Perpétua Pinto-do-Ó

Human umbilical cord tissue-derived mesenchymal stromal cells attenuate remodeling after myocardial infarction by proangiogenic, antiapoptotic, and endogenous cell-activation mechanisms.

IntroductionAmong the plethora of cells under investigation to restore a functional myocardium, mesenchymal stromal cells (MSCs) have been granted considerable interest. However, whereas the beneficial effects of bone marrow MSCs (BM-MSCs) in the context of the diseased heart are widely reported, data are still scarce on MSCs from the umbilical cord matrix (UCM-MSCs). Herein we report on the effect of UCM-MSC transplantation to the infarcted murine heart, seconded by the dissection of the molecular mechanisms at play.MethodsHuman umbilical cord tissue-derived MSCs (UCX®), obtained by using a proprietary technology developed by ECBio, were delivered via intramyocardial injection to C57BL/6 females subjected to permanent ligation of the left descending coronary artery. Moreover, medium produced by cultured UCX® preconditioned under normoxia (CM) or hypoxia (CMH) was collected for subsequent in vitro assays.ResultsEvaluation of the effects upon intramyocardial transplantation shows that UCX® preserved cardiac function and attenuated cardiac remodeling subsequent to myocardial infarction (MI). UCX® further led to increased capillary density and decreased apoptosis in the injured tissue. In vitro, UCX®-conditioned medium displayed (a) proangiogenic activity by promoting the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), and (b) antiapoptotic activity in HL-1 cardiomyocytes subjected to hypoxia. Moreover, in adult murine cardiac Sca-1+ progenitor cells (CPCs), conditioned medium enhanced mitogenic activity while activating a gene program characteristic of cardiomyogenic differentiation.ConclusionsUCX® preserve cardiac function after intramyocardial transplantation in a MI murine model. The cardioprotective effects of UCX® were attributed to paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis, augment proliferation, and activate a pool of resident CPCs. Overall, these results suggest that UCX® should be considered an alternative cell source when designing new therapeutic approaches to treat MI.

Hippo pathway effectors control cardiac progenitor cell fate by acting as dynamic sensors of substrate mechanics and nanostructure

Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full clarification. Here we demonstrate that the paralog proteins YAP and TAZ exert a crucial role in adult cardiac progenitor cell mechano-sensing and fate decision. Cardiac progenitors respond to dynamic modifications in substrate rigidity and nanopattern by promptly changing YAP/TAZ intracellular localization. We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.

Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing

IntroductionThe secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds.MethodsA UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively.ResultsUCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles.ConclusionsThis work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

Assessment of renal dopaminergic system activity during cyclosporine A administration in the rat.

1 Administration of cyclosporine A (CsA; 50 mg kg−1 day−1, s.c.) for 14 days produced an increase in both systolic (SBP) and diastolic (DBP) blood pressure by 60 and 25 mmHg, respectively. The urinary excretion of dopamine, DOPAC and HVA was reduced from day 5–6 of CsA administration onwards (dopamine from 19 to 46%, DOPAC from 16 to 48%; HVA from 18 to 42%). In vehicle‐treated rats, the urinary excretion of dopamine and DOPAC increased (from 7 to 60%) from day 5 onwards; by contrast, the urinary excretion of HVA was reduced (from 27 to 60%) during the second week. 2 No significant difference was observed between the Vmax and Km values of renal aromatic L‐amino acid decarboxylase (AAAD) in rats treated with CsA for 7 and 14 days or with vehicle. 3 Km and Vmax of monoamine oxidase types A and B did not differ significantly between rats treated with CsA for 7 and 14 days or with vehicle. 4 Maximal catechol‐O‐methyltransferase activity (Vmax) in homogenates of renal tissues obtained from rats treated with CsA for 7 or 14 days was significantly higher than that in vehicle‐treated rats; Km(22.3 ± 1.5 μm) values for COMT did not differ between the three groups of rats. 5 The accumulation of newly‐formed dopamine and DOPAC in cortical tissues of rats treated with CsA for 14 days was three to four times higher than in controls. The outflow of both dopamine and DOPAC declined progressively with time and reflected the amine and amine metabolite tissue contents. No significant difference was observed between the DOPAC/dopamine ratios in the perifusate of renal tissues obtained from CsA‐ and vehicle‐treated rats. In addition, no significant differences were observed in k values or in the slope of decline of both DA and DOPAC between experiments performed with CsA and vehicle‐treated animals. 6 The Vmax for the saturable component of L‐3, 4‐dihydroxyphenylalanine (L‐DOPA) uptake in renal tubules from rats treated with CsA was twice that of vehicle‐treated animals. Km in CsA‐ and vehicle‐treated rats did not differ. 7 The decrease in the urinary excretion of sodium and an increase in blood pressure during CsA treatment was accompanied by a reduction in daily urinary excretion of dopamine. This appears to result from a reduction in the amount of L‐DOPA made available to the kidney and does not involve changes in tubular AAAD, the availability of dopamine to leave the renal cells and dopamine metabolism. The enhanced ability of the renal tissues of CsA‐treated animals to synthesize dopamine, when exogenous L‐DOPA is provided, results from an enhanced activity of the uptake process of L‐DOPA in renal tubular cells.

TNF-α Regulates the Effects of Irradiation in the Mouse Bone Marrow Microenvironment

Background Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-α, known to regulate cell apoptosis, could modulate the onset of secondary MDS. Principal Findings We show that TNF-α is induced by irradiation and regulates BM cells apoptosis in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-α deficient (TNF-α KO) mice or WT mice treated with a TNF-α-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-α KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (≥5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression. Conclusion Taken together, our data shows that TNF-α induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression.

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: increase in Na+, K(+)-ATPase activity in renal proximal tubules via activation of 5-HT1A receptors.

1 5‐Hydroxytryptamine (5‐HT) is antinatriuretic. Since this effect of 5‐HT is not accomplished by changes in glomerular haemodynamics, we have examined in this study whether 5‐HT may influence sodium excretion by affecting the Na+, K+‐ATPase activity in renal cortical tubules. 2 Na+, K+‐ATPase activity was determined as the rate of [32P]‐ATP hydrolysis in renal cortical tubules in suspension. Basal Na+,K+‐ATPase activity in renal tubules was 4.8 ± 0.4 μmol Pi mg−1 protein h−1 (n = 8). The 5‐HT1A receptor agonist, (±)‐8‐hydroxy‐2‐(di‐n‐propylamino) tetraline (8‐OH‐DPAT) (10 to 3000 nM) induced a concentration‐dependent increase (P < 0.05) in Na+,K+‐ATPase activity with an EC50 value of 355 nM (95% confidence limits: 178, 708). Maximal stimulation elicited by 3000 nM of 8‐ OH‐DPAT was antagonized by the selective 5‐HT1A receptor antagonist, (+)‐WAY 100135 (10 to 1000 nM) with an IC50 value of 20 nM (14, 29); 0.3 μm (+)‐WAY 100135 completely abolished (P < 0.01) the stimulatory effect of 8‐OH‐DPAT. The stimulatory effect of 8‐OH‐DPAT was found to be time‐ dependent (15 ± 2% and 66 ± 7% increase at 2.5 and 5.0 min, respectively). The 5‐HT2 receptor agonist α‐methyl‐5‐HT (100 to 3000 nM) did not induce any significant changes in Na+‐ATPase activity (5.0 ± 1.5 μmol Pi mg−1 protein h−1; n = 4). 3 The stimulatory effect 8‐OH‐DPAT was absent when homogenates were used. Stimulation occurred at a Vmax concentration (70 mM) of sodium supporting the notion that stimulation occurs independently of increasing sodium permeability. 4 The inhibitory effect of dopamine (P < 0.05) on Na+,K+‐ATPase activity was blunted by coincubation with 8‐OH‐DPAT (0.5 μm). 5 It is concluded that activation of 5‐HT1A receptors increases Na+,K+‐ATPase activity in renal cortical tubules; this effect may represent an important cellular mechanism, at the tubule level, responsible for the antinatriuretic effect of 5‐HT.

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: effects of amine precursors on the cell inward transfer and decarboxylation.

1 The present work was designed to examine the interference of L‐3,4‐dihydroxyphenylalanine (L‐DOPA) on the cell inward transport of L‐5‐hydroxytryptophan (L‐5‐HTP) and on its decarboxylation by aromatic L‐amino acid decarboxylase (AAAD) in rat isolated renal tubules. 2 The accumulation of both L‐5‐HTP and L‐DOPA in renal tubules was found to occur through non‐saturable and saturable mechanisms. The kinetics of the saturable component L‐5‐HTP and L‐DOPA uptake in renal tubules were as follows: L‐5‐HTP, Vmax = 24.9 ± 4.5 nmol mg−1 protein h−1 and Km = 121 (95% confidence limits: 75, 193) μm (n = 5); L‐DOPA, Vmax = 58.0 ± 4.3 nmol mg−1 protein h−1 and Km = 135 (97, 188) μm (n = 5). When the saturation curve of L‐5‐HTP tubular uptake was performed in the presence of L‐DOPA (250 μm), the maximal rate of accumulation of L‐5‐HTP in renal tubules was found to be markedly (P < 0.01) reduced (Vmax = 10.5 ± 1.7 nmol mg−1 protein h−1, n = 4); this was accompanied by a significant (P < 0.05) increase in Km values (325 [199, 531] μm, n = 4). 3 L‐DOPA (50 to 2000 μm) was found to produce a concentration‐dependent decrease (38% to 91% reduction) in the tubular uptake of 5‐HTP; the Ki value (in μm) of L‐DOPA for inhibition of L‐5‐HTP uptake was found to be 29.1 (13.8, 61.5) (n = 6). 4 At the highest concentration tested the organic anion inhibitor, probenecid (10 μm) produced no significant (P = 0.09) changes in L‐5‐HTP and L‐DOPA uptake (18% and 22% reduction, respectively). The organic cation inhibitor, cyanine 863 (1‐ethyl‐2‐[1,4‐dimethyl‐2‐phenyl‐6‐pyrimidinylidene)methyl]‐quinolinium) produced a potent inhibitory effect on the tubular uptake of L‐5‐HTP (Ki = 212 [35, 1289] nM, n = 8), being slightly less effective against L‐DOPA uptake (Ki = 903 [584, 1396] nM, n = 5). The cyanine derivatives 1,1‐diethyl‐2,4‐cyanine (decynium 24) and 1,1‐diethyl‐2,2‐cyanine (decynium 22) potently inhibited the tubular uptake of both L‐5‐HTP (Ki = 100 [49, 204] and 120 [26, 561] nM, n = 4–6, respectively) and L‐DOPA (Ki = 100 [40, 290] and 415 [157, 1094] nM, n = 5, respectively). 5 The Vmax and Km values for AAAD using L‐DOPA as the substrate (Vmax = 479.9 ± 74.0 nmol mg−1 protein h−1; Km = 2380 [1630, 3476] μm; n = 4) were both found to be significantly (P < 0.01) higher than those observed when using L‐5‐HTP (Vmax = 81.4 ± 5.2 nmol mg−1 protein h−1, Km = 97 [87, 107] μm, n = 10). The addition of 5 mM L‐DOPA to the incubation medium reduced by 30% (P < 0.02) the maximal rate of decarboxylation of L‐5‐HTP (Vmax = 56.7 ± 3.1 nmol mg−1 protein h−1, n = 10) and resulted in a significant (P < 0.05) increase in Km values (249 [228, 270] μm, n = 10). 6 The results presented suggest that L‐5‐HTP and L‐DOPA are using the same transporter (most probably, the organic cation transporter) in order to be taken up into renal tubular cells; L‐DOPA exerts a competitive type of inhibition upon the tubular uptake and decarboxylation of L‐5‐HTP. The decrease in the formation of 5‐HT as induced by L‐DOPA may also depend on a decrease in the rate of its decarboxylation by AAAD.

Sca-1+ Cardiac Progenitor Cells and Heart-Making: A Critical Synopsis

The identification, in the adult, of cardiomyocyte turnover events and of cardiac progenitor cells (CPCs) has revolutionized the field of cardiovascular medicine. However, the low rate of CPCs differentiation events reported both in vitro and in vivo, even after injury, raised concerns on the biological significance of these subsets. In this Comprehensive Review, we discuss the current understanding of cardiac Lin(-)Sca-1(+) cells in light of what is also known for cellular compartments with similar phenotypes in other organs. The Lin(-)Sca-1(+) heart subset is heterogeneous and displays a mesenchymal profile, characterized by a limited ability to generate cardiomyocytes in vitro and in vivo, even after injury. There is no evidence for Sca-1 expression in embryonic cardiovascular progenitors. In other organs, Sca-1 expression is mainly observed on mesoderm-derived cells, although it is not restricted to stem/progenitor cell populations. It is urgent to determine, at a single cell level, to which extent cardiac Lin(-)Sca-1(+) cells overlap with the fibroblast compartment.

Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe's disease.

In Krabbes disease, a demyelinating disorder, add‐on strategies targeting the peripheral nervous system (PNS) are needed, as it is not corrected by bone‐marrow (BM) transplantation. To circumvent this limitation of BM transplantation, we assessed whether i.v. delivery of immortalized EGFP+ BM‐derived murine mesenchymal stromal cells (BM‐MSCTERT‐EGFP) targets the PNS of a Krabbes disease model, the Twitcher mouse. In vitro, BM‐MSCTERT‐EGFP retained the phenotype of primary BM‐MSC and did not originate tumors upon transplantation in nude mice. In vivo, undifferentiated EGFP+ cells grafted the Twitcher sciatic nerve where an increase in Schwann cell precursors and axonal number was detected. The same effect was observed on BM‐MSCTERT‐EGFP i.v. delivery following sciatic nerve crush, a model of axonal regeneration. Reiterating the in vivo findings, in a coculture system, BM‐MSCTERT‐EGFP induced the proliferation of Twitcher‐derived Schwann cells and the neurite outgrowth of both Twitcher‐derived neurons and wild‐type neurons grown in the presence of psychosine, the toxic substrate that accumulates in Krabbes disease. In vitro, this neuritogenic effect was blocked by K252a, an antagonist of Trk receptors, and by antibody blockage of brain derived neurotrophic factor, a neurotrophin secreted by BM‐MSCTERT‐EGFP and induced in neighboring Schwann cells. In vivo, BM‐MSCTERT‐EGFP surmounted the effect of K252a, indicating their ability to act through a neurotrophin‐independent mechanism. In summary, i.v. delivery of BM‐MSCTERT‐EGFP exerts a multilevel effect targeting neurons and Schwann cells, coordinately diminishing neuropathology. Therefore, to specifically target the PNS, MSC should be considered an add‐on option to BM transplantation in Krabbes disease and in other disorders where peripheral axonal loss occurs. STEM CELLS 2011;29:1738–1751

Short-Term vs. Sustained Inhibition of Proximal Tubule Na, K-ATPase Activity by Dopamine: Cellular Mechanisms

Dopamine (DA) produces a natriuresis attributed in part to inhibition of Na,K-ATPase activity (NKA) in the proximal tubule (PCT), and impairment in this inhibition has been linked to several forms of hypertension in animals. Here we examined whether the intracellular signaling mechanisms involved are the same in the early and late phases of this phenomenon. DA (1 microM) inhibited NKA similarly after 15 min (by 38%) or 180 min (by 36%) incubation, taken to represent short-term (ST) and sustained (Sd) pump regulation, respectively. Calphostin C, a specific inhibitor of protein kinase C (PKC), completely blocked the ST action of DA on NKA, whereas IP20, a specific inhibitor of protein kinase (PKA), had no effect. In contrast, IP20 completely abolished the Sd (180 min) inhibition by DA, whereas calphostin C had only a partial or variable effect. The DA-1 agonist fenoldopam (which does not activate PKC but increases cAMP) alone failed to inhibit the pump at 180 min (as it does also in the short-term in PCT), suggesting that ST inhibition is required for the Sd effect to occur. Furthermore, PTH1-34, a known ST inhibitor of NKA suppressed the pump at 180 min (by 46%), but unlike in the short-term, this effect was completely prevented by IP20. In contrast, PTH3-34, which does not stimulate adenylyl cyclase or activate PKA, caused only a small (19%) and variable Sd inhibition. In conclusion, short-term inhibition of the PCT pump by dopamine is mediated via PKC, whereas the sustained inhibition requires the PKA pathway in addition to the ongoing PKC-mediated effect.