NMDA-Type Glutamate Receptor Activation Promotes Vascular Remodeling and Pulmonary Arterial Hypertension.

BACKGROUND: Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N-methyl-d-aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown. METHODS: We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers. RESULTS: We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. Kv channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries. CONCLUSIONS: These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.

Concise Review: Pluripotent Stem Cell-Derived Cardiac Cells, A Promising Cell Source for Therapy of Heart Failure: Where Do We Stand?

Heart failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential proarrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials.

Repression of CMIP transcription by WT1 is relevant to podocyte health.

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.

G protein-coupled receptor kinase 2 and group I metabotropic glutamate receptors mediate inflammation-induced sensitization to excitotoxic neurodegeneration.

OBJECTIVE: The concept of inflammation-induced sensitization is emerging in the field of perinatal brain injury, stroke, Alzheimer disease, and multiple sclerosis. However, mechanisms underpinning this process remain unidentified. METHODS: We combined in vivo systemic lipopolysaccharide-induced or interleukin (IL)-1ß-induced sensitization of neonatal and adult rodent cortical neurons to excitotoxic neurodegeneration with in vitro IL-1ß sensitization of human and rodent neurons to excitotoxic neurodegeneration. Within these inflammation-induced sensitization models, we assessed metabotropic glutamate receptors (mGluR) signaling and regulation. RESULTS: We demonstrate for the first time that group I mGluRs mediate inflammation-induced sensitization to neuronal excitotoxicity in neonatal and adult neurons across species. Inflammation-induced G protein-coupled receptor kinase 2 (GRK2) downregulation and genetic deletion of GRK2 mimicked the sensitizing effect of inflammation on excitotoxic neurodegeneration. Thus, we identify GRK2 as a potential molecular link between inflammation and mGluR-mediated sensitization. INTERPRETATION: Collectively, our findings indicate that inflammation-induced sensitization is universal across species and ages and that group I mGluRs and GRK2 represent new avenues for neuroprotection in perinatal and adult neurological disorders.

Upregulation of c-mip is closely related to podocyte dysfunction in membranous nephropathy.

Membranous nephropathy is a glomerular disease typified by a nephrotic syndrome without infiltration of inflammatory cells or proliferation of resident cells. Although the cause of the disease is unknown, the primary pathology involves the generation of autoantibodies against antigen targets on the surface of podocytes. The mechanisms of nephrotic proteinuria, which reflect a profound podocyte dysfunction, remain unclear. We previously found a new gene, c-mip (c-maf-inducing protein), that was associated with the pathophysiology of idiopathic nephrotic syndrome. Here we found that c-mip was not detected in the glomeruli of rats with passive-type Heymann nephritis given a single dose of anti-megalin polyclonal antibody, yet immune complexes were readily present, but without triggering of proteinuria. Rats reinjected with anti-megalin develop heavy proteinuria a few days later, concomitant with c-mip overproduction in podocytes. This overexpression was associated with the downregulation of synaptopodin in patients with membranous nephropathy, rats with passive Heymann nephritis, and c-mip transgenic mice, while the abundance of death-associated protein kinase and integrin-linked kinase was increased. Cyclosporine treatment significantly reduced proteinuria in rats with passive Heymann nephritis, concomitant with downregulation of c-mip in podocytes. Thus, c-mip has an active role in the podocyte disorders of membranous nephropathy.

Activation of microglial N-methyl-D-aspartate receptors triggers inflammation and neuronal cell death in the developing and mature brain.

OBJECTIVE: Activated microglia play a central role in the inflammatory and excitotoxic component of various acute and chronic neurological disorders. However, the mechanisms leading to their activation in the latter context are poorly understood, particularly the involvement of N-methyl-D-aspartate receptors (NMDARs), which are critical for excitotoxicity in neurons. We hypothesized that microglia express functional NMDARs and that their activation would trigger neuronal cell death in the brain by modulating inflammation. METHODS AND RESULTS: We demonstrate that microglia express NMDARs in the murine and human central nervous system and that these receptors are functional in vitro. We show that NMDAR stimulation triggers microglia activation in vitro and secretion of factors that induce cell death of cortical neurons. These damaged neurons are further shown to activate microglial NMDARs and trigger a release of neurotoxic factors from microglia in vitro, indicating that microglia can signal back to neurons and possibly induce, aggravate, and/or maintain neurologic disease. Neuronal cell death was significantly reduced through pharmacological inhibition or genetically induced loss of function of the microglial NMDARs. We generated Nr1 LoxP(+/+) LysM Cre(+/-) mice lacking the NMDAR subunit NR1 in cells of the myeloid lineage. In this model, we further demonstrate that a loss of function of the essential NMDAR subunit NR1 protects from excitotoxic neuronal cell death in vivo and from traumatic brain injury. INTERPRETATION: Our findings link inflammation and excitotoxicity in a potential vicious circle and indicate that an activation of the microglial NMDARs plays a pivotal role in neuronal cell death in the perinatal and adult brain.

Differential effects of cyclosporin A and tacrolimus on magnesium influx in Caco2 cells.

PURPOSE: Hypomagnesemia with urinary magnesium (Mg) wasting is a well acknowledged side effect of cyclosporin A (CsA) and tacrolimus (FK506) treatments. TRPM6, TRPM7 and MagT1 are involved in the active transcellular Mg transport processes in intestine and kidney. Since Mg homeostasis is tightly controlled by the dynamic action of intestinal absorption of dietary Mg and renal excretion of Mg, we investigated whether CsA and FK506 in commercially available solutions for clinical use decrease the expression and the function of TRPM6, TRPM7 or MagT1 in the intestinal epithelial cell line Caco2. METHODS: Changes of intracellular free Mg concentrations were measured by Mag-fura-2 imaging in Mg-free medium after the addition of 1 mM MgCl2. TRPM6, TRPM7 and MagT1 were evidenced in cells by immunofluorescence. Proteins and mRNAs were quantified after 18 hours of treatment with CsA or FK506 by western-blot and real-time RT-PCR analyses, respectively. RESULTS: TRPM6 and MagT1 were evidenced on all cell membranes, TRPM7 only on the inner membranes. CsA was responsible for a profound decrease in Mg2+ influx in intestinal epithelial cells, which may result in a decrease of intestinal Mg absorption, whereas FK506 was responsible for a marked increase in Mg2+ influx. Neither CsA nor FK506 altered TRPM6, TRPM7 or MagT1 mRNA levels or MagT1 protein level. CONCLUSIONS: In Caco2 cells, Mg2+ influx was inhibited by CsA solutions whereas enhanced by FK506 solutions, without alteration of MagT1, TRPM6 and TRPM7 expression, leading to the conclusion that CsA and FK506 have opposite effects in the functional activity of the Mg transporters herein examined. In clinical use, FK506 should be preferred for patients at risk for hypomagnesemia.

Regulation of T-type Cav3.1 channels expression by synthetic glucocorticoid dexamethasone in neonatal cardiac myocytes.

The effect of the dexamethasone (Dex) on the regulation of the T-type Ca(2+) channel expressions was investigated in primary cultures of neonatal rat ventricular myocytes. We found that Dex (1 microM) increases the T-type Ca(2+) current (I(CaT)) associated with an increase in Ca(v)3.1 mRNA amount. We isolated the upstream region from Ca(v)3.1 encoding gene and tested the activity of the promoter in transfected ventricular myocytes. We found a minimal Dex-responsive region that displayed putative glucocorticoid receptor (GR) and nuclear factor kappa-B (NFkappaB) targets. The GR selective antagonist, RU38486 (10 microM), nearly turned off the transcriptional activity of Ca(v)3.1 encoding gene, and an NFkappaB inhibitor, pyrrolodine dithiocarbonate (10 microM), completely abolished the Dex-induced mRNA increase. However, Dex-induced GR and NFkappaB synthesis and nuclear translocation were not timely related to Ca(v)3.1 mRNA increase. These results indicate that both GR and NFkappaB were necessary, but not sufficient, to trigger the increase in Ca(v)3.1 mRNA amount. This study showed the relationship between glucocorticoid and T-type channels up-regulation that may be involved in cardiac development and pathology.

Standard and Strain Measurements by Echocardiography Detect Early Overloaded Right Ventricular Dysfunction: Validation against Hemodynamic and Myocyte Contractility Changes in a Large Animal Model.

BACKGROUND: Early detection of right ventricular (RV) failure is required to improve the management of patients with congenital heart diseases. The aim of this study was to validate echocardiography for the early detection of overloaded RV dysfunction, compared with hemodynamic and myocyte contractility assessment. METHODS: Using a porcine model reproducing repaired tetralogy of Fallot, RV function was evaluated over 4 months using standard echocardiography and speckle-tracking compared with hemodynamic parameters (conductance catheter). Sarcomere shortening and calcium transients were recorded in RV isolated myocytes. Contractile reserve (ΔEmax) was assessed by ß-adrenergic stimulation in vivo (dobutamine 5 µg/kg) and ex vivo (isoproterenol 100 nM). RESULTS: Six operated animals were compared with four age- and sex-matched controls. In the operated group, hemodynamic RV efficient ejection fraction was significantly decreased (29.7% [26.2%-34%] vs 42.9% [40.7%-48.6%], P < .01), and inotropic responses to dobutamine were attenuated (ΔEmax was 51% vs 193%, P < .05). Echocardiographic measurements of fraction of area change, tricuspid annular plane systolic excursion, tricuspid annular peak systolic velocity (S') and RV free wall longitudinal systolic strain and strain rate were significantly decreased. Strain rate, S', and tricuspid annular plane systolic excursion were correlated with ΔEmax (r = 0.75, r = 0.78, and r = 0.65, respectively, P < .05). These alterations were associated in RV isolated myocytes with the decrease of sarcomere shortening in response to isoproterenol and perturbations of calcium homeostasis assessed by the increase of spontaneous calcium waves. CONCLUSIONS: In this porcine model, both standard and strain echocardiographic parameters detected early impairments of RV function and cardiac reserve, which were associated with cardiomyocyte excitation-contraction coupling alterations.

Right ventricular failure secondary to chronic overload in congenital heart diseases: benefits of cell therapy using human embryonic stem cell-derived cardiac progenitors.

OBJECTIVE: Despite the increasing incidence of right ventricular (RV) failure in adult patients with congenital heart disease, current therapeutic options are still limited. By contrast to left-heart diseases, cell-based myocardial regeneration applied to the right ventricle is poorly studied, even though it may be a therapeutic solution. As human embryonic stem cell-derived cardiac progenitors seem to be good candidates owing to their proliferation capacity, our aim was to assess, in a large animal model of overloaded RV dysfunction, the feasibility and effects of such a cell therapy. METHODS: Human MesP1(+)/SSEA-1(+) cardiogenic mesodermal cells were administered using multiple intramyocardial injections 4 months after a surgical procedure mimicking the repaired tetralogy of Fallot, and their effects were observed 3 months later on hemodynamic, rhythmic, and histologic parameters. RESULTS: All pigs (sham n = 6, treated n = 6) survived without complication, and cell therapy was clinically well tolerated. Although functional, contractility, and energetics parameters evolved similarly in both groups, benefits regarding arrhythmic susceptibility were observed in the treated group, associated with a significant decrease of peri-myocyte fibrosis (5.71% ± 2.49% vs 12.12% ± 1.85%; P < .01) without interstitial fibrosis change (5.18% ± 0.81% vs 5.49% ± 1.01%). Such a decrease could be related to paracrine effects, as no human cells could be detected within the myocardium. CONCLUSIONS: Cell therapy using intramyocardial injections of human MesP1(+)/SSEA-1(+) cardiogenic mesodermal cells seems to have benefits regarding overloaded RV tissue remodeling and arrhythmic susceptibility, but this mode of administration is not sufficient to obtain a significant improvement in RV function.

Renal ischemia-reperfusion injury in the rat is prevented by a novel immune modulation therapy.

BACKGROUND: Vasogen Inc.'s (Mississauga, Ontario, Canada) immune modulation therapy (IMT) is a therapy in which cells from the patient's own blood are modified by ex vivo exposure to specific physicochemical stressors, including oxidation, ultraviolet (UV) light, and an elevated temperature. The therapy has been shown to have a beneficial effect in models of inflammation and vascular diseases. This study tested the hypothesis that IMT can prevent renal ischemia-reperfusion (I/R) injury in rats. METHODS: Whole blood was collected from syngeneic age-matched donors by cardiac puncture. It was treated with a combination of controlled physiochemical stressors consisting of elevated temperature, a gas mixture of medical oxygen containing ozone, and UV light. The treated blood (150 microL) was injected in the gluteal muscle. Control animals received the same volume of untreated blood or physiological saline. Transient (45 or 60 minutes) left-renal ischemia was produced with simultaneous contralateral nephrectomy in treated and control spontaneously hypertensive rats (SHR). Young and old male and female rats were studied. Plasma creatinine, diuresis, and the survival rates of each group were compared. Renal apoptosis-necrosis was estimated by DNA laddering, histology, and in situ terminal deoxynucleotidyl transferase assay. mRNA levels of several regulators of apoptosis-regeneration were determined in control and postischemic kidneys by Northern blotting. RESULTS: IMT pretreatment of SHR significantly reduced renal I/R injury compared with equivalent placebo treatments consisting of untreated blood- or saline-injected SHR, as evidenced by a significant increase of the survival rate curves in young and old male SHR, which correlated with 24-hour postischemic diuresis. The increases in plasma creatinine following renal I/R were significantly lower in IMT-treated young male and old female SHR compared with saline or untreated blood-injected controls. Dilution analysis showed that the protective effect of treated blood was lost by dilution. Loss of epithelial cells was reduced in IMT-treated rats, with a significant decline in the peak of apoptosis 12 hours after acute ischemic renal injury. IMT did not modify the pattern of mRNA levels of several genes involved in the inflammation and regeneration processes. CONCLUSION: Our data demonstrate that IMT prevents the destruction of kidney tissue and the resulting animal death caused by renal I/R injury.

Right ventricular failure secondary to chronic overload in congenital heart disease: an experimental model for therapeutic innovation.

OBJECTIVE: Mortality and morbidity related to right ventricular failure remain a problem for the long-term outcome of congenital heart diseases. Therapeutic innovation requires establishing an animal model reproducing right ventricular dysfunction secondary to chronic pressure-volume overload. METHODS: Right ventricular tract enlargement by transvalvular patch and pulmonary artery banding were created in 2-month-old piglets (n = 6) to mimic repaired tetralogy of Fallot. Age-matched piglets were used as controls (n = 5). Right ventricular function was evaluated at baseline and 3 and 4 months of follow-up by hemodynamic parameters and electrocardiography. Right ventricular tissue remodeling was characterized using cellular electrophysiologic and histologic analyses. RESULTS: Four months after surgery, right ventricular peak pressure increased to 75% of systemic pressure and pulmonary regurgitation significantly progressed, end-systolic and end-diastolic volumes significantly increased, and efficient ejection fraction significantly decreased compared with controls. At 3 months, the slope of the end-systolic pressure-volume relationship was significantly elevated compared with baseline and controls; a significant rightward shift of the slope, returning to the baseline value, was observed at 4 months, whereas stroke work progressed at each step and was significantly higher than in controls. Four months after surgery, QRS duration was significantly prolonged as action potential duration. Significant fibrosis and myocyte hypertrophy without myolysis and inflammation were observed in the operated group at 4 months. CONCLUSION: Various aspects of early right ventricular remodeling were analyzed in this model. This model reproduced evolving right ventricular alterations secondary to chronic volumetric and barometric overload, as observed in repaired tetralogy of Fallot with usual sequelae, and can be used for therapeutic innovation.

A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates.

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

Functional interaction of CaV channel isoforms with ryanodine receptors studied in dysgenic myotubes.

The L-type Ca(2+) channels Ca(V)1.1 (alpha(1S)) and Ca(V)1.2 (alpha(1C)) share properties of targeting but differ by their mode of coupling to ryanodine receptors in muscle cells. The brain isoform Ca(V)2.1 (alpha(1A)) lacks ryanodine receptor targeting. We studied these three isoforms in myotubes of the alpha(1S)-deficient skeletal muscle cell line GLT under voltage-clamp conditions and estimated the flux of Ca(2+) (Ca(2+) input flux) resulting from Ca(2+) entry and release. Surprisingly, amplitude and kinetics of the input flux were similar for alpha(1C) and alpha(1A) despite a previously reported strong difference in responsiveness to extracellular stimulation. The kinetic flux characteristics of alpha(1C) and alpha(1A) resembled those in alpha(1S)-expressing cells but the contribution of Ca(2+) entry was much larger. alpha(1C) but not alpha(1A)-expressing cells revealed a distinct transient flux component sensitive to sarcoplasmic reticulum depletion by 30 microM cyclopiazonic acid and 10 mM caffeine. This component likely results from synchronized Ca(2+)-induced Ca(2+) release that is absent in alpha(1A)-expressing myotubes. In cells expressing an alpha(1A)-derivative (alpha(1)Aas(1592-clip)) containing the putative targeting sequence of alpha(1S), a similar transient component was noticeable. Yet, it was considerably smaller than in alpha(1C), indicating that the local Ca(2+) entry produced by the chimera is less effective in triggering Ca(2+) release despite similar global Ca(2+) inward current density.