Elisabetta Cervio

Mesenchymal stem cell therapy for heart disease

Mesenchymal stem cells (MSC) are adult stem cells with capacity for self-renewal and multi-lineage differentiation. Initially described in the bone marrow, MSC are also present in other organs and tissues. From a therapeutic perspective, because of their easy preparation and immunologic privilege, MSC are emerging as an extremely promising therapeutic agent for tissue regeneration and repair. Studies in animal models of myocardial infarction have demonstrated the ability of transplanted MSC to engraft and differentiate into cardiomyocytes and vascular cells. Most importantly, engrafted MSC secrete a wide array of soluble factors that mediate beneficial paracrine effects and may greatly contribute to cardiac repair. Together, these properties can be harnessed to both prevent and reverse remodeling in the ischemically injured ventricle. In proof-of-concept and phase I clinical trials, MSC therapy improved left ventricular function, induced reverse remodeling, and decreased scar size. In this review we will focus on the current understanding of MSC biology and MSC mechanism of action in cardiac repair.

Conditioned Medium From Human Amniotic Mesenchymal Stromal Cells Limits Infarct Size and Enhances Angiogenesis

The paracrine properties of human amniotic membrane‐derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC‐CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC‐CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC‐CM by cytokine array. Our results strongly support the concept that the administration of hAMC‐CM favors the repair process after acute myocardial infarction.

Role of galanin receptor 1 in peristaltic activity in the guinea pig ileum

Galanin effects are mediated by distinct receptors, galanin receptor 1 (GAL-R1), GAL-R2 and GAL-R3. Here, we analyzed 1) the role of GAL-R1 in cholinergic transmission and peristalsis in the guinea-pig ileum using longitudinal muscle-myenteric plexus preparations and intact segments of the ileum in organ bath, and 2) the distribution of GAL-R1 immunoreactivity in the myenteric plexus with immunohistochemistry and confocal microscopy. Galanin inhibited electrically stimulated contractions of longitudinal muscle-myenteric plexus preparations with a biphasic curve. Desensitization with 1 microM galanin suppressed the high potency phase of the curve, whereas the GAL-R1 antagonist, RWJ-57408 (1 microM), inhibited the low potency phase. Galanin (3 microM) reduced the longitudinal muscle contraction and the peak pressure, and decreased the compliance of the circular muscle. All these effects were antagonized by RWJ-57408 (1 or 10 microM). RWJ-57408 (10 microM) per se did not affect peristalsis parameters in normal conditions, nor when peristalsis efficiency was reduced by partial nicotinic transmission blockade with hexamethonium. In the myenteric plexus, GAL-R1 immunoreactivity was localized to neurons and to fibers projecting within the plexus and to the muscle. GAL-R1 was expressed mostly by cholinergic neurons and by some neurons containing vasoactive intestinal polypeptide or nitric oxide synthase. This study indicates that galanin inhibits cholinergic transmission to the longitudinal muscle via two separate receptors; GAL-R1 mediates the low potency phase. The reduced peristalsis efficiency could be explained by inhibition of the cholinergic drive, whereas the decreased compliance is probably due to inhibition of descending neurons and/or to the activation of an excitatory muscular receptor. Endogenous galanin does not appear to affect neuronal pathways subserving peristalsis in physiologic conditions via GAL-R1.

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over‐expressed in P19 cells individually or in different combinations. The over‐expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real‐time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation‐contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over‐expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. Stem Cells 2015;33:1187–1199

Review of the implications of dietary tryptophan intake in patients with irritable bowel syndrome and psychiatric disorders

In this review, we address the possible role of the essential amino acid L-tryptophan or its metabolic derivative 5-hydroxytryptophan in the modulation of serotonin (5-hydroxytryptamine) synthesis and thereby in affecting the pathophysiology of central and peripheral nervous system disorders, including depression and irritable bowel syndrome. L-Tryptophan may represent a link between apparently disparate functional disorders and is of interest for general gastroenterologists, neurogastroenterologists, and neurologists. On the basis of estimates showing that approximately 20% of patients with functional bowel disorders seeking care in referral centres have psychiatric comorbidity, we attempt to provide a conceptual framework for defining the possible role of L-tryptophan in this population.

Allogeneic lethally irradiated cord blood mononuclear cells in no-option critical limb ischemia: a "box of rain".

Critical limb ischemia (CLI) is burdened by a 40% major amputation rate, and a 5-year life expectancy <50%. We report the first in-human injection of lethally γ-irradiated non-human leukocyte antigen (HLA)-matched cord blood (CB)-derived mononuclear cells in a no-option CLI patient, to induce therapeutic neo-angiogenesis, with evidence of successful outcome supported by clinical findings (ulcer healing and pain relief), instrumental assessment (transcutaneous O2 pressure, ankle/brachial index, and contrast-enhanced ultrasonography), and histological demonstration of muscular tissue repair and capillary network expansion. If our approach will be confirmed, the huge number of CB units currently discarded might be redirected toward regenerative medicine purposes, leading to cutting-edge solutions for important unmet clinical needs, such as ischemic diseases, which remain the main cause of disability and mortality in western countries.

Role of galanin receptor 1 in gastric motility in rat.

Abstract  Galanin actions are mediated by distinct galanin receptors (GAL‐R), GAL‐R1, ‐R2 and ‐R3. We investigated the role of GAL‐R1 in gastric motility and the expression of GAL‐R1 in the rat stomach. In vivo, in urethane‐anaesthetized rats, galanin (equipotent for all GAL‐Rs) induced a short inhibition of gastric motility, followed by increase in tonic and phasic gastric motility; the latter was significantly reduced by the GAL‐R1 antagonist, RWJ‐57408. Galanin 1–16 (high affinity for GAL‐R1 and ‐R2) induced a long‐lasting decrease of intragastric pressure, which was not modified by RWJ‐57408. In vitro, galanin and galanin 1–16 induced increase of intragastric pressure that was not affected by RWJ‐57408. Tetrodotoxin (TTX) did not suppress the galanin excitatory effect, whereas the effect of galanin 1–16 on gastric contraction was increased by TTX‐ or N‐nitro‐L‐arginine, an inhibitor of nitric oxide synthase. GAL–R1 immunoreactivity was localized to cholinergic and tachykinergic neurons and to neurons immunoreactive for nitric oxide synthase or vasoactive intestinal polypeptide. This study suggests that an extrinsic GAL‐R1 pathway mediates the galanin excitatory action, whereas an extrinsic, non GAL‐R1 pathway is likely to mediate the galanin inhibitory effect in vivo. GAL‐R1 intrinsic neurons do not appear to play a major role in the control of gastric motility.

Identification of galanin receptor 1 on excitatory motor neurons in the guinea pig ileum.

Abstract  Exogenously administered galanin inhibits cholinergic transmission to the longitudinal muscle and reduces peristaltic efficiency in the guinea pig ileum with a mechanism partially mediated by galanin receptor 1 (GAL‐R1). We investigated the effect of exogenous galanin 1–16, which has high affinity for GAL‐R1, on the ascending excitatory reflex of the circular muscle elicited by radial distension in isolated segments of guinea pig ileum. We used a three‐compartment bath that allows dissecting the ascending pathway into the oral (site of excitatory motor neurons), intermediate (site of ascending interneurons) and caudal compartment (site of intrinsic primary afferent neurons). Galanin 1–16 (0.3–3 μmol L−1) applied to the oral compartment inhibited in a concentration‐dependent manner the ascending excitatory reflex elicited by the wall distension in the caudal compartment. This effect was antagonized by the GAL‐R1 antagonist, RWJ‐57408 (1 and 10 μmol L−1). By contrast, galanin 1–16 was ineffective when added to the intermediate or caudal compartment up to 3 μmol L−1. GAL‐R1 immunoreactive neurons did not contain neuron‐specific nuclear protein, a marker for intrinsic primary afferent neurons. These findings indicate that GAL‐R1s are present on motor neurons responsible for the ascending excitatory reflex, but not on ascending interneurons and intrinsic primary afferent neurons.

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea

Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non‐adrenergic, non‐cholinergic (NANC) relaxations in guinea‐pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase‐2 (HO‐2), the CO‐producing enzyme, in tracheal neurons.

ALDH1A3 Is the Key Isoform That Contributes to Aldehyde Dehydrogenase Activity and Affects in Vitro Proliferation in Cardiac Atrial Appendage Progenitor Cells

High aldehyde dehydrogenase (ALDHhi) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDHhi cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDHhi and ALDHlo atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role. Methods and Results: Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDHhi activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34+). Depending on their ALDH activity, RT-PCR analysis of ALDHhi and ALDHlo cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDHhi cells, but not ALDHlo cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDHhi cells gave rise to a higher number of cardiomyocytes compared with ALDHlo cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDHhi atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDHhi cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDHlo cells. Conclusions: ALDH1A3 is the key isoform responsible for ALDH activity in ALDHhi atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects in vitro proliferation of these cells.