Daniele Bani

Relaxin : A pleiotropic hormone

1. Relaxin is a peptide hormone of about 6000 Da belonging to the insulin family. Like insulin, relaxin is composed by two disulfide-linked chains, termed the A and B chains, the B chain bearing the receptor interaction site. 2. Relaxin is produced primarily by the corpus luteum, in both pregnant and nonpregnant females. It attains the highest plasma levels during pregnancy. In this condition, relaxin is also produced by the decidua and placenta. In males, relaxin is synthesized in the prostate and released in the seminal fluid. An additional source of relaxin has recently been identified in the heart atria. 3. Relaxin has a broad range of biologic activities, some of which have been known for a long time. These latter ones include: (a) the induction of collagen remodeling and consequent softening of the tissues of the birth canal in view of delivery; (b) the inhibition of uterine contractile activity; (c) the stimulation of growth and differentiation of the mammary gland. 4. In more recent years, novel sites of relaxin action have been recognized. In particular, it has been shown that relaxin: (a) regulates growth and differentiation of breast cancer cells in culture; (b) promotes dilation of blood vessels in several organs and tissues, including the uterus, the mammary gland, the lung and the heart; (c) has a chronotropic action on the heart; (d) inhibits the release of histamine by mast cells, thus being able to counteract experimental allergic asthma; (d) depresses aggregation of platelets and their release by megakaryocytes; (e) influences the secretion of hormones by the pituitary gland; and (f) contributes to the regulation of fluid balance. 5. Concerning the mechanisms of action of relaxin, stimulation of nitric oxide generation, with consequent rise in intracellular cyclic GMP levels, and stimulation of cyclic AMP production have been demonstrated to occur in the target cells and organs. 6. It may be expected that the next decade will provide answers about the utility of relaxin, in terms of insight into the actual physiologic functions of relaxin in the animal kingdom and especially in man, in view of possible therapeutic use of relaxin or relaxin-derived drugs in human disease, especially considering that human recombinant relaxin is now available for clinical experimentation.

Oxidative Stress by Monoamine Oxidase Mediates Receptor-Independent Cardiomyocyte Apoptosis by Serotonin and Postischemic Myocardial Injury

Background— Serotonin (5-hydroxytryptamine [5-HT]), released by activated platelets during cardiac ischemia, is metabolized by the mitochondrial enzyme monoamine oxidase A (MAO-A). Because hydrogen peroxide is one of the byproducts of 5-HT degradation by MAO-A, we investigated the potential role of reactive oxygen species generated by MAOs in 5-HT-dependent cardiomyocyte death and post-ischemia-reperfusion cardiac damage. Methods and Results— Treatment of isolated adult rat cardiomyocytes with 5-HT induced intracellular oxidative stress and cell apoptosis. The apoptotic cascade triggered by 5-HT involves release of cytochrome c, upregulation of proapoptotic Bax protein, and downregulation of antiapoptotic Bcl-2 protein. These effects were prevented by inhibition of amine transporter or MAO, antioxidants, or iron chelation. In contrast, cardiomyocyte apoptosis was only slightly affected by the 5-HT2B receptor antagonist SB 206553. In vivo, inhibition of MAO-A largely reduced myocardial ultrastructural damage induced by 30 minutes of ischemia followed by 60 minutes of reperfusion in the rat heart. Cardioprotective effects of MAO inhibitors were associated with the prevention of postischemic oxidative stress, neutrophil accumulation, and mitochondrial-dependent cell death and were not reverted by SB 206553. Administration of MAO-A inhibitors during ischemia was still effective in preventing cardiac damage. Conclusions— Our results supply the first direct evidence that oxidative stress induced by MAO is responsible for receptor-independent apoptotic effects of 5-HT in cardiomyocytes and postischemic myocardial injury. These findings provide new insight into the mechanisms of 5-HT action in the heart and may constitute the basis for novel therapies.

Relaxin‐induced increased coronary flow through stimulation of nitric oxide production

1 Relaxin (RLX) is a multifunctional hormone which, besides its role in pregnancy and parturition, has also been shown to influence the cardiovascular system. In this study, we investigated the effect of RLX on coronary flow of rat and guinea‐pig hearts, isolated and perfused in a Langendorff apparatus. RLX was either added to the perfusion fluid at a concentration of 5 × 10−9 M for a 20‐min perfusion, or given as a bolus into the aortic cannula at concentrations of 10−9 M, 5 × 10−9 M and 10−8 M dissolved in 1 ml of perfusion fluid. 2 RLX, given either for a 20‐min perfusion or as a bolus in the aortic cannula to guinea‐pig and rat isolated hearts, increased the coronary flow and the amount of nitrite, a stable end‐product of nitric oxide (NO) metabolism, that appeared in the perfusates in a concentration‐dependent fashion. 3 The increase in coronary flow and in nitrite in the perfusates induced by RLX was significantly reduced by pretreatment with the nitric oxide synthase (NOS) inhibitor, NG‐monomethyl‐L‐arginine (L‐NMMA, 10−4 M). 4 The effects of RLX on coronary flow and nitrite amounts in the perfusates were compared with those induced by the endothelium‐dependent vasodilator agent, acetylcholine (ACh, 10−8 − 10−7 M), and by the endothelium‐independent vasodilator agent, sodium nitroprusside (SNP, 10−7‐10−6M). The results obtained show that RLX is more effective than ACh and SNP in increasing coronary flow. 5 The results of this study show that RLX increases coronary flow through stimulation of NO production; hence this hormone should be regarded as a novel agent capable of improving myocardial perfusion.

Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue

Adipose tissue is a dynamic endocrine organ with a central role in metabolism regulation. Functional differences in adipose tissue seem associated with the regional distribution of fat depots, in particular in subcutaneous and visceral omental pads. Here, we report for the first time the isolation of human adipose‐derived adult stem cells from visceral omental and subcutaneous fat (V‐ASCs and S‐ASCs, respectively) from the same subject. Immunophenotyping shows that plastic culturing selects homogeneous cell populations of V‐ASCs and S‐ASCs from the corresponding stromal vascular fractions (SVFs), sharing typical markers of mesenchymal stem cells. Electron microscopy and electrophysiological and real‐time RT‐PCR analyses confirm the mesenchymal stem nature of both V‐ASCs and S‐ASCs, while no significant differences in a limited pattern of cytokine/chemokine expression can be detected. Similar to S‐ASCs, V‐ASCs can differentiate in vitro toward adipogenic, osteogenic, chondrogenic, muscular, and neuronal lineages, as demonstrated by histochemical, immunofluorescence, real‐time RT‐PCR, and electrophysiological analyses, suggesting the multipotency of such adult stem cells. Our data demonstrate that both visceral and subcutaneous adipose tissues are a source of pluripotent stem cells with multigermline potential. However, the visceral rather than the subcutaneous ASC could represent a more appropriate in vitro cell model for investigating the molecular mechanisms implicated in the pathophysiology of metabolic disorders such as obesity.—Baglioni, S., Francalanci, M., Squecco, R., Lombardi, A., Cantini, G., Angeli, R., Gelmini, S., Guasti, D., Benvenuti, S., Annunziato, F., Bani, D., Liotta, F., Francini, F., Perigli, G., Serio, M., Luconi, M. Characterization of human adult stem‐cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J. 23, 3494–3505 (2009). www.fasebj.org

Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders

Glomerular diseases account for 90% of end‐stage kidney disease. Podocyte loss is a common determining factor for the progression toward glomerulosclerosis. Mature podocytes cannot proliferate, but recent evidence suggests that they can be replaced by renal progenitors localized within the Bowmans capsule. Here, we demonstrate that Notch activation in human renal progenitors stimulates entry into the S‐phase of the cell cycle and cell division, whereas its downregulation is required for differentiation toward the podocyte lineage. Indeed, a persistent activation of the Notch pathway induced podocytes to cross the G2/M checkpoint, resulting in cytoskeleton disruption and death by mitotic catastrophe. Notch expression was virtually absent in the glomeruli of healthy adult kidneys, while a strong upregulation was observed in renal progenitors and podocytes in patients affected by glomerular disorders. Accordingly, inhibition of the Notch pathway in mouse models of focal segmental glomerulosclerosis ameliorated proteinuria and reduced podocyte loss during the initial phases of glomerular injury, while inducing reduction of progenitor proliferation during the regenerative phases of glomerular injury with worsening of proteinuria and glomerulosclerosis. Taken altogether, these results suggest that the severity of glomerular disorders depends on the Notch‐regulated balance between podocyte death and regeneration provided by renal progenitors. STEM CELLS 2010; 28:1674–1685.

Relaxin Activates the l-Arginine–Nitric Oxide Pathway in Vascular Smooth Muscle Cells in Culture

The peptide hormone relaxin (RLX) has been shown to elicit a powerful vasodilatory response in several target organs. This response is mediated by the stimulation of intrinsic nitric oxide (NO) generation. The present study was designed to clarify whether RLX directly promotes the relaxation of vascular smooth muscle cells through stimulation of NO generation. Vascular smooth muscle cells from bovine aortas were incubated with RLX at concentrations ranging from 1 nmol/L to 1 micromol/L. The expression and activity of NO synthase, production of NO, and the intracellular levels of cGMP and Ca2+ were determined. The cell morphology and signal transduction mechanisms of these bovine aortic smooth muscle cells in response to RLX were also studied. RLX stimulated the expression of immunoreactive inducible NO synthase and increased significantly and in a concentration-related fashion inducible NO synthase activity, NO generation, and intracellular cGMP levels. Concurrently, RLX significantly decreased cytosolic Ca2+ concentrations and caused changes in cell shape and the actin cytoskeleton that were consistent with cell relaxation. The signal transduction mechanisms leading to the enhanced expression of inducible NO synthase protein and activity caused by RLX involve the activation of tyrosine kinase, phosphatidylcholine-phospholipase C, and the transcription factor nuclear factor-kappaB, similar to bacterial endotoxins and proinflammatory cytokines. This study suggests that RLX is an endogenous agent capable of regulating vascular tone by activation of the L-arginine-NO pathway in vascular smooth muscle cells.

Telocytes as supporting cells for myocardial tissue organization in developing and adult heart.

Recent evidence indicates that the adult heart contains sub‐epicardial cardiogenic niches where cardiac stem cells and stromal supporting cells reside together. Such stromal cells include a special population, previously identified as interstitial Cajal‐like cells and recently termed telocytes because of their long, slender processes (telopodes) embracing the myocardial precursors. Specific stromal cells, presumptively originated from the epicardium, have been postulated to populate the developing heart where they are thought to play a role in its morphogenesis. This study is designed to investigate the occurrence of telocytes in the developing heart and provide clues to better understand their role as supporting cells involved in the architectural organization of the myocardium during heart development. Our results showed that stromal cells with the immunophenotypical (vimentin, CD34) and ultrastructural features of telocytes were present in the mouse heart since early embryonic to adult life, as well as in primary cultures of neonatal mouse cardiac cells. These cells formed an extended network of telopodes which closely embraced the growing cardiomyocytes and appeared to contribute to the aggregation of cardiomyocyte clusters in vitro. In conclusion, the present findings strongly suggest that, during heart development, stromal cells identifiable as telocytes could play a nursing and guiding role for myocardial precursors to form the correct three‐dimensional tissue pattern and contribute to compaction of the embryonic myocardial trabeculae. It is tempting to speculate that telocytes could be a novel, possible target for therapeutic strategies aimed at potentiating cardiac repair and regeneration after ischemic injury.

Relaxin up-regulates inducible nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial cells

Relaxin (RLX) is a reproductive hormone with vasodilatatory properties on several organs, including the heart. RLX‐induced vasodilatation appears to depend on the stimulation of endogenous NO production. Here, we investigate whether RLX acts on rat coronary endothelial (RCE) cells in vitro by inducing changes of NO generation and, if so, to clarify the possible mechanism of action. RCE cells were treated for 24 h with vehicle (controls) or RLX, alone or in association with inhibitors of NO synthesis or dexamethasone, which inhibits transcription of NO synthase gene. In some experiments, inactivated RLX was given in the place of authentic RLX. Expression of NO synthase isozymes II and III was analyzed by immunocytochemistry, Western blot, and RT‐PCR. NO production was evaluated by the Griess reaction for nitrite and the NO‐sensitive fluorophore DAF‐2/DA. Agonist‐induced changes of intracellular Ca2+ transient were studied with the Ca2+‐sensitive fluorophore Fura 2‐AM. RLX was found to up regulate NOS II mRNA and protein and to stimulate intrinsic NO generation, likely through the activation of a dexamethasone‐sensitive transcription factor, and to decrease agonist‐induced intracellular Ca2+ transient. Conversely, RLX had negligible effects on NOS III expression. By these biological effects, RLX may afford significant protection against cardiovascular disease.

Novel drug development opportunity for relaxin in acute myocardial infarction: evidences from a swine model

The hormone relaxin has been shown to cause coronary vasodilation and to prevent ischemia/reperfusion‐induced cardiac injury in rodents. This study provides evidence that relaxin, used as an adjunctive drug to coronary reperfusion, reduces the functional, biochemical, and histopathological signs of myocardial injury in an in vivo swine model of heart ischemia/reperfusion, currently used to test cardiotropic drugs for myocardial infarction. Human recombinant relaxin, given at reperfusion at doses of 1.25, 2.5, and 5 µg/kg b.wt. after a 30‐min ischemia, caused a dose‐related reduction of key markers of myocardial damage (serum myoglobin, CK‐MB, troponin T) and cardiomyocyte apoptosis (caspase 3, TUNEL assay), as well as of cardiomyocyte contractile dysfunction (myofibril hypercontraction). Compared with the controls, relaxin also increased the uptake of the viability tracer 201Thallium and improved ventricular performance (cardiac index). Relaxin likely acts by reducing oxygen free radical‐induced myocardial injury (malondialdehyde, tissue calcium overload) and inflammatory leukocyte recruitment (myeloperoxidase). The present findings show that human relaxin, given as a drug to counteract reperfusion‐induced cardiac injury, affords a clear‐cut protection to the heart of swine with induced myocardial infarction. The findings also provide background to future clinical trials with relaxin as adjunctive therapy to catheter‐based coronary angioplasty in patients with acute myocardial infarction.

Effects of relaxin on mast cells. In vitro and in vivo studies in rats and guinea pigs.

The results of the current study demonstrate that relaxin inhibits histamine release by mast cells. This effect is related to the peptide concentrations, and could be observed in both isolated rat serosal mast cells stimulated with compound 48/80 or calcium ionophore A 23187, and in serosal mast cells isolated from sensitized guinea pigs and challenged with the antigen. The morphological findings agree with the functional data, revealing that relaxin attenuates calcium ionophore-induced granule exocytosis by isolated rat serosal mast cells. Similar effects of relaxin have also been recognized in vivo by light microscopic and densitometric analysis of the mesenteric mast cells of rats which received the hormone intraperitoneally 20 min before local treatment of the mesentery with calcium ionophore. Moreover, evidence is provided that relaxin stimulates endogenous production of nitric oxide and attenuates the rise of intracellular Ca2+ concentration induced by calcium ionophore. The experiments with drugs capable of influencing nitric oxide production also provide indirect evidence that the inhibiting effect of relaxin on mast cell histamine release is related to an increased generation of nitric oxide. It is suggested that relaxin may have a physiological role in modulating mast cell function through the L-arginine-nitric oxide pathway.