Role of the RhoA/ROCK pathway in high-altitude associated neonatal pulmonary hypertension in lambs.

Exposure to high-altitude chronic hypoxia during pregnancy may cause pulmonary hypertension in neonates, as a result of vasoconstriction and vascular remodeling. We hypothesized that susceptibility to pulmonary hypertension, due to an augmented expression and activity of the RhoA/Rho-kinase (ROCK) pathway in these neonates, can be reduced by daily administration of fasudil, a ROCK inhibitor. We studied 10 highland newborn lambs with conception, gestation, and birth at 3,600 m in Putre, Chile. Five highland controls (HLC) were compared with 5 highland lambs treated with fasudil (HL-FAS; 3 mg·kg(-1)·day(-1) iv for 10 days). Ten lowland controls were studied in Lluta (50 m; LLC). During the 10 days of fasudil daily administration, the drug decreased pulmonary arterial pressure (PAP) and resistance (PVR), basally and during a superimposed episode of acute hypoxia. HL-FAS small pulmonary arteries showed diminished muscular area and a reduced contractile response to the thromboxane analog U46619 compared with HLC. Hypoxia, but not fasudil, changed the protein expression pattern of the RhoA/ROCKII pathway. Moreover, HL-FAS lungs expressed less pMYPT1(T850) and pMYPT1T(696) than HLC, with a potential increase of the myosin light chain phosphatase activity. Finally, hypoxia induced RhoA, ROCKII, and PKG mRNA expression in PASMCs of HLC, but fasudil reduced them (HL-FAS) similarly to LLC. We conclude that fasudil decreases the function of the RhoA/ROCK pathway, reducing the PAP and PVR in chronically hypoxic highland neonatal lambs. The inhibition of ROCKs by fasudil may offer a possible therapeutic tool for the pulmonary hypertension of the neonates.

Entrenamiento de la capacidad aeróbica por medio de la terapia acuática en niños con parálisis cerebral tipo diplejía espástica / Training aerobic capacity through the aquatic therapy in children with cerebral palsy type spastic diplegia

Antecedentes. La parálisis cerebral tipo diplejía espática genera cambios en el sistema cardiovascular que afectan la capacidad aeróbica. La terapia acuática es una estrategia terapéutica óptima tanto para el manejo de la población como para el entrenamiento de la capacidad aeróbica, por las respuestas fisiológicas que genera y porque brinda la facilidad de generar mayores cargas al sistema cardiovascular con menores riesgos que en tierra. Objetivo. Identificar las características que debe tener una propuesta de intervención fisioterapéutica para el entrenamiento de la capacidad aérobica en niños y niñas entre los 8 y los 12 años, con parálisis cerebral tipo diplejía espástica, empleando la terapia acuática. Materiales y métodos. Se desarrolló un estudio de tipo descriptivo-propositivo, en el cual se formuló una propuesta de intervención basada en información recolectada a través de referencias bibliográficas. Resultados. Se presentan en forma de propuesta de intervención, describiendo en detalle las fases del entrenamiento de la capacidad aeróbica, mediante los principios del entrenamiento y la prescripción de ejercicio físico, teniendo en cuenta las respuestas fisiológicas ante la carga, así como las características propias de la población. Conclusión. La parálisis cerebral tipo diplejía espástica, genera cambios en la capacidad aeróbica; por esto, el fisioterapeuta debe incluirla en los procesos de rehabilitación como uno de sus objetivos. Para lograrlo, la terapia acuática es una modalidad de tratamiento óptima, puesto que genera mayor seguridad de movimiento y respuestas fisiológicas favorables en el sistema cardiovascular.

Background. The cerebral palsy is a condition that generates changes in the cardiovascular system affecting the aerobic capacity. Aquatic therapy is an optimal therapeutic strategy for the management of both the population and the aerobic capacity training because of the physiological responses generated and the ease of generating higher loads on the cardiovascular system with lower risk than on land. Objective. The aim of this research was to identify the characteristics of a physiotherapy intervention proposal for children aged 8 to 12 years-old with spastic diplegia cerebral palsy, using aquatic therapy for aerobic capacity training. Materials and methods. The research reflects a descriptive-proposal study, in which an intervention proposal was made based on information collected through references. Results. Results are presented in the form of intervention proposal that seeks to describe in detail the stages of aerobic training based on the principles of training and prescribing exercise, taking into account the physiological responses to the load and the characteristics of the population. Conclusion. The cerebral palsy caused changes in the aerobic capacity therefore it must be included in the physiotherapist rehabilitation processes as one of its main objectives. The findings indicate that aquatic therapy is an excellent treatment method for training aerobic capacity in this population, because of the greater security of movement and the favorable physiological responses that are generated in the cardiovascular system.

Extracellular Trypanosoma cruzi calreticulin in the host-parasite interplay.

Calreticulin (CRT) from vertebrates is a calcium-binding protein present mainly in the endoplasmic reticulum (ER). There, it directs the conformation of proteins and controls calcium levels. This review will focus on several extracellular roles of Trypanosoma cruzi CRT (TcCRT) in relation to its capacity to inhibit the complement system, mediate parasite infectivity, interfere with angiogenesis and, as a possible consequence, with tumor growth. The TcCRT antiangiogenic effect parallels with the capacity of T. cruzi infection to inhibit tumor development in vivo. Thus, the TcCRT, complement, and endothelial cell interactions seem to be an evolutionary adaptation to promote prolonged parasite-host relationships.

Trypanosoma cruzi calreticulin: a novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity.

In Trypanosoma cruzi, calreticulin (TcCRT) translocates from the endoplasmic reticulum (ER) to the area of flagellum emergence. We propose herein that the parasite uses this molecule to capture complement C1, in an infective apoptotic mimicry strategy. Thus, TcCRT/C1 interactions, besides inhibiting the classical pathway of complement activation as previously shown in our laboratories, will also promote infectivity. This fact correlates with significant increases in TcCRT mRNA levels during early infection stages of a VERO cell line. In vitro, the collagenous and globular C1q domains simultaneously bind TcCRT and antigen aggregated Igs, respectively. Accordingly, mouse immunizations with TcCRT induced humoral responses that, after challenge, correlated with increased parasitemia. Thus, on the parasite surface, whole Igs anti-TcCRT promote C1 deposits on trypomastigotes while, as expected, F(ab')2 fragments decrease it. Likewise, pretreatment of the parasites with whole anti-TcCRT antibodies augmented parasitemia and mortality in mice. In contrast, pretreatment with F(ab')2 fragments anti-TcCRT, devoid of their capacity to provide additional C1q binding sites, was protective. Most important, while pretreatment of trypomastigotes with C1q increased infectivity in the RAW murine cell line, as well as mice mortality and parasitemia, the F(ab')2 fragments significantly interfered with the C1q-dependent infectivity. Differently from other surface molecules involved in infectivity, TcCRT uses C1 as an adaptor molecule to recognize host cells. As expected, since TcCRT is one of several cell surface parasite molecules participating in infectivity, attempts to interfere with the C1/TcCRT interactions with F(ab')2 fragments, were moderately but significantly effective, both in vitro and in vivo.

Antiangiogenic and antitumor effects of Trypanosoma cruzi Calreticulin.

BACKGROUND: In Latin America, 18 million people are infected with Trypanosoma cruzi, the agent of Chagas' disease, with the greatest economic burden. Vertebrate calreticulins (CRT) are multifunctional, intra- and extracellular proteins. In the endoplasmic reticulum (ER) they bind calcium and act as chaperones. Since human CRT (HuCRT) is antiangiogenic and suppresses tumor growth, the presence of these functions in the parasite orthologue may have consequences in the host/parasite interaction. Previously, we have cloned and expressed T. cruzi calreticulin (TcCRT) and shown that TcCRT, translocated from the ER to the area of trypomastigote flagellum emergence, promotes infectivity, inactivates the complement system and inhibits angiogenesis in the chorioallantoid chicken egg membrane. Most likely, derived from these properties, TcCRT displays in vivo inhibitory effects against an experimental mammary tumor. METHODOLOGY AND PRINCIPAL FINDINGS: TcCRT (or its N-terminal vasostatin-like domain, N-TcCRT) a) Abrogates capillary growth in the ex vivo rat aortic ring assay, b) Inhibits capillary morphogenesis in a human umbilical vein endothelial cell (HUVEC) assay, c) Inhibits migration and proliferation of HUVECs and the human endothelial cell line Eahy926. In these assays TcCRT was more effective, in molar terms, than HuCRT: d) In confocal microscopy, live HUVECs and EAhy926 cells, are recognized by FITC-TcCRT, followed by its internalization and accumulation around the host cell nuclei, a phenomenon that is abrogated by Fucoidin, a specific scavenger receptor ligand and, e) Inhibits in vivo the growth of the murine mammary TA3 MTXR tumor cell line. CONCLUSIONS/SIGNIFICANCE: We describe herein antiangiogenic and antitumor properties of a parasite chaperone molecule, specifically TcCRT. Perhaps, by virtue of its capacity to inhibit angiogenesis (and the complement system), TcCRT is anti-inflammatory, thus impairing the antiparasite immune response. The TcCRT antiangiogenic effect could also explain, at least partially, the in vivo antitumor effects reported herein and the reports proposing antitumor properties for T. cruzi infection.

Molecular mechanisms involved in the inactivation of the first component of human complement by Trypanosoma cruzi calreticulin.

Trypanosoma cruzi (T. cruzi), the agent of Chagas' disease, the sixth most important neglected tropical disease worldwide, causes 50,000 deaths per year in Latin America. T. cruzi calreticulin (TcCRT), a highly pleiotropic chaperone molecule, plays important roles in several host/parasite interactions. Among other functions, we have previously shown that TcCRT, translocated from the endoplasmic reticulum to the area of flagellar emergence, binds human C1q and inhibits activation of the classical pathway in vitro. Based on a series of in vitro experiments, we propose here two mechanisms to explain how TcCRT inhibits the classical pathway at the initial stages of C1 (q, r, s) activation. First, TcCRT interacts in vitro with both solid phase bound active C1s and C1, but impairment of C4 activating capacity is evident only when the serine proteases are within the structural context of the macromolecular first component. Although C1s activity, in this context, is inhibited by TcCRT, the serine protease is not displaced from the C1 complex. Second, TcCRT prevents C1 formation, by interfering with the ability of the (C1r-C1s)(2) tetramer to bind C1q. These complement inhibitory effects are better explained by direct interaction of the parasite protein with C1, rather than by the TcCRT capacity to bind calcium, an essential element for the functional integrity of C1.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens.

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

Trypanosoma cruzi calreticulin: a possible role in Chagas' disease autoimmunity.

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas' disease, an endemic and chronic illness that affects 18 million people in Latin America. The mechanisms underlying its pathogenesis are controversial. There is a growing body of evidence supporting the view that T. cruzi infection elicits severe autoimmune responses in the host, which significantly contribute to the pathogenesis of Chagas' disease, and several recent studies have reported the presence of autoantibodies and effector T lymphocytes against parasite and self antigens in infected patients and experimentally infected animals. T. cruzi calreticulin (TcCRT) is a 45kDa protein, immunogenic in humans, rabbits and mice. It has a high degree of homology with human (HuCRT) and mouse calreticulin (MoCRT), which would explain why an immune response to TcCRT could contribute to autoimmune reactions in Chagas' disease. Anti-TcCRT antibodies generated in A/J mice immunized with recombinant TcCRT (rTcCRT) reacted with rHuCRT and bound to neonatal and adult isogenic cardiomyocytes cultured in vitro. Interestingly, histological alterations, such as edema formation and cell infiltrates, which include CD3(+) cells, were detected in heart sections from immunized animals. Therefore, in rTcCRT-immunized mice, an autoimmune reaction against host CRT, paralleled by histological cardiac alterations, suggests a role of the parasite molecule in the induction of immunologically mediated heart tissue damage. The data presented here propose that TcCRT participates in the induction of cardiac autoimmunity in Chagas' disease.

High-salt diet inhibits expression of angiotensin type 2 receptor in resistance arteries.

Recent studies suggested that type 2 angiotensin receptor (AT2R) could contribute to regulation of blood pressure and/or vascular remodeling. A key question relates to the effects of potential modulators of vascular AT2R expression. In the present work, we evaluated if high salt intake (70 mmol/L NaCl in drinking water) could modulate rat mesenteric artery AT2R function and expression. Angiotensin II dose-response curves were studied in rat perfused pressurized small-diameter arteries in the presence of losartan (AT1R antagonist). Arteries were precontracted with phenylephrine, yielding approximately 30% decrease in resting diameter. AT2R activation by angiotensin-induced dose-dependent relaxation of precontracted arteries (60.1+/-9.1% of phenylephrine-induced contraction, P<0.05). In contrast, AT2R-dependent relaxation was not observed in arteries obtained from rats on high-salt diet. Semi-quantitative reverse-transcription polymerase chain reaction experiments demonstrated reduced amount of AT2R mRNA in arteries of rats on high-salt diet (65.5+/-7.5% of control levels, P<0.05). Western blot studies demonstrated decreased AT2R in mesenteric artery protein fractions of high-salt diet rats (60.0+/-18.0 of control levels, P<0.05). In a second set of experiments, adrenalectomy (4 days) blunted AT2R-mediated vasorelaxation and decreased AT2R mRNA (72.0+/-11.0% of control levels, P<0.05). AT2R abundance in protein fractions of mesenteric arteries of ADX rats was also diminished (64.0+/-13% of control levels, P<0.05). Both, AT2R mRNA and protein downregulation were prevented by mineralocorticoid replacement therapy. Finally, physiological concentrations of aldosterone caused a dose-dependent increase in AT2R mRNA of small diameter mesenteric artery explants. The results are consistent with aldosterone-mediated upregulation AT2R.

Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors.

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.

Interleukin-1beta increases spinal cord wind-up activity in normal but not in monoarthritic rats.

Cytokines produced by spinal cord glia after peripheral inflammation, infection or trauma have a relevant role in the maintenance of pain states. The effect of intrathecally administered interleukin-1beta (IL-1beta) on spinal cord nociceptive transmission was studied in normal and monoarthritic rats by assessing wind-up activity in a C-fiber-mediated reflex paradigm evoked by repetitive (1 Hz) electric stimulation. Low i.t. doses of IL-1beta (0.03, 0.12, 0.5 and 2.0 ng) dose-dependently enhanced wind-up activity in normal rats, while higher doses (8.0 ng) only produced a marginal unsignificant effect. IL-1beta administration to monoarthritic rats did not significantly change wind-up scores at any dose. Adaptive changes developed in the spinal cord during chronic pain may underlie the ineffectiveness of exogenous IL-1beta to up-regulate nociceptive transmission.