TRIM35 Monoubiquitinates H2B in Cardiac Cells, Implications for Heart Failure.

BACKGROUND: The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. METHODS: Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. RESULTS: Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. CONCLUSIONS: These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.

TRIM35-mediated degradation of nuclear PKM2 destabilizes GATA4/6 and induces P53 in cardiomyocytes to promote heart failure.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that translocates to the nucleus to regulate transcription factors in different tissues or pathologic states. Although studied extensively in cancer, its biological role in the heart remains unresolved. PKM1 is more abundant than the PKM2 isoform in cardiomyocytes, and thus, we speculated that PKM2 is not genetically redundant to PKM1 and may be critical in regulating cardiomyocyte-specific transcription factors important for cardiac survival. Here, we showed that nuclear PKM2 (S37P-PKM2) in cardiomyocytes interacts with prosurvival and proapoptotic transcription factors, including GATA4, GATA6, and P53. Cardiomyocyte-specific PKM2-deficient mice (Pkm2 Mut Cre+) developed age-dependent dilated cardiac dysfunction and had decreased amounts of GATA4 and GATA6 (GATA4/6) but increased amounts of P53 compared to Control Cre+ hearts. Nuclear PKM2 prevented caspase-1-dependent cleavage and degradation of GATA4/6 while also providing a molecular platform for MDM2-mediated reduction of P53. In a preclinical heart failure mouse model, nuclear PKM2 and GATA4/6 were decreased, whereas P53 was increased in cardiomyocytes. Loss of nuclear PKM2 was ubiquitination dependent and associated with the induction of the E3 ubiquitin ligase TRIM35. In mice, cardiomyocyte-specific TRIM35 overexpression resulted in decreased S37P-PKM2 and GATA4/6 along with increased P53 in cardiomyocytes compared to littermate controls and similar cardiac dysfunction to Pkm2 Mut Cre+ mice. In patients with dilated left ventricles, increase in TRIM35 was associated with decreased S37P-PKM2 and GATA4/6 and increased P53. This study supports a previously unrecognized role for PKM2 as a molecular platform that mediates cell signaling events essential for cardiac survival.

Loss of the fructose transporter SLC2A5 inhibits cancer cell migration.

Metastasis is the primary cause of cancer patient death and the elevation of SLC2A5 gene expression is often observed in metastatic cancer cells. Here we evaluated the importance of SLC2A5 in cancer cell motility by silencing its gene. We discovered that CRISPR/Cas9-mediated inactivation of the SLC2A5 gene inhibited cancer cell proliferation and migration in vitro as well as metastases in vivo in several animal models. Moreover, SLC2A5-attenuated cancer cells exhibited dramatic alterations in mitochondrial architecture and localization, uncovering the importance of SLC2A5 in directing mitochondrial function for cancer cell motility and migration. The direct association of increased abundance of SLC2A5 in cancer cells with metastatic risk in several types of cancers identifies SLC2A5 as an important therapeutic target to reduce or prevent cancer metastasis.

MFN2-driven mitochondria-to-nucleus tethering allows a non-canonical nuclear entry pathway of the mitochondrial pyruvate dehydrogenase complex.

The mitochondrial pyruvate dehydrogenase complex (PDC) translocates into the nucleus, facilitating histone acetylation by producing acetyl-CoA. We describe a noncanonical pathway for nuclear PDC (nPDC) import that does not involve nuclear pore complexes (NPCs). Mitochondria cluster around the nucleus in response to proliferative stimuli and tether onto the nuclear envelope (NE) via mitofusin-2 (MFN2)-enriched contact points. A decrease in nuclear MFN2 levels decreases mitochondria tethering and nPDC levels. Mitochondrial PDC crosses the NE and interacts with lamin A, forming a ring below the NE before crossing through the lamin layer into the nucleoplasm, in areas away from NPCs. Effective blockage of NPC trafficking does not decrease nPDC levels. The PDC-lamin interaction is maintained during cell division, when lamin depolymerizes and disassembles before reforming daughter nuclear envelopes, providing another pathway for nPDC entry during mitosis. Our work provides a different angle to understanding mitochondria-to-nucleus communication and nuclear metabolism.

A reversible metabolic stress-sensitive regulation of CRMP2A orchestrates EMT/stemness and increases metastatic potential in cancer.

An epithelial-to-mesenchymal transition (EMT) phenotype with cancer stem cell-like properties is a critical feature of aggressive/metastatic tumors, but the mechanism(s) that promote it and its relation to metabolic stress remain unknown. Here we show that Collapsin Response Mediator Protein 2A (CRMP2A) is unexpectedly and reversibly induced in cancer cells in response to multiple metabolic stresses, including low glucose and hypoxia, and inhibits EMT/stemness. Loss of CRMP2A, when metabolic stress decreases (e.g., around blood vessels in vivo) or by gene deletion, induces extensive microtubule remodeling, increased glutamine utilization toward pyrimidine synthesis, and an EMT/stemness phenotype with increased migration, chemoresistance, tumor initiation capacity/growth, and metastatic potential. In a cohort of 27 prostate cancer patients with biopsies from primary tumors and distant metastases, CRMP2A expression decreases in the metastatic versus primary tumors. CRMP2A is an endogenous molecular brake on cancer EMT/stemness and its loss increases the aggressiveness and metastatic potential of tumors.

SNPs for Genes Encoding the Mitochondrial Proteins Sirtuin3 and Uncoupling Protein 2 Are Associated With Disease Severity, Type 2 Diabetes, and Outcomes in Patients With Pulmonary Arterial Hypertension and This Is Recapitulated in a New Mouse Model Lacking Both Genes.

Background Isolated loss-of-function single nucleotide polymorphisms (SNPs) for SIRT3 (a mitochondrial deacetylase) and UCP2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with both pulmonary arterial hypertension (PAH) and insulin resistance, but their collective role in animal models and patients is unknown. Methods and Results In a prospective cohort of patients with PAH (n=60), we measured SNPs for both SIRT3 and UCP2, along with several clinical features (including invasive hemodynamic data) and outcomes. We found SIRT3 and UCP2 SNPs often both in the same patient in a homozygous or heterozygous manner, correlating positively with PAH severity and associated with the presence of type 2 diabetes and 10-year outcomes (death and transplantation). To explore this mechanistically, we generated double knockout mice for Sirt3 and Ucp2 and found increasing severity of PAH (mean pulmonary artery pressure, right ventricular hypertrophy/dilatation and extensive vascular remodeling, including inflammatory plexogenic lesions, in a gene dose-dependent manner), along with insulin resistance, compared with wild-type mice. The suppressed mitochondrial function (decreased respiration, increased mitochondrial membrane potential) in the double knockout pulmonary artery smooth muscle cells was associated with apoptosis resistance and increased proliferation, compared with wild-type mice. Conclusions Our work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PAH (without environmental or chemical triggers) that mimics human PAH and may explain the findings in our patient cohort. Our study offers a new mouse model of PAH, with several features of human disease that are typically absent in other PAH mouse models.

Myocardial Functional Decline During Prolonged Ex Situ Heart Perfusion.

BACKGROUND: Myocardial function declines in a time-dependent fashion during ex situ heart perfusion. Cell death and metabolic alterations may contribute to this phenomenon, limiting the safe perfusion period and the potential of ex situ heart perfusion to expand the donor pool. Our aim was to investigate the etiology of myocardial functional decline in ex situ perfused hearts. METHODS: Cardiac function, apoptosis, effectors and markers of cell death, and metabolic function were assessed in healthy pig hearts perfused for 12 hours. These hearts were perfused in nonworking mode or working mode. RESULTS: Cardiac function declined during ex situ heart perfusion regardless of perfusion mode but was significantly better preserved in the hearts perfused in working mode (11-hour cardiac index/1-hour cardiac index: working mode, 33%; nonworking mode, 10%; p = 0.025). The rate of apoptosis was higher in the ex situ perfused hearts compared with in vivo samples (apoptotic cells: in vivo, 0.13%; working mode, 0.54%; nonworking mode, 0.88%; p < 0.001), but the absolute values were low and out of proportion to the decline in function in either group. Myocardial dysfunction at the end of the perfusion interval was partially rescued by delivery of a pyruvate bolus. CONCLUSIONS: A significant decline in myocardial function occurs over time in hearts preserved ex situ that is out of proportion to the magnitude of myocyte cell death present in dysfunctional hearts. Alterations in myocardial substrate utilization during prolonged ex situ heart perfusion may contribute to this phenomenon and represent an avenue to improve donor heart preservation.

A Leukocyte Filter Does Not Provide Further Benefit During Ex Vivo Lung Perfusion.

Normothermic ex vivo lung perfusion (EVLP) allows for assessment and reconditioning of donor lungs. Although a leukocyte filter (LF) is routinely incorporated into the EVLP circuit; its efficacy remains to be determined. Twelve pig lungs were perfused and ventilated ex vivo in a normothermic state for 12 hours. Lungs (n = 3) were allocated to four groups according to perfusate composition and the presence or absence of a LF in the circuit (acellular ± LF, cellular ± LF). Acceptable physiologic lung parameters were achieved during EVLP; however, increased amounts of pro-inflammatory cytokines (TNF-α and IL-6) and leukocytes in the perfusate were observed despite the presence or absence of a LF. Analysis of cells washed off the LF demonstrates that it trapped leukocytes although being ineffective throughout perfusion as it became saturated over 12 hours of EVLP. We conclude that there is no objective evidence to support the routine incorporation of a LF during EVLP as it does not provide further benefit and its removal does not appear to cause harm. The lack of hypothesized benefit to a LF may be because of the saturation of the LF with donor leukocytes, leading to similar amounts of circulating leukocytes still present in the perfusate with and without a LF.

Effect of fatty acids on human bone marrow mesenchymal stem cell energy metabolism and survival.

Successful stem cell therapy requires the optimal proliferation, engraftment, and differentiation of stem cells into the desired cell lineage of tissues. However, stem cell therapy clinical trials to date have had limited success, suggesting that a better understanding of stem cell biology is needed. This includes a better understanding of stem cell energy metabolism because of the importance of energy metabolism in stem cell proliferation and differentiation. We report here the first direct evidence that human bone marrow mesenchymal stem cell (BMMSC) energy metabolism is highly glycolytic with low rates of mitochondrial oxidative metabolism. The contribution of glycolysis to ATP production is greater than 97% in undifferentiated BMMSCs, while glucose and fatty acid oxidation combined only contribute 3% of ATP production. We also assessed the effect of physiological levels of fatty acids on human BMMSC survival and energy metabolism. We found that the saturated fatty acid palmitate induces BMMSC apoptosis and decreases proliferation, an effect prevented by the unsaturated fatty acid oleate. Interestingly, chronic exposure of human BMMSCs to physiological levels of palmitate (for 24 hr) reduces palmitate oxidation rates. This decrease in palmitate oxidation is prevented by chronic exposure of the BMMSCs to oleate. These results suggest that reducing saturated fatty acid oxidation can decrease human BMMSC proliferation and cause cell death. These results also suggest that saturated fatty acids may be involved in the long-term impairment of BMMSC survival in vivo.

A miR-208-Mef2 axis drives the decompensation of right ventricular function in pulmonary hypertension.

RATIONALE: Right ventricular (RV) failure is a major cause of morbidity and mortality in pulmonary hypertension, but its mechanism remains unknown. Myocyte enhancer factor 2 (Mef2) has been implicated in RV development, regulating metabolic, contractile, and angiogenic genes. Moreover, Mef2 regulates microRNAs that have emerged as important determinants of cardiac development and disease, but for which the role in RV is still unclear. OBJECTIVE: We hypothesized a critical role of a Mef2-microRNAs axis in RV failure. METHODS AND RESULTS: In a rat pulmonary hypertension model (monocrotaline), we studied RV free wall tissues from rats with normal, compensated, and decompensated RV hypertrophy, carefully defined based on clinically relevant parameters, including RV systolic and end-diastolic pressures, cardiac output, RV size, and morbidity. Mef2c expression was sharply increased in compensating phase of RVH tissues but was lost in decompensation phase of RVH. An unbiased screening of microRNAs in our model resulted to a short microRNA signature of decompensated RV failure, which included the myocardium-specific miR-208, which was progressively downregulated as RV failure progressed, in contrast to what is described in left ventricular failure. With mechanistic in vitro experiments using neonatal and adult RV cardiomyocytes, we showed that miR-208 inhibition, as well as tumor necrosis factor-α, activates the complex mediator of transcription 13/nuclear receptor corepressor 1 axis, which in turn promotes Mef2 inhibition, closing a self-limiting feedback loop, driving the transition from compensating phase of RVH toward decompensation phase of RVH. In our model, serum tumor necrosis factor-α levels progressively increased with time while serum miR-208 levels decreased, mirroring its levels in RV myocardium. CONCLUSIONS: We describe an RV-specific mechanism for heart failure, which could potentially lead to new biomarkers and therapeutic targets.

Sirtuin 3 deficiency is associated with inhibited mitochondrial function and pulmonary arterial hypertension in rodents and humans.

Suppression of mitochondrial function promoting proliferation and apoptosis suppression has been described in the pulmonary arteries and extrapulmonary tissues in pulmonary arterial hypertension (PAH), but the cause of this metabolic remodeling is unknown. Mice lacking sirtuin 3 (SIRT3), a mitochondrial deacetylase, have increased acetylation and inhibition of many mitochondrial enzymes and complexes, suppressing mitochondrial function. Sirt3KO mice develop spontaneous PAH, exhibiting previously described molecular features of PAH pulmonary artery smooth muscle cells (PASMC). In human PAH PASMC and rats with PAH, SIRT3 is downregulated, and its normalization with adenovirus gene therapy reverses the disease phenotype. A loss-of-function SIRT3 polymorphism, linked to metabolic syndrome, is associated with PAH in an unbiased cohort of 162 patients and controls. If confirmed in large patient cohorts, these findings may facilitate biomarker and therapeutic discovery programs in PAH.

A nuclear pyruvate dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation.

DNA transcription, replication, and repair are regulated by histone acetylation, a process that requires the generation of acetyl-coenzyme A (CoA). Here, we show that all the subunits of the mitochondrial pyruvate dehydrogenase complex (PDC) are also present and functional in the nucleus of mammalian cells. We found that knockdown of nuclear PDC in isolated functional nuclei decreased the de novo synthesis of acetyl-CoA and acetylation of core histones. Nuclear PDC levels increased in a cell-cycle-dependent manner and in response to serum, epidermal growth factor, or mitochondrial stress; this was accompanied by a corresponding decrease in mitochondrial PDC levels, suggesting a translocation from the mitochondria to the nucleus. Inhibition of nuclear PDC decreased acetylation of specific lysine residues on histones important for G1-S phase progression and expression of S phase markers. Dynamic translocation of mitochondrial PDC to the nucleus provides a pathway for nuclear acetyl-CoA synthesis required for histone acetylation and epigenetic regulation.

Targeting cell motility in pulmonary arterial hypertension.

Pulmonary artery smooth muscle cells (PASMC), in pulmonary arterial hypertension (PAH), contribute to obliterative vascular remodelling and are characterised by enhanced proliferation, suppressed apoptosis and, a much less studied, increased migration potential. One of the major proteins that regulate cell migration is focal adhesion kinase (FAK), but its role in PAH is not fully understood. We hypothesised that targeting cell migration by FAK inhibition may be a new therapeutic strategy in PAH. In vivo, inhalation of FAK-siRNA (n=5) or oral delivery of PF-228 (FAK inhibitor PF-573 228; n=5) inhibited rat monocrotaline induced PAH, improving the haemodynamics, vascular remodelling (media thickness), and right ventricular hypertrophy. In vitro, FAK was activated in PAH human lungs (n=8) or PASMC when compared to those form healthy subjects (Western blot, n=5), in a Src-dependent manner, as it was reversed by the specific Src inhibitor PP2. The degree of FAK phosphorylation at Y576 correlated positively with pulmonary vascular resistance in PAH patients. FAK inhibition (siRNA, PF-228 and PP2) in PAH-PASMCs induced a fivefold increase in apoptosis (percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling), a 2.5-fold decrease in proliferation (%Ki67), an 18% decrease in cell migration (colorimetric assay) and a 50% decrease in cell invasion (wound healing). Suppressing PASMC migration by FAK inhibition inhibits PAH progression and may open a new therapeutic window in PAH.

A metabolic remodeling in right ventricular hypertrophy is associated with decreased angiogenesis and a transition from a compensated to a decompensated state in pulmonary hypertension.

UNLABELLED: Right ventricular (RV) failure is an important clinical problem with no available therapies, largely because its molecular mechanisms are unknown. Mitochondrial remodeling resulting to a metabolic shift toward glycolysis has been described in RV hypertrophy (RVH), but it is unknown whether this is beneficial or detrimental. While clinically RV failure follows a period of compensation, the transition from a compensated (cRVH) to a decompensated hypertrophied RV (dRVH) is not studied in animal models. We modeled the natural history of RVH and failure in the monocrotaline rat model of pulmonary hypertension by serially assessing clinically relevant parameters in the same animal. We defined dRVH as the stage in which RV systolic pressure started decreasing, along with the cardiac output, while the RV continued to remodel. dRVH was characterized by ascites, weight loss, and high mortality, compared to cRVH. A cRVH myocardium had hyperpolarized mitochondria and low production of mitochondria-derived reactive oxygen species (mROS), activated hypoxia-inducible factor 1α (HIF1α), and increased levels of glucose transporter 1, vascular endothelial growth factor, and stromal-derived factor 1, promoting increased glucose uptake (measured by positron emission tomography-computed tomography) and angiogenesis measured by lectin imaging in vivo. The transition to dRVH was marked by a sharp rise in mROS, inhibition of HIF1α, and activation of p53, both of which contributed to down-regulation of pyruvate dehydrogenase kinase and decreased glucose uptake. This transition was also associated with a sharp decrease in angiogenic factors and angiogenesis. We show that the previously described metabolic shift, promoting HIF1α activation and angiogenesis, is not sustained during the progression of RV failure. The loss of this beneficial remodeling may be triggered by a rise in mROS resulting in HIF1α inhibition and suppressed angiogenesis. The resultant ischemia may contribute to the rapid deterioration of RV function upon entrance to a decompensation phase. The use of clinical criteria and techniques to define and study dRVH facilitates clinical translation of our findings with direct implications for RV therapeutic and biomarker discovery programs. KEY MESSAGE: Decreased RV angiogenesis marks the transition from a cRVH to a dRVH. The RVs in cRVH animals are associated with decreased mROS and increased HIF1α activity compared to dRVH. The RVs in cRVH animals have increased GLUT1 levels and increased glucose uptake compared to the dRVH.

Attenuating endoplasmic reticulum stress as a novel therapeutic strategy in pulmonary hypertension.

BACKGROUND: Evidence suggestive of endoplasmic reticulum (ER) stress in the pulmonary arteries of patients with pulmonary arterial hypertension has been described for decades but has never been therapeutically targeted. ER stress is a feature of many conditions associated with pulmonary arterial hypertension like hypoxia, inflammation, or loss-of-function mutations. ER stress signaling in the pulmonary circulation involves the activation of activating transcription factor 6, which, via induction of the reticulin protein Nogo, can lead to the disruption of the functional ER-mitochondria unit and the increasingly recognized cancer-like metabolic shift in pulmonary arterial hypertension that promotes proliferation and apoptosis resistance in the pulmonary artery wall. We hypothesized that chemical chaperones known to suppress ER stress signaling, like 4-phenylbutyrate (PBA) or tauroursodeoxycholic acid, will inhibit the disruption of the ER-mitochondrial unit and prevent/reverse pulmonary arterial hypertension. METHODS AND RESULTS: PBA in the drinking water both prevented and reversed chronic hypoxia-induced pulmonary hypertension in mice, decreasing pulmonary vascular resistance, pulmonary artery remodeling, and right ventricular hypertrophy and improving functional capacity without affecting systemic hemodynamics. These results were replicated in the monocrotaline rat model. PBA and tauroursodeoxycholic acid improved ER stress indexes in vivo and in vitro, decreased activating transcription factor 6 activation (cleavage, nuclear localization, luciferase, and downstream target expression), and inhibited the hypoxia-induced decrease in mitochondrial calcium and mitochondrial function. In addition, these chemical chaperones suppressed proliferation and induced apoptosis in pulmonary artery smooth muscle cells in vitro and in vivo. CONCLUSIONS: Attenuating ER stress with clinically used chemical chaperones may be a novel therapeutic strategy in pulmonary hypertension with high translational potential.

Phosphatidylethanolamine deficiency in Mammalian mitochondria impairs oxidative phosphorylation and alters mitochondrial morphology.

Mitochondrial dysfunction is implicated in neurodegenerative, cardiovascular, and metabolic disorders, but the role of phospholipids, particularly the nonbilayer-forming lipid phosphatidylethanolamine (PE), in mitochondrial function is poorly understood. Elimination of mitochondrial PE (mtPE) synthesis via phosphatidylserine decarboxylase in mice profoundly alters mitochondrial morphology and is embryonic lethal (Steenbergen, R., Nanowski, T. S., Beigneux, A., Kulinski, A., Young, S. G., and Vance, J. E. (2005) J. Biol. Chem. 280, 40032-40040). We now report that moderate <30% depletion of mtPE alters mitochondrial morphology and function and impairs cell growth. Acute reduction of mtPE by RNAi silencing of phosphatidylserine decarboxylase and chronic reduction of mtPE in PSB-2 cells that have only 5% of normal phosphatidylserine synthesis decreased respiratory capacity, ATP production, and activities of electron transport chain complexes (C) I and CIV but not CV. Blue native-PAGE analysis revealed defects in the organization of CI and CIV into supercomplexes in PE-deficient mitochondria, correlated with reduced amounts of CI and CIV proteins. Thus, mtPE deficiency impairs formation and/or membrane integration of respiratory supercomplexes. Despite normal or increased levels of mitochondrial fusion proteins in mtPE-deficient cells, and no reduction in mitochondrial membrane potential, mitochondria were extensively fragmented, and mitochondrial ultrastructure was grossly aberrant. In general, chronic reduction of mtPE caused more pronounced mitochondrial defects than did acute mtPE depletion. The functional and morphological changes in PSB-2 cells were largely reversed by normalization of mtPE content by supplementation with lyso-PE, a mtPE precursor. These studies demonstrate that even a modest reduction of mtPE in mammalian cells profoundly alters mitochondrial functions.

Human internal mammary artery (IMA) transplantation and stenting: a human model to study the development of in-stent restenosis.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches (1,2). The commonly used animal models include mice, rats, rabbits, and pigs (3-5). However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models (6,7). In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents. This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Pyruvate dehydrogenase inhibition by the inflammatory cytokine TNFα contributes to the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMC). This apoptosis resistance is characterized by PASMC mitochondrial hyperpolarization [in part, due to decreased pyruvate dehydrogenase (PDH) activity], decreased mitochondrial reactive oxygen species (mROS), downregulation of Kv1.5, increased [Ca(++)](i), and activation of the transcription factor nuclear factor of activated T cells (NFAT). Inflammatory cells are present within and around the remodeled arteries and patients with PAH have elevated levels of inflammatory cytokines, including tumor necrosis factor-α (TNFα). We hypothesized that the inflammatory cytokine TNFα inhibits PASMC PDH activity, inducing a PAH phenotype in normal PASMC. We exposed normal human PASMC to recombinant human TNFα and measured PDH activity. In TNFα-treated cells, PDH activity was significantly decreased. Similar to exogenous TNFα, endogenous TNFα secreted from activated human CD8(+) T cells, but not quiescent T cells, caused mitochondrial hyperpolarization, decreased mROS, decreased K(+) current, increased [Ca(++)](i), and activated NFAT in normal human PASMC. A TNFα antibody completely prevented, while recombinant TNFα mimicked the T cell-induced effects. In vivo, the TNFα antagonist etanercept prevented and reversed monocrotaline (MCT)-induced PAH. In a separate model, T cell deficient rats developed less severe MCT-induced PAH compared to their controls. We show that TNFα can inhibit PASMC PDH activity and induce a PAH phenotype. Our work supports the use of anti-inflammatory therapies for PAH.

The role of Nogo and the mitochondria-endoplasmic reticulum unit in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is caused by excessive proliferation of vascular cells, which occlude the lumen of pulmonary arteries (PAs) and lead to right ventricular failure. The cause of the vascular remodeling in PAH remains unknown, and the prognosis of PAH remains poor. Abnormal mitochondria in PAH PA smooth muscle cells (SMCs) suppress mitochondria-dependent apoptosis and contribute to the vascular remodeling. We hypothesized that early endoplasmic reticulum (ER) stress, which is associated with clinical triggers of PAH including hypoxia, bone morphogenetic protein receptor II mutations, and HIV/herpes simplex virus infections, explains the mitochondrial abnormalities and has a causal role in PAH. We showed in SMCs from mice that Nogo-B, a regulator of ER structure, was induced by hypoxia in SMCs of the PAs but not the systemic vasculature through activation of the ER stress-sensitive transcription factor ATF6. Nogo-B induction increased the distance between the ER and mitochondria and decreased ER-to-mitochondria phospholipid transfer and intramitochondrial calcium. In addition, we noted inhibition of calcium-sensitive mitochondrial enzymes, increased mitochondrial membrane potential, decreased mitochondrial reactive oxygen species, and decreased mitochondria-dependent apoptosis. Lack of Nogo-B in PASMCs from Nogo-A/B-/- mice prevented these hypoxia-induced changes in vitro and in vivo, resulting in complete resistance to PAH. Nogo-B in the serum and PAs of PAH patients was also increased. Therefore, triggers of PAH may induce Nogo-B, which disrupts the ER-mitochondria unit and suppresses apoptosis. This could rescue PASMCs from death during ER stress but enable the development of PAH through overproliferation. The disruption of the ER-mitochondria unit may be relevant to other diseases in which Nogo is implicated, such as cancer or neurodegeneration.

Fatty acid oxidation and malonyl-CoA decarboxylase in the vascular remodeling of pulmonary hypertension.

Pulmonary arterial hypertension is caused by excessive growth of vascular cells that eventually obliterate the pulmonary arterial lumen, causing right ventricular failure and premature death. Despite some available treatments, its prognosis remains poor, and the cause of the vascular remodeling remains unknown. The vascular smooth muscle cells that proliferate during pulmonary arterial hypertension are characterized by mitochondrial hyperpolarization, activation of the transcription factor NFAT (nuclear factor of activated T cells), and down-regulation of the voltage-gated potassium channel Kv1.5, all of which suppress apoptosis. We found that mice lacking the gene for the metabolic enzyme malonyl-coenzyme A (CoA) decarboxylase (MCD) do not show pulmonary vasoconstriction during exposure to acute hypoxia and do not develop pulmonary arterial hypertension during chronic hypoxia but have an otherwise normal phenotype. The lack of MCD results in an inhibition of fatty acid oxidation, which in turn promotes glucose oxidation and prevents the shift in metabolism toward glycolysis in the vascular media, which drives the development of pulmonary arterial hypertension in wild-type mice. Clinically used metabolic modulators that mimic the lack of MCD and its metabolic effects normalize the mitochondrial-NFAT-Kv1.5 defects and the resistance to apoptosis in the proliferated smooth muscle cells, reversing the pulmonary hypertension induced by hypoxia or monocrotaline in mice and rats, respectively. This study of fatty acid oxidation and MCD identifies a critical role for metabolism in both the normal pulmonary circulation (hypoxic pulmonary vasoconstriction) and pulmonary hypertension, pointing to several potential therapeutic targets for the treatment of this deadly disease.