A central role for hsa-miR-138-5p and the Mitochondrial Calcium Uniporter (MCU) in human and experimental PAH
Dr. Stephen Archer, Queen’s University
Mark Ormiston, Queen’s University
Pulmonary arterial hypertension (PAH) is an obstructive, arterial vasculopathy in which disorders of endothelial cells, smooth muscle cells (PASMC), fibroblasts and inflammatory cells cause loss of vascular lumen, vascular stiffening and vasoconstriction1. This adverse vascular remodelling increases right ventricular afterload, ultimately causing death from right ventricular failure. Although vasoconstriction is a major pathophysiologic feature in only ~5% of patients, the approved PAH-targeted therapeutics are primarily vasodilators. Consequently, prognosis remains poor with survival rates of ~50% at 5-years2. This suggests the need to better understand and therapeutically target non-vasospastic pathways, such as PAH’s cancer-like phenotype, which includes excess proliferation and apoptosis-resistance. Underlying this phenotype are acquired ionic abnormalities (notably increases in intracellular calcium3) and mitochondrial abnormalities, including changes in mitochondrial metabolism1,4 and dynamics5,6. In PAH PASMC7,8 and endothelial cells9, glucose metabolism is altered so that glycolysis is uncoupled from glucose oxidation. This metabolic shift, first observed in cancer cells by Warburg10, suppresses apoptosis while increasing proliferation1,4. The mitochondrial network in PAH is also fragmented due to increased fission, caused by posttranslational activation of dynamin related protein 1 (Drp1)5, and impaired fusion, caused by downregulation of mitofusin-211.
Although the regulation of intracellular calcium and mitochondrial bioenergetics are closely linked, the molecular mediators of inflow12,13 and egress14,15 of mitochondrial calcium have only recently been identified. Calcium enters the mitochondria via the mitochondrial calcium uniporter complex (MCUC), an inwardly rectifying, Ca2+-selective, ion channel in the inner mitochondrial membrane16. The MCUC’s high Ca2+- selectivity at the low [Ca2+]cyto16 allows mitochondrial uptake and release of calcium to dynamically buffer[Ca2+]cyto17. The MCUC has 5 components: mitochondrial calcium uptake protein 1 (MICU1)18, which inhibits the MCUC, the 40kD protein pore-forming MCU subunit12,19, and other regulatory subunits (MICU2, MCUb and the essential MCU regulator, EMRE)20,21. Knockdown of MCU reduces mitochondrial matrix calcium in vitro12 and causes reduced PDH activity in the skeletal muscle of MCU-/- mice22. Relevant to PAH, overexpression of MCU increases [Ca2+]mito and sensitizes cells to apoptotic stimuli12.
In this project CVN members at Queen’s University will collaborate with investigators at Laval University to assess the role that decreased MCUC function plays in the etiology of PAH. We will assess whether decreased expression of MCU and increased expression of MICU1 underlie the key ionic and mitochondrial-metabolic phenotypic features of PAH. Our preliminary evidence suggests that expression of the MCU subunit may be post-transcriptionally downregulated by several microRNAs (miR), notably miR-138-5P and miR-25-3p. These miRs may also inhibit CREB1, a transcriptional regulator of MCU. The proposed research will capitalize on the extensive library of human control and PAH cells and tissue in the laboratories of Drs. Bonnet and Provencher (Laval). In vitro studies will assess the ability of chemical or molecular inhibition of MCUC to recapitulate the PAH phenotype in normal PASMC, as well as the therapeutic effects of restoring MCU expression in human PAH cells. Finally, we will assess the therapeutic role of this pathway in vivo, using nebulized anti-miRs in an attempt to reverse severe, established monocrotaline-induced PAH.
Note: Contact the CVN directly for all reference information.
Novel Biomarkers of Small Vessel Disease and Cognitive Impairment in End-Stage Renal Disease
Dr. Jason Fish, Toronto General Research Institute
Mansoor Husain, Toronto General Research Institute
Rulan Parekh, Hospital for Sick Children
Carmela Tartaglia, Toronto Western Hospital
Clifford Librach, University of Toronto
A common complication of end-stage renal disease (ESRD) is the accelerated development of cognitive impairment and dementia. The molecular mechanisms responsible are poorly understood. Unfortunately there are no effective treatments for cognitive decline in these patients. In addition, no prognostic tests are available to identify patients that will develop cognitive deficits. Microvascular dysfunction in ESRD patients may contribute to vascular cognitive impairment (VCI). ESRD patients have elevated vascular inflammation and calcification, and cardiovascular (CV) and cerebrovascular pathologies are common in these patients. We will seek to identify functional biomarkers of VCI in ESRD patients by utilizing a large clinical cohort from the Predictors of Arrhythmic and Cardiovascular Risk in ESRD (PACE) study. This is a completed prospective study with a rich repository of self-reported, clinical, CV, cognitive and dialysis-related data as well as a biobank for biomarker assessment in 571 incident dialysis patients (< 6 months on regular outpatient dialysis) recruited in Baltimore and surrounding area, with a comprehensive collection of CV imaging and clinical specimens at baseline (n=400) with repeated measures over 4 years (n=218). This cohort includes primarily younger adults with less confounding of age-related disease and comorbidities.
Our recent work has identified a crucial role for circulating microRNAs (miRs) in regulating vascular inflammation (PMID: 25838349). We found that healthy endothelial cells secrete extracellular vesicles (EVs) that contain anti-inflammatory miRs that can be transferred to monocytes to suppress their activation.
Interestingly, in a diabetic mouse model of VCI we found that circulating EVs have altered miR contents and can induce inflammatory/calcification pathways in recipient endothelial cells. This suggests that EVs and the miRs that they contain may contribute to vascular inflammation/calcification and may therefore be implicated in VCI pathogenesis. We will identify circulating miR and calcification biomarkers associated with microvascular dysfunction, vascular calcification and the development of VCI. We will also determine whether an increase in brain-derived EVs in the blood is indicative of VCI brain pathology. These studies will aid in the early diagnosis and prognosis of disease and these biomarkers may be used to monitor the effectiveness of therapeutic interventions. Our studies will also assess miR function in inflammation and calcification, which may uncover novel therapeutic targets.
Our project will combine the expertise of the Fish lab in vascular inflammation, extracellular vesicle and microRNA biology, the expertise of the Husain lab in cardiovascular pathology, together with the expertise of the Parekh lab in clinical epidemiology and biomarker analysis in ESRD. Our studies will address the devastating impact of VCI in a high-risk population. This unmet clinical need has been identified as a priority area of the Canadian Vascular Network. We are poised to make progress in this research area.
Cognitive Impairment Following Pre-eclampsia: Of Mice and Women
Dr. Louise Pilote, McGill University
Marilyn Cipolla, University of Vermont
We will use existing databases from a large cohort study, Coronary Artery Risk Development in Young Adults (CARDIA), to determine the impact of pre-eclampsia on cognitive function in women. These data will be acquired through the National Heart, Lung, and Blood Institute Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC). In leveraging this database, we can begin to explore the relationship between vascular complications in pregnancy and how these may impact cognitive function later in life which can eventually lead to more focused intervention studies in women. In collaboration with Dr. Hamel, and with expertise from Dr. Cipolla (University of Vermont), an animal study will be simultaneously undertaken to test whether a history of pre-eclampsia in female mice is associated with cognitive decline post-partum, in middle and in old age, and exploration of possible cerebrovascular and white matter disease biomarkers will be initiated.
Mesenchymal stromal cell exosomes as a therapy for microvascular hyperpermeability in sepsis
Dr. Manoj Lalu, Ottawa Hospital Research Institute
Duncan Stewart, Ottawa Hospital Research Institute
Septic shock is a devastating condition with a mortality rate of 30-40% despite modern therapy and care. It is now recognized that sepsis is vasculopathic disease of the endothelium – the unchecked inflammatory response to an infection results in endothelial microvascular injury and reversible/irreversible injury to the microcirculation. This injury contributes to hyperpermeability of small vessels, organ dysfunction, and death. Organs that are particularly affected are the brain (cognitive dysfunction), kidney, and lungs.
We believe exosomes (microvesicles) from mesenchymal stromal cells (MSCs) may be vascular protective and ameliorate the negative effects of sepsis. Specifically, we hypothesize that MSC-derived exosomes (MEX) will decrease small vessel damage/leak (as assessed by microcomputed tomography), decrease organ dysfunction, and reduce death in an experimental model of septic shock. We will further investigate the effects of MEX using in vitro techniques, including the transendothelial electrical resistance assay.
We believe this research fits within the mandate of the Vascular Network. Sepsis represents an important and unique cardiovascular disease that primarily affects small vessels of many organs and is underrepresented within current Network research. Sepsis is well known to precipitate and aggravate vascular cognitive impairment (VCI). The importance of sepsis research has been recognized by the AHA as it is a disease that increasing numbers of vascular patients are affected by. Moreover, patients with preexisting vascular disease (e.g. chronic cognitive dysfunction) fair particularly poorly when affected by sepsis. Thus, there is a critical need for increased research in this area.
Microvascular Angina and Oxygenation Sensitive CMR
Dr. Todd Anderson, University of Calgary
James White, University of Calgary
The reduction of oxygen supplied to the heart due to the obstruction of coronary arteries is thought to be one of the leading causes of angina chest pain. However, a significant portion of patients (10-20%) who present with symptoms of angina also appear to have minimal coronary artery disease (CAD) (Kemp et al., 1986). The disease is far more prevalent in females, with up to 50% of women presenting with angina appearing to have non-obstructive CAD (Sharaf et al., 2001). Once termed “cardiac syndrome X”, this illness is now referred to as microvascular angina (MA), endothelial dysfunction or chest pain with non-obstructive CAD depending on the phenotype of the patient. The pathogenesis of MA, as the name suggests, is rooted in the dysfunction of the coronary microvasculature (CMV). With over 80% of total vascular resistance attributed to the CMV, any dysfunction of these small vessels has the potential to impact myocardial perfusion. Due to the nature of MA as a non-obstructive cardiac disease, traditional means of diagnosing coronary dysfunction are largely ineffective (Kuruvilla and Kramer, 2014). Compounding matters is the small size of the coronary microvasculature, which makes direct imaging impossible (Mumma and Flacke, 2016).
Cardiac magnetic resonance imaging (CMR) is a promising, newer means of CMV assessment. CMR is one of the few imaging techniques which has sufficient resolution to visualize subendocardial ischemia (Kwong et al., 2003); as such, CMR has been used to characterize the nature of MA cases as ischemic or non-ischemic (Panting et al., 2002; Vermeltfoort et al., 2007), informing the treatment approach (Cannon, 2009) and providing a means of evaluating treatment efficacy (Mehta et al., 2011). CMR has also been used as a non-invasive alternative to coronary reactivity testing in women with CMV dysfunction (Thomson et al., 2015) and is frequently employed as a means of examining CMV function as evidenced by myocardial perfusion (Loffler and Bourque, 2016; Shufelt et al., 2013; Park et al., 2014; Lanza et al., 2008). Although vasoactive substances such as adenosine or dipyridamole are often used to induce hyperemia for CMR imaging, calculated changes in breathing pattern are capable of affecting coronary perfusion. Indeed, oxygenation sensitive CMR (OS-CMR) employing prolonged breath holds and hyperventilation, resulted in greater changes in myocardial oxygenation than through adenosine administration (Fischer et al., 2015), suggesting OS-CMR may be a useful surrogate marker of CMV function. Although perfusion CMR has been employed to assess general CMV dysfunction (Thompson et al., 2015; Shufelt et al., 2013) and as a diagnostic tool for cardiac syndrome X (Lanza et at., 2008; Vermeltfoort et al., 2007; Panting et al., 2002), OS-CMR has yet to be studied as a means of evaluating women who present with non-obstructive CAD and anginal chest pain. Given the less invasive nature of OS-CMR compared to perfusion imaging, research in this area could yield a valuable new technique for the assessment of microvascular angina.