Monica Salio

Isolation and Expansion of Adult Cardiac Stem Cells From Human and Murine Heart

Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy. However, in the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential. Recent studies have even surmised the existence of resident cardiac stem cells, endothelial cells generating cardiomyocytes by cell contact or extracardiac progenitors for cardiomyocytes, but these findings are still controversial. We describe the isolation of undifferentiated cells that grow as self-adherent clusters (that we have termed “cardiospheres”) from subcultures of postnatal atrial or ventricular human biopsy specimens and from murine hearts. These cells are clonogenic, express stem and endothelial progenitor cell antigens/markers, and appear to have the properties of adult cardiac stem cells. They are capable of long-term self-renewal and can differentiate in vitro and after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse to yield the major specialized cell types of the heart: myocytes (ie, cells demonstrating contractile activity and/or showing cardiomyocyte markers) and vascular cells (ie, cells with endothelial or smooth muscle markers).

Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling.

Erythropoietin (EPO), originally identified for its critical hormonal role in promoting erythrocyte survival and differentiation, is a member of the large and diverse cytokine superfamily. Recent studies have identified multiple paracrine/autocrine functions of EPO that coordinate local responses to injury by maintaining vascular autoregulation and attenuating both primary (apoptotic) and secondary (inflammatory) causes of cell death. Experimental evidence also supports a role for EPO in repair and regeneration after brain and spinal cord injury, including the recruitment of stem cells into the region of damage. Tissue expression of the EPO receptor is widespread, especially during development, and includes the heart. However, it is currently unknown as to whether EPO plays a physiological function in adult myocardial tissue. We have assessed the potential protective role of EPO in vitro with adult rat cardiomyocytes, and in vivo in a rat model of myocardial infarction with reperfusion. The results show that EPO markedly prevents the apoptosis of cultured adult rat myocardiocytes subjected to 28 h of hypoxia (≈3% normal oxygen). Additional studies employing a rat model of coronary ischemia–reperfusion showed that the administration of recombinant human EPO (5,000 units/kg of body weight; i.p. daily for 7 days) reduces cardiomyocyte loss by ≈50%, an extent sufficient to normalize hemodynamic function within 1 week after reperfusion. These observations not only suggest a potential therapeutic role for recombinant human EPO in the treatment of myocardial ischemia and infarction by preventing apoptosis and attenuating postinfarct deterioration in hemodynamic function, but also predict that EPO is likely a tissue-protective cytokine in other organs as well.

Disruption of the Ang II type 1 receptor promotes longevity in mice

The renin-angiotensin system plays a role in the etiology of hypertension and the pathophysiology of cardiac and renal diseases in humans. Ang II is the central product of this system and is involved in regulating immune responses, inflammation, cell growth, and proliferation by acting through Ang II type 1 receptors (AT1 and AT2). Here, we show that targeted disruption of the Agtr1a gene that encodes AT1A results in marked prolongation of life span in mice. Agtr1a-/- mice developed less cardiac and vascular injury, and multiple organs from these mice displayed less oxidative damage than wild-type mice. The longevity phenotype was associated with an increased number of mitochondria and upregulation of the prosurvival genes nicotinamide phosphoribosyltransferase (Nampt) and sirtuin 3 (Sirt3) in the kidney. In cultured tubular epithelial cells, Ang II downregulated Sirt3 mRNA, and this effect was inhibited by an AT1 antagonist. These results demonstrate that disruption of AT1 promotes longevity in mice, possibly through the attenuation of oxidative stress and overexpression of prosurvival genes, and suggests that the Ang II/AT1 pathway may be targeted to influence life span in mammals.

Effects of Valsartan on Circulating Brain Natriuretic Peptide and Norepinephrine in Symptomatic Chronic Heart Failure The Valsartan Heart Failure Trial (Val-HeFT)

Background—Brain natriuretic peptide (BNP) and norepinephrine (NE) are strongly related to severity of and are independent predictors of outcome in heart failure. The long-term effects of angiotensin receptor blockers on BNP and NE in heart failure patients are not known. Methods and Results—Both BNP and NE were measured in 4284 patients randomized to valsartan or placebo in the Valsartan Heart Failure Trial (Val-HeFT) at baseline and 4, 12, and 24 months after randomization. The effects of valsartan were tested by ANCOVA, controlling for baseline values and concomitant ACE inhibitors and/or &bgr;-blockers. BNP and NE concentrations were similar at baseline in the 2 groups and were decreased by valsartan starting at 4 months and up to 24 months. BNP increased over time in the placebo group. At the end point, least-squares mean (±SEM) BNP increased from baseline by 23±5 pg/mL in the placebo group (n=1979) but decreased by 21±5 pg/mL (n=1940) in the valsartan group (P <0.0001). NE increased by 41±6 pg/mL (n=1979) and 12±6 pg/mL (n=1941) for placebo and valsartan, respectively (P =0.0003). Concomitant therapy with both ACE inhibitors and &bgr;-blockers significantly reduced the effect of valsartan on BNP but not on NE (P for interaction=0.0223 and 0.2289, respectively). Conclusions—In Val-HeFT, the largest neurohormone study in patients with symptomatic chronic heart failure, BNP and NE rose over time in the placebo group. Valsartan caused sustained reduction in BNP and attenuated the increase in NE over the course of the study. These neurohormone effects of valsartan are consistent with the clinical benefits reported in Val-HeFT.

Cardioprotective Function of the Long Pentraxin PTX3 in Acute Myocardial Infarction

Background— Despite widespread clinical use as a prognostic marker in ischemic heart disorders, the actual pathogenetic role of the short pentraxin, C-reactive protein, has not undergone stringent genetic testing because of evolutionary divergence between mouse and humans. The long pentraxin PTX3 is conserved in evolution, is expressed in the heart under inflammatory conditions, and is a candidate prognostic marker in acute myocardial infarction. It was therefore important to assess whether PTX3 plays a pathogenetic role in acute myocardial infarction. Methods and Results— In a model of acute myocardial infarction caused by coronary artery ligation and reperfusion, tissue mRNA expression and circulating levels of PTX3 increased. The interleukin-1R–MyD88 pathway plays a pivotal role in the induction of PTX3 transcript after ischemia. ptx3-deficient mice showed exacerbated heart damage (33% larger infarcts in null mice; P=0.0047). Increased myocardial damage in ptx3-deficient mice was associated with a greater no-reflow area, increased neutrophil infiltration, decreased number of capillaries, and increased number of apoptotic cardiomyocytes. In addition, ptx3-deficient mice with acute myocardial infarction showed higher circulating levels of interleukin-6 and increased C3 deposition in lesional tissue. The phenotype was reversed by exogenous PTX3. Conclusions— Thus, PTX3 plays a nonredundant, regulatory, cardioprotective role in acute myocardial infarction in mice. Our results suggest that modulation of the complement cascade contributes to the cardioprotective function of PTX3.

Comparative measurement of N-terminal pro-brain natriuretic peptide and brain natriuretic peptide in ambulatory patients with heart failure.

Abstract It is not clear whether brain natriuretic peptide (BNP) or N-terminal proBNP (NT-proBNP) is superior as a diagnostic and prognostic indicator in cardiac diseases. Here, we compare the clinical correlations of both peptides in a population of 92 ambulatory patients with heart failure, using a well-established immunoradiometric assay (IRMA) for BNP and an automated electrochemiluminescence immunoassay for NT-proBNP. The analytical correlation between the two peptides was satisfactory over a wide range of concentrations (1–686 pM for BNP) with the equation: NT-proBNP = 3.48 × BNP–19 and a correlation coefficient r2=0.94. In addition, the concentration of both peptides increased in a similar fashion according to the severity of the disease New York Heart Association (NYHA) functional class, left ventricular ejection fraction, etiology) and age; for instance, the ratios between median levels measured in NYHA class III vs. class II patients were comparable for BNP (383 vs. 16 pM, ratio 24) and NT-proBNP (1306 vs. 57 pM, ratio 23). We conclude that N-terminal proBNP, as assayed in the present study, correlates equally to BNP with clinical variables in patients with heart

Mutant Prion Protein Expression Causes Motor and Memory Deficits and Abnormal Sleep Patterns in a Transgenic Mouse Model

A familial form of Creutzfeldt-Jakob disease (CJD) is linked to the D178N/V129 prion protein (PrP) mutation. Tg(CJD) mice expressing the mouse homolog of this mutant PrP synthesize a misfolded form of the mutant protein, which is aggregated and protease resistant. These mice develop clinical and pathological features reminiscent of CJD, including motor dysfunction, memory impairment, cerebral PrP deposition, and gliosis. Tg(CJD) mice also display electroencephalographic abnormalities and severe alterations of sleep-wake patterns strikingly similar to those seen in a human patient carrying the D178N/V129 mutation. Neurons in these mice show swelling of the endoplasmic reticulum (ER) with intracellular retention of mutant PrP, suggesting that ER dysfunction could contribute to the pathology. These results establish a transgenic animal model of a genetic prion disease recapitulating cognitive, motor, and neurophysiological abnormalities of the human disorder. Tg(CJD) mice have the potential for giving greater insight into the spectrum of neuronal dysfunction in prion diseases.

Tetracycline and its analogues protect Caenorhabditis elegans from β amyloid-induced toxicity by targeting oligomers.

The accumulation and deposition of amyloid beta (Aβ) peptide in extracellular dense plaques in the brain is a key phase in Alzheimers disease (AD). Small oligomeric forms of Aβ are responsible for the toxicity and the early cognitive impairment observed in patients before the amyloid plaque deposits appear. It is essential for the development of an efficient cure for AD to identify compounds that interfere with Aβ aggregation, counteracting the molecular mechanisms involved in conversion of the monomeric amyloid protein into oligomeric and fibrillar forms. Tetracyclines have been proposed for AD therapy, although their effects on the aggregation of Aβ protein, particularly their ability to interact in vivo with the Aβ oligomers and/or aggregates, remain to be understood. Using transgenic Caenorhabditis elegans as a simplified invertebrate model of AD, we evaluated the ability of tetracyclines to interfere with the sequence of events leading to Aβ proteotoxicity. The drugs directly interact with the Aβ assemblies in vivo and reduce Aβ oligomer deposition, protecting C. elegans from oxidative stress and the onset of the paralysis phenotype. These effects were specific, dose-related and not linked to any antibiotic activity, suggesting that the drugs might offer an effective therapeutic strategy to target soluble Aβ aggregates.

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

Distinct cardiac and renal effects of ETA receptor antagonist and ACE inhibitor in experimental type 2 diabetes.

Diabetic nephropathy is associated with cardiovascular morbidity. Angiotensin-converting enzyme (ACE) inhibitors provide imperfect renoprotection in advanced type 2 diabetes, and cardiovascular risk remains elevated. Endothelin (ET)-1 has a role in renal and cardiac dysfunction in diabetes. Here, we assessed whether combination therapy with an ACE inhibitor and ET(A) receptor antagonist provided reno- and cardioprotection in rats with overt type 2 diabetes. Four groups of Zucker diabetic fatty (ZDF) rats were treated orally from 4 (when proteinuric) to 8 mo with vehicle, ramipril (1 mg/kg), sitaxsentan (60 mg/kg), and ramipril plus sitaxsentan. Lean rats served as controls. Combined therapy ameliorated proteinuria and glomerulosclerosis mostly as a result of the action of ramipril. Simultaneous blockade of ANG II and ET-1 pathways normalized renal monocyte chemoattractant protein-1 and interstitial inflammation. Cardiomyocyte loss, volume enlargement, and capillary rarefaction were prominent abnormalities of ZDF myocardium. Myocyte volume was reduced by ramipril and sitaxsentan, which also ameliorated heart capillary density. Drug combination restored myocardial structure and reestablished an adequate capillary network in the presence of increased cardiac expression of VEGF/VEGFR-1, and significant reduction of oxidative stress. In conclusion, in type 2 diabetes concomitant blockade of ANG II synthesis and ET-1 biological activity through an ET(A) receptor antagonist led to substantial albeit not complete renoprotection, almost due to the ACE inhibitor. The drug combination also showed cardioprotective properties, which however, were mainly dependent on the contribution of the ET(A) receptor antagonist through the action of VEGF.