Angela Chiavegato

An Excitatory Loop with Astrocytes Contributes to Drive Neurons to Seizure Threshold

Studies in rodent brain slices suggest that seizures in focal epilepsies are sustained and propagated by the reciprocal interaction between neurons and astroglial cells

Myosin Heavy-Chain Isoform Composition and Distribution in Developing and Adult Human Aortic Smooth Muscle

The myosin heavy-chain (MHC) composition of developing and adult human aortic smooth muscle (SM) was studied by SDS-polyacrylamide gel electrophoresis, Western blotting and indirect immunofluorescence using a panel of anti-MHC antibodies. On 5% SDS gels, three bands of 204, 200 and 196 kDa apparent molecular mass were identified in fetal, infant and adult stages of development. In the extracts from thoracic aorta (upper level), the 204, and 200-kDa bands (designated as SM-1 and SM-2, respectively) were recognized by SM-G4 and SMMS-1 antibodies, raised against a SM antigen, whereas the 196-kDa band was reactive with nonmuscle (NM)-F6 and NM-G2 antiplatelet MHC antibodies. Western blotting and immunofluorescence tests performed on bovine brain and other human NM tissues using NM-F6 and NM-G2 indicated that antigenic targets of the two antibodies resembled that of so-called IIB and IIA NM myosin found in the bovine system, respectively. In the aortic media, SM-1 was expressed throughout development, while SM-2 was upregulated during late fetal and postnatal development. Similarly, the 196-kDa band showed two distinct patterns of immunoreactivity with the anti-NM-MHC antibodies: with NM-G2, antigenicity was equal at all the developmental stages examined, whereas with NM-F6, it diminished during postnatal development. In the upper level, the cellular distribution of NM-G2 and NM-F6 immunoreactivities was similar in the early fetus but quite distinct at later stages of development. In infant and adult subjects, SM cells (SMC) reactive with NM-F6 accumulated predominantly within the intimal layer as well as in some areas of the underlying media as cell foci, whereas NM-G2 homogeneously stained the two layers. In the aorta near the diaphragm (lower level), both antibodies stained the thickened intima but not the underlying media. These data are consistent with the existence of developmental, stage-specific molecular and cellular transitions during vascular SMC maturation in human aortic media. In addition, these data suggest that IIB-like myosin may be expressed in SMC involved specifically in intimal thickening.

Fast spiking interneuron control of seizure propagation in a cortical slice model of focal epilepsy

In focal epilepsy the propagation of seizure discharges arising at restricted brain sites is opposed by feedforward inhibition. Failure of this inhibition marks focal seizure propagation to distant neurons. The cellular source of inhibition and the mechanism of inhibition failure are, however, undefined. Here we reveal that a subclass of GABAergic interneurons, i.e. the parvalbumin‐expressing, fast‐spiking interneurons, are a main source of the inhibitory signal that locally restrains seizures. Furthermore, a firing impairment in these interneurons, probably due to a drastic membrane depolarization, is an important event that by reducing the overall strength of local inhibition allows seizures to propagate across the cortex. Our data suggest that modulation of fast‐spiking interneuron activity may represent a new therapeutic strategy to prevent generalization of focal epilepsies.

Myosin Isoform Expression in Smooth Muscle Cells during Physiological and Pathological Vascular Remodeling

There is substantial evidence indicating that the study of cytoskeletal and cytocontractile protein composition in vascular smooth muscle cells (SMCs) can be valuable in tracing structural changes during vascular remodeling. Recent nucleic acid and protein investigations suggest that myosin can be used as a new specific marker for the identification of SMC phenotypes in some pathological conditions affecting the vascular wall. In view of this new information, it would seem timely to review the structural bases of myosin isoform expression in the vascular smooth muscle system as well as the factors involved in its regulation. A puzzling feature has arisen in recent studies on this topic: the presence of non-muscle myosin variants in SMCs during physiological and pathological vascular remodeling. In the response to injury caused by mechanical, chemical and hormonal factors in animals, characterized by proliferation and migration of vascular SMCs from the media to the intima, there is a partial or complete recapitulation of a myosin isoform pattern pertinent to developing vascular smooth muscle tissue. Analysis of myosin isoform content in the vascular wall also demonstrates that: (1) changes in SMC composition may occur independent of medial SMC migration into intima, and (2) the presence of fetal-type SMCs in the neointima is not necessarily related to specific positional changes of medial SMCs.

In Vitro and In Vivo Cardiomyogenic Differentiation of Amniotic Fluid Stem Cells

Cell therapy has developed as a complementary treatment for myocardial regeneration. While both autologous and allogeneic uses have been advocated, the ideal candidate has not been identified yet. Amniotic fluid-derived stem (AFS) cells are potentially a promising resource for cell therapy and tissue engineering of myocardial injuries. However, no information is available regarding their use in an allogeneic context. c-kit-sorted, GFP-positive rat AFS (GFP-rAFS) cells and neonatal rat cardiomyocytes (rCMs) were characterized by cytocentrifugation and flow cytometry for the expression of mesenchymal, embryonic and cell lineage-specific antigens. The activation of the myocardial gene program in GFP-rAFS cells was induced by co-culture with rCMs. The stem cell differentiation was evaluated using immunofluorescence, RT-PCR and single cell electrophysiology. The in vivo potential of Endorem-labeled GFP-rAFS cells for myocardial repair was studied by transplantation in the heart of animals with ischemia/reperfusion injury (I/R), monitored by magnetic resonance imaging (MRI). Three weeks after injection a small number of GFP-rAFS cells acquired an endothelial or smooth muscle phenotype and to a lesser extent CMs. Despite the low GFP-rAFS cells count in the heart, there was still an improvement of ejection fraction as measured by MRI. rAFS cells have the in vitro propensity to acquire a cardiomyogenic phenotype and to preserve cardiac function, even if their potential may be limited by poor survival in an allogeneic setting.

Different cardiovascular potential of adult- and fetal-type mesenchymal stem cells in a rat model of heart cryoinjury

Efficacy of adult (bone marrow, BM) versus fetal (amniotic fluid, AF) mesenchymal stem cells (MSCs) to replenish damaged rat heart tissues with new cardiovascular cells has not yet been established. We investigated on the differentiation potential of these two rat MSC populations in vitro and in a model of acute necrotizing injury (ANI) induced by cryoinjury. Isolated BM-MSCs and AF-MSCs were characterized by flow cytometry and cytocentrifugation and their potential for osteogenic, adipogenic, and cardiovascular differentiation assayed in vitro using specific induction media. The left anterior ventricular wall of syngeneic Fisher 344 (n = 48) and athymic nude (rNu) rats (n = 6) was subjected to a limited, nontransmural epicardial ANI in the approximately one third of wall thickness without significant hemodynamic effects. The time window for in situ stem cell transplantation was established at day 7 postinjury. Fluorochrome (CMTMR)-labeled BM-MSCs (2 × 106) or AF-MSCs (2 × 106) were injected in syngeneic animals (n = 26) around the myocardial lesion via echocardiographic guidance. Reliability of CMTMR cell tracking in this context was ascertained by transplanting genetically labeled BM-MSCs or AF-MSCs, expressing the green fluorescent protein (GFP), in rNu rats with ANI. Comparison between the two methods of cell tracking 30 days after cell transplantation gave slightly different values (1420,58 ± 129,65 cells/mm2 for CMTMR labeling and 1613.18 ± 643.84 cells/mm2 for genetic labeling; p = NS). One day after transplantation about one half CMTMR-labeled AF-MSCs engrafted to the injured heart (778.61 ± 156.28 cells/mm2) in comparison with BM-MSCs (1434.50± 173.80 cells/mm2, p < 0.01). Conversely, 30 days after cell transplantation survived MSCs were similar: 1275.26 ± 74.51/mm2 (AF-MSCs) versus 1420.58 ± 129.65/mm2 for BM-MSCs (p = NS). Apparent survival gain of AF-MSCs between the two time periods was motivated by the cell proliferation rate calculated at day 30, which was lower for BM-MSCs (6.79 ± 0.48) than AF-MSCs (10.83 ± 3.50; p < 0.01), in the face of a similar apoptotic index (4.68 ± 0.20 for BM-MSCs and 4.16 ± 0.58 for AF-MSCs; p = NS). These cells were also studied for their expression of markers specific for endothelial cells (ECs), smooth muscle cells (SMCs), and cardiomyocytes (CMs) using von Willebrand factor (vWf), smooth muscle (SM) α-actin, and cardiac troponin T, respectively. Grafted BM-MSCs or AF-MSCs were found as single cell/small cell clusters or incorporated in the wall of microvessels. A larger number of ECs (227.27 ± 18.91 vs. 150.36 ± 24.08 cells/mm2, p < 0.01) and CMs (417.91 ± 100.95 vs. 237.43 ± 79.99 cells/mm2, p < 0.01) originated from AF-MSCs than from BM-MSCs. Almost no SMCs were seen with AF-MSCs, in comparison to BM-MSCs (98.03 ± 40.84 cells/mm2), in concordance with lacking of arterioles, which, instead, were well expressed with BM-MSCs (71.30 ± 55.66 blood vessels/mm2). The number of structurally organized capillaries was slightly different with the two MSCs (122.49± 17.37/mm2 for AF-MSCs vs. 148.69 ± 54.41/mm2 for BM-MSCs; p = NS). Collectively, these results suggest that, in the presence of the same postinjury microenvironment, the two MSC populations from different sources are able to activate distinct differentiation programs that potentially can bring about a myocardial-capillary or myocardial-capillary-arteriole re-constitution.

Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain

Myotoxins play a major role in the pathogenesis of the envenomations caused by snake bites in large parts of the world where this is a very relevant public health problem. We show here that two myotoxins that are major constituents of the venom of Bothrops asper, a deadly snake present in Latin America, induce the release of large amounts of K+ and ATP from skeletal muscle. We also show that the released ATP amplifies the effect of the myotoxins, acting as a “danger signal,” which spreads and causes further damage by acting on purinergic receptors. In addition, the release of ATP and K+ well accounts for the pain reaction characteristic of these envenomations. As Bothrops asper myotoxins are representative of a large family of snake myotoxins with phospholipase A2 structure, these findings are expected to be of general significance for snake bite envenomation. Moreover, they suggest potential therapeutic approaches for limiting the extent of muscle tissue damage based on antipurinergic drugs.

Human Bone Marrow-Derived CD133+ Cells Delivered to a Collagen Patch on Cryoinjured Rat Heart Promote Angiogenesis and Arteriogenesis:

Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133+-derived cells (BM-CD133+ cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133+ cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133+cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo—and in both groups of nude rats transplanted with BM-CD133+—there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133+cells into the injured heart. Although chimeric human–rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5–2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133+ cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133+ cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the “traditional” method of cell injection into the myocardium.

Phenotypic changes in the regenerating rabbit bladder muscle. Role of interstitial cells and innervation on smooth muscle cell differentiation.

Abstract We have studied the phenotypic changes in regenerating smooth muscle (SM) tissue of detrusor muscle after local application of a necrotizing, freeze–thaw injury to the serosal surface of rabbit bladder. Bromo-deoxyuridine (BrdU) incorporation and immunofluorescence studies were performed on bladder cryosections from day 2 up to day 15 after surgery with monoclonal antibodies specific for some cytoskeletal markers [desmin, vimentin, non-muscle (NM) myosin] and for SM-specific proteins (α-actin, myosin, and SM22). Four days after lesion, some clls incorporated in regenerating SM bundles are BrdU positive, but all display a phenotypic pattern identical to that of the interstitial, highly proliferating cells, i.e., expression of vimentin. By days 7–15 the differentiation profile of regenerating SM returns to that of uninjured SM tissue (appearance of desmin, SM-type α-actin, and SM myosin). A chemical denervation achieved by 6-hydroxydopamine treatment for 2 weeks induces the formation of vimentin/SM α-actin/NM myosin/SM22-containing myofibroblasts in the interstitial, fibroblast-like cells of uninjured bladder. In the bladder wall, alteration of reinnervation during the regenerating SM process produces: (1) in the outer region, the activation of vimentin/SM α-actin/desmin myofibroblasts in the de novo SM cell bundles; and (2) in the inner region of bladder, including the muscularis mucosae, the formation of proliferating, fully differentiated SM cells peripherally to newly formed SM cell bundles. These findings suggest that: (1) the de novo SM tissue formation in the bladder can occur via incorporation of interstitial cells into growing SM bundles; and (2) the alteration of reinnervation during the regenerating process induces a spatial-specific differentiation of interstitial myofibroblasts in SM cells before SM cell bundling.

Transforming growth factor beta1 involvement in the conversion of fibroblasts to smooth muscle cells in the rabbit bladder serosa.

In an attempt to identify the growth factors or cytokines involved in the serosal thickening that occurs in rabbit bladder subjected to partial outflow obstruction, the following growth factors – transforming growth factor β1, platelet-derived growth factor, epidermal growth factor, granulocyte colony-stimulating factor and granulocyte–monocyte colony-stimulating factor – were delivered separately onto the serosal surface of the intact bladder via osmotic minipumps. The proliferative/differentiative cellular response of the rabbit bladder wall was evaluated by bromodeoxyuridine incorporation and immunofluorescence staining with a panel of monoclonal antibodies to cytoskeletal proteins (desmin, vimentin, keratins 8 and 18 and non-muscle myosin) and to smooth muscle (α-actin, myosin and SM22) proteins. Administration of the transforming growth factor, but not of the other growth factors/cytokines, was effective in inducing serosal thickening. Accumulating cells in this tissue were identified as myofibroblasts, i.e. cells showing a mixed fibroblast–smooth muscle cell differentiation profile. The phenotypic pattern of myofibroblasts changed in a time-dependent manner: 21 days after the growth factor delivery, small bundles of smooth muscle cells were found admixed with myofibroblasts, as occurs in the obstructed bladder. These ‘ectopic’ muscle structures displayed a variable proliferating activity and expressed an immature smooth muscle cell phenotype. The complete cellular conversion to smooth muscle cells was not achieved if transforming growth factor β1 was delivered to fibroblasts of subcutaneous tissue. These findings suggest a tissue-specific role for this growth factor in the cellular conversion from myofibroblast to smooth muscle cells.