Nestin expression is lost in ventricular fibroblasts during postnatal development of the rat heart and re-expressed in scar myofibroblasts.

Studies have reported that the intermediate filament protein nestin was expressed in various non-stem/progenitor cells during development, downregulated during postnatal growth and re-expressed following injury. The present study tested the hypothesis that an analogous paradigm was prevalent for ventricular fibroblasts. In the neonatal rat heart, nestin protein levels were significantly higher than the adult heart and the isolation of cardiac cells revealed a selective expression in ventricular fibroblasts. In adult ventricular fibroblasts, nestin protein expression was markedly lower compared to neonatal ventricular fibroblasts. Following ischemic damage to the rat heart, nestin staining was detected in a subpopulation of scar myofibroblasts (37%) and the percentage of immunoreactive cells was greater than adult ventricular fibroblasts (7%) but significantly lower than neonatal ventricular fibroblasts (86%). Moreover, dissimilar rates of (3)H-thymidine uptake were observed among the fibroblast populations and may be related in part to the disparate percentage of nestin(+) cells. To assess the role of nestin in DNA synthesis, neonatal ventricular fibroblasts were infected with a lentivirus containing a shRNAmir directed against the intermediate filament protein. The partial depletion of nestin expression in neonatal ventricular fibroblasts significantly reduced basal DNA synthesis, in the absence of an apoptotic response. Thus, postnatal development of the rat heart was associated with a selective loss of nestin expression in ventricular fibroblasts and subsequent induction in a subpopulation of myofibroblasts following ischemic injury. The re-expression of nestin in scar myofibroblasts may represent an adaptive response to enhance their proliferative rate and accelerate the healing process.

The inflammatory preatherosclerotic remodeling induced by intermittent hypoxia is attenuated by RANTES/CCL5 inhibition.

RATIONALE: The highly prevalent obstructive sleep apnea syndrome (OSA) with its main component intermittent hypoxia (IH) is a risk factor for cardiovascular mortality. The poor knowledge of its pathophysiology has limited the development of specific treatments, whereas the gold standard treatment, continuous positive airway pressure, may not fully reverse the chronic consequences of OSA and has limited acceptance in some patients. OBJECTIVES: To examine the contribution of IH-induced inflammation to the cardiovascular complications of OSA. METHODS: We investigated systemic and vascular inflammatory changes in C57BL6 mice exposed to IH (21-5% Fi(O(2)), 60-s cycle) or normoxia 8 hours per day up to 14 days. Vascular alterations were reassessed in mice treated with a blocking antibody of regulated upon activation, normal T-cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) signaling pathway, or with the IgG isotype control throughout the IH exposure. MEASUREMENTS AND MAIN RESULTS: IH induced systemic inflammation combining increased splenic lymphocyte proliferation and chemokine expression, with early and predominant RANTES/CCL5 alterations, and enhanced splenocyte migration toward RANTES/CCL5. IH also induced structural and inflammatory vascular alterations. Leukocyte-endothelium adhesive interactions were increased, attested by leukocyte rolling and intercellular adhesion molecule-1 expression in mesenteric vessels. Aortas had increased intima-media thickness with elastic fiber alterations, mucoid depositions, nuclear factor-κB-p50 and intercellular adhesion molecule-1 overexpression, hypertrophy of smooth-muscle cells overexpressing RANTES/CCL5, and adventitial-periadventitial T-lymphocyte infiltration. RANTES/CCL5 neutralization prevented both intima-media thickening and inflammatory alterations, independently of the IH-associated proatherogenic dyslipidemia. CONCLUSIONS: Inflammation is a determinant mechanism for IH-induced preatherosclerotic remodeling involving RANTES/CCL5, a key chemokine in atherogenesis. Characterization of the inflammatory response could allow identifying at-risk patients for complications, and its pharmacologic manipulation may represent a potential complementary treatment of sleep apnea consequences.

Post-translational modifications, a key process in CD36 function: lessons from the spontaneously hypertensive rat heart.

CD36, a multifunctional protein, is involved in cardiac long chain fatty acid (LCFA) metabolism and in the etiology of heart diseases, yet the functional impact of Cd36 gene variants remains unclear. In 7-week-old spontaneously hypertensive rats (SHR), which, like humans, carry numerous mutations in Cd36, we tested the hypothesis that their restricted cardiac LCFA utilization occurs prior to hypertrophy due to defective CD36 post-translational modifications (PTM), as assessed by ex vivo perfusion of (13)C-labeled substrates and biochemical techniques. Compared to their controls, SHR hearts displayed a lower (i) contribution of LCFA to ß-oxidation (-40%) and triglycerides (+2.8 folds), which was not explained by transcriptional changes or malonyl-CoA level, a recognized ß-oxidation inhibitor, and (ii) membrane-associated CD36 protein level, but unchanged distribution. Other results demonstrate alterations in CD36 PTM in SHR hearts, specifically by N-glycosylation, and the importance of O-linked-ß-N-acetylglucosamine for its membrane recruitment and role in LCFA use in the heart.

Nestin (+) stem cells independently contribute to neural remodelling of the ischemic heart.

Recent studies have revealed the existence of multipotent nestin-immunoreactive cells in the adult mammalian heart. These cells were recruited to infarct site following ischemic injury and differentiated to a vascular lineage leading to de novo blood vessel formation. Here, we show that a sub-population of cardiac resident nestin((+)) cells can further differentiate to a neuronal-like fate in vivo following myocardial infarction. In the ischemically damaged rat heart, neurofilament-M((+)) fibres were detected innervating the peri-infarct/infarct region and the preponderance of these fibres were physically associated with processes emanating from nestin((+)) cells. One week after isogenic heterotopic cardiac transplantation, the beating transplanted rat heart was devoid of neurofilament-M((+)) fibre staining. The superimposition of an ischemic insult to the transplanted heart led to the de novo synthesis of neurofilament-M((+)) fibres by cardiac resident nestin((+)) cells. Nerve growth factor infusion and the exposure of normal rats to intermittent hypoxia significantly increased the density of neurofilament-M((+)) fibres in the heart. However, these newly formed neurofilament-M((+)) fibres were not physically associated with nestin((+)) processes. These data highlight a novel paradigm of reparative fibrosis as a subpopulation of cardiac resident nestin((+)) cells directly contributed to neural remodelling of the peri-infarct/infarct region of the ischemically damaged rat heart via the de novo synthesis of neurofilament-M fibres.

The rat heart contains a neural stem cell population; role in sympathetic sprouting and angiogenesis.

Nestin-expressing cells were identified in the normal rat heart characterized by a small cell body and numerous processes and following an ischemic insult migrated to the infarct region. The present study was undertaken to identify the phenotype, origin and biological role of nestin-expressing cells during reparative fibrosis. A neural stem cell phenotype was identified based on musashi-1 expression, growth as a neurosphere, and differentiation to a neuronal cell. Using the Wnt1-cre; Z/EG transgenic mouse model, which expresses EGFP in embryologically-derived neural crest cells, the reporter signal was detected in nestin-expressing cells residing in the heart. In infarcted human hearts, nestin-expressing cells were detected in the viable myocardium and the scar and morphologically analogous to the population identified in the rat heart. Following either an ischemic insult or the acute administration of 6-hydroxydopamine, sympathetic sprouting was dependent on the physical association of neurofilament-M immunoreactive fibres with nestin-positive processes emanating from neural stem cells. To specifically study the biological role of the subpopulation in the infarct region, neural stem cells were isolated from the scar, fluorescently labelled and transplanted in the heart of 3-day post-MI rats. Injected scar-derived neural stem cells migrated to the infarct region and were used as a substrate for de novo blood vessel formation. These data have demonstrated that the heart contains a resident population of neural stem cells derived from the neural crest and participate in reparative fibrosis. Their manipulation could provide an alternative approach to ameliorate the healing process following ischemic injury.

Delayed myocardial preconditioning induced by cobalt chloride in the rat: HIF-1α and iNOS involvement.

We previously reported that acute exposure to intermittent hypoxia results in delayed cardioprotection against ischemia/reperfusion (I/R) injury and that the hypoxia-inducible factor (HIF)-1α, a transcriptional factor stabilized by hypoxia, as well as inducible nitric oxide synthase (iNOS) play a key role in this form of preconditioning. As cobalt chloride (CoCl(2)) is known to promote HIF-1α stabilization by inhibiting prolyl hydroxylase activity, we hypothesized that CoCl(2) could mimic the cardioprotective effects of hypoxia. Two groups of rats were administered 30 mg/kg twice of CoCl(2) or sterile water. Twenty-four hours later, hearts were perfused in Langendorff mode and subjected to an I/R protocol. Infarct size and functional recovery were studied. The role of iNOS was assessed by measuring myocardial iNOS content and by observing the effects of the iNOS inhibitor aminoguanidine (Ag, 100 µm, prior to ischemia). The role of HIF-1α was investigated by preventing its stabilization using cadmium chloride (CdCl(2), 1 mg/kg), administered 1 h before cobalt treatment. Treatment by CoCl(2) significantly reduced myocardial infarction by 33% and increased coronary flow (CF) at reperfusion by 27% compared with control rats, and this was accompanied by a threefold increase in myocardial iNOS content. CdCl(2) pretreatment and Ag perfusion abolished the beneficial effects on both infarct size and CF. Thus, the hypoxia-sensitive transcription factor HIF-1α and iNOS appear to play a pivotal role in the delayed pharmacological myocardial preconditioning induced by cobalt, thus mimicking the effects of hypoxic preconditioning. These results underscore the importance of prolyl hydroxylases as potential therapeutic targets for cardioprotection.

The phenotype and potential origin of nestin+ cardiac myocyte-like cells following infarction.

Nestin+ cardiac myocyte-like cells were detected in the peri-infarct/infarct region of the ischemically damaged heart. The present study was undertaken to elucidate the phenotype and potential origin of nestin+ cardiac myocyte-like cells and identify stimuli implicated in their appearance. In the infarcted human and rat heart, nestin+ cardiac myocyte-like cells were morphologically and structurally immature, exhibited a desmin-immunoreactive striated phenotype, expressed the beta(1)-adrenergic receptor, and associated with an aberrant pattern of connexin-43 expression and/or organization. Nestin+ cardiac myocyte-like cells were detected 24 h postischemic injury and persisted in the infarcted rat heart for 9 mo. In the normal rat heart, cardiac progenitor transcriptional factors Nkx2.5/GATA4 were detected in a subpopulation of nestin+ neural stem cells. Following an ischemic insult, nestin+/Nkx2.5+ neural stem cells migrated to the peri-infarct/infarct region and appeared to be in a primordial state of differentiation to a nestin+ cardiac myocyte-like cell. The exposure of adult male rats to normobaric hypoxia (12% O2) for 10 days failed to promote the appearance of nestin+ cardiac myocyte-like cells. Following osmotic pump delivery of isoproterenol to normal adult rats, nestin+ cardiac myocyte-like cells were detected, albeit the response was modest and secondary to tissue loss. Thus ischemia-induced appearance of nestin+ cardiac myocyte-like cells apparently represents an adaptive response to heal the infarcted heart. Nkx2.5/GATA4 expression in a subpopulation of resident neural stem cells provides the appropriate phenotype for their potential differentiation to a nestin+ cardiac myocyte-like cell.

Prevention of HIF-1 activation and iNOS gene targeting by low-dose cadmium results in loss of myocardial hypoxic preconditioning in the rat.

This study aimed to underline the interaction between hypoxia-inducible factor-1 (HIF-1) and the inducible nitric oxide synthase (iNOS) gene in vivo and their contribution to the delayed myocardial preconditioning induced by acute intermittent hypoxia (IH) in the rat using chromatin immunoprecipitation and pharmacological inhibition by low-dose cadmium. Langendorff-perfused hearts of Wistar rats exposed to normoxia or IH 24 h earlier were submitted to global ischemia and reperfusion. Effects of iNOS inhibition by aminoguanidine (100 microM) before ischemia or of low-dose injection of cadmium chloride (1 mg/kg) before normoxia or IH were tested. Myocardial HIF-1 and iNOS quantification and in vivo chromatin immunoprecipitation of HIF-1 bound to the iNOS gene promoter were performed. IH-induced delayed cardioprotection resulted in an improvement in coronary flow and functional recovery at reperfusion and a decrease in infarct size. Myocardial HIF-1 activity was increased with resulting targeting of the iNOS gene. Aminoguanidine abolished the cardioprotective effects of IH. Cadmium chloride treatment before IH prevented myocardial HIF-1 activation (72.3 +/- 4.0 vs. 42.1 +/- 9.7 arbitrary units after cadmium chloride; P < 0.05), targeting of the iNOS gene, iNOS expression, and preconditioning (infarct size: 15.9 +/- 5.6 vs. 30.1 +/- 5.4% after cadmium chloride; P < 0.05). This study is the first to demonstrate the interaction of HIF-1 with the myocardial iNOS gene in situ after hypoxic preconditioning. Prevention of HIF-1 activation and iNOS gene targeting by a single low dose of cadmium abolished the delayed cardioprotective effects, bringing insight into the cardiovascular consequences of cadmium exposure.

Intermittent hypoxia-induced delayed cardioprotection is mediated by PKC and triggered by p38 MAP kinase and Erk1/2.

We previously reported that acute intermittent hypoxia (IH) confers delayed cardioprotection against a prolonged ischemic insult in the rat, via the involvement of nitric oxide synthase and K(ATP) channels. In the present study, we investigated the role of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K), stress activated p38 MAP kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2) using selective inhibitors of these pathways. Adult male rats were exposed to 1-min cycles of IH (10% O(2), 40 s)/normoxia (21% O(2), 20 s) during 4 h or to normoxic cycles. 24 h later, isolated hearts were perfused in Langendorff mode and subjected to a 30-min global ischemia followed by 120 min of reperfusion. Compared to normoxic conditions, IH significantly reduced infarct size (22.2+/-2.4% vs. 33.8+/-2.6%, p<0.05), improved coronary flow and decreased the contracture at reperfusion. When administered before sustained ischemia, chelerythrine (a PKC inhibitor) abolished both the IH-induced reduction in infarct size (36.1+/-4.9%) and improvement in hemodynamic parameters. In contrast, chelerythrine administration 10 min before IH, did not modify the delayed cardioprotective response. Similarly, wortmannin (a PI3K inhibitor) administration 10 min before IH was unable to block the cardioprotective effects. However, administration of SB203580 (a p38 MAPK inhibitor) and PD98059 (an Erk1/2 inhibitor), 30 min before IH abolished its delayed infarct-sparing effect (32.2+/-3.4% and 33.9+/-2.9%, respectively). In addition, 24 h after IH, a significant increase in p38 MAPK and Erk1/2 phosphorylation was observed by Western blot. These results suggest that the delayed preconditioning induced by intermittent hypoxia does not involve the PI3K signalling pathway and that is mediated by PKC and triggered by p38 MAPK and Erk1/2.