the effect of the combination of the PDK1 inhibitor BX795, cisplatin and radiotherapy in targeting it. Results: Immunohistochemical analysis revealed that higher PDK1 expression is associated with a poor prognosis in OSCC. The immunoprecipitation assay indicated PDK1/CD47 binding. PDK1 ligation significantly impaired OSCC orosphere formation and downregulated Sox2, Oct4, and CD133 expression. The combination of BX795 and cisplatin markedly reduced in OSCC cell’s epithelial-mesenchymal transition, implying its synergistic effect. p-PDK1, CD47, Akt, PFKP, PDK3 and LDHA protein expression were significantly reduced, with the strongest inhibition in the combination group. Chemo/radiotherapy together with abrogation of PDK1 inhibits the oncogenic (Akt/CD47) and glycolytic (LDHA/PFKP/PDK3) signaling and, enhanced or sensitizes OSCC to the anticancer drug effect through inducing apoptosis and DNA damage together with metabolic reprogramming. Conclusions: Therefore, the results from our current study may serve as a basis for developing new therapeutic strategies against chemo/radioresistant OSCC.     

Pyruvate Dehydrogenase Kinase Inhibition by Dichloroacetate in Melanoma Cells Unveils Metabolic Vulnerabilities

Melanoma is characterized by high glucose uptake, partially mediated through elevated pyruvate dehydrogenase kinase (PDK), making PDK a potential treatment target in melanoma. We aimed to reduce glucose uptake in melanoma cell lines through PDK inhibitors dichloroacetate (DCA) and AZD7545 and through PDK knockdown, to inhibit cell growth and potentially unveil metabolic co-vulnerabilities resulting from PDK inhibition. MeWo cells were most sensitive to DCA, while SK-MEL-2 was the least sensitive, with IC₅₀ values ranging from 13.3 to 27.0 mM. DCA strongly reduced PDH phosphorylation and increased the oxygen consumption rate:extracellular acidification rate (OCR:ECAR) ratio up to 6-fold. Knockdown of single PDK isoforms had similar effects on PDH phosphorylation and OCR:ECAR ratio as DCA but did not influence sensitivity to DCA. Growth inhibition by DCA was synergistic with the glutaminase inhibitor CB-839 (2- to 5-fold sensitization) and with diclofenac, known to inhibit monocarboxylate transporters (MCTs) (3- to 8-fold sensitization). CB-839 did not affect the OCR:ECAR response to DCA, whereas diclofenac strongly inhibited ECAR and further increased the OCR:ECAR ratio. We conclude that in melanoma cell lines, DCA reduces proliferation through reprogramming of cellular metabolism and synergizes with other metabolically targeted drugs.