Regulatory CD4+ CD25+ Foxp3+ T cells expand during experimental Plasmodium infection but do not prevent cerebral malaria

Pathogenic CD8+ T cells are implicated in the physiopathological mechanisms leading to experimental cerebral malaria (CM) in Plasmodium berghei ANKA (PbA) infected mice. Therefore, we hypothesised that in CM susceptible mice the neuropathology could be, at least in part, the result of an inefficient control of pathogenic effector T cells by CD4+ CD25+ Treg cells. Remarkably, the number of CD4+ CD25high T cells expressing Foxp3 increased in the spleen during the course of infection. These cells displayed an activated phenotype and consistent with that, CD4+ CD25high Treg cells isolated from PbA-infected mice showed an enhanced regulatory activity in vitro. Surprisingly, these cells do not migrate to the brain at the time of neurological symptoms as the conventional CD4+ T cells do. CM was not exacerbated in anti-CD25 treated mice when infected with PbA one month after treatment, even if splenic CD8+ T cells expressing CD69 increased in these mice. Taken together, these results show that P. berghei infection leads to an increase of the number of splenic CD4+ CD25high Treg cells exhibiting in vitro suppressive function, but they do not seem to be involved in vivo in the protection against CM.     

Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions

Pulmonary veins show greater sensitivity to endothelin (ET)-1-induced vasoconstriction than pulmonary arteries, and remodeling was observed in pulmonary veins under hypoxic conditions. We examined, using an immunohistochemical method, the expression of Big ET-1, ET-converting enzyme (ECE), and ET(A) and ET(B) receptors in rat pulmonary veins under normoxic and hypoxic conditions. In control rats, Big ET-1 and ECE were coexpressed in the intima and media of the pulmonary veins, with an even distribution along the axial pathway. ET(A) and ET(B) receptors were expressed in the pulmonary veins, with a predominant distribution in the proximal segments. The expression of Big ET-1 was more abundant in the pulmonary veins than in the pulmonary arteries. After exposure to hypoxia for 7 or 14 days, the expression of Big ET-1, ECE, and ET receptors increased in small pulmonary veins. Increases in the medial thickness, wall thickness, and immunoreactivity for alpha-smooth muscle actin were also observed in the small pulmonary veins under hypoxic conditions. The upregulation of ET-1 and ET receptors in the small pulmonary veins is associated with vascular remodeling, which may lead to the development of hypoxic pulmonary hypertension.     

Cognitive Dysfunction Is Sustained after Rescue Therapy in Experimental Cerebral Malaria,and Is Reduced by Additive Antioxidant Therapy

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.     

Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells.

Growing evidence suggests that a pressure-induced increase in the synthesis of endothelin (ET-1) is involved in arterial remodeling and, as a consequence, in the manifestation of chronic hypertension. To study potential stretch-induced changes in gene expression and their functional consequences, we have cultured rat aortic smooth muscle cells (raSMC) and porcine aortic endothelial cells (PAEC) on flexible elastomer membranes. The cells were periodically stretched (up to 20% elongation, 0.5 Hz, 6 h) and the expression of prepro-ET-1 and that of the endothelin A and B receptors (ET(A)-R and ET(B)-R) were analyzed by semi-quantitative RT-PCR analysis and ELISA (ET-1). In contrast to PAEC where ET-1 synthesis was up-regulated up to eightfold on exposure to cyclic stretch, ET-1 synthesis in raSMC was decreased by more than 80% under these conditions. ET(A) R -mRNA expression in stretched raSMC declined to 50% whereas ET(B) R -mRNA levels were increased up to 10-fold. One functional consequence of this apparent shift in receptor abundance was an apoptosis-promoting action of exogenous ET-1 (10 nM), as judged by the appearance of subdiploid peaks during FACS analysis, caspase-3 activation and chromatin condensation. This ET-1-induced apoptosis appeared to be ET(B)-R mediated, as it was completely suppressed by the ET(B)-R antagonist BQ 788 but not by the ET(A)-R antagonist BQ 123. Moreover, raSMC derived from homozygous spotting lethal rats, which lack a functional ET(B)-R, showed no signs of apoptosis after exposure to cyclic strain and exogenous ET-1. These findings suggest a central role for the endothelin system in the onset of hypertension-induced remodeling in conduit arteries, which may proceed via an initial stretch-induced apoptosis of the smooth muscle cells.     

Perforin mediated apoptosis of cerebral microvascular endothelial cells during experimental cerebral malaria

Cerebral malaria is a serious complication of Plasmodium falciparum infection. We have investigated the role of perforin in the pathogenesis of cerebral malaria in a murine model (Plasmodium berghei ANKA (PbA) infection). C57BL/6 mice demonstrated the typical neuropathological symptoms of experimental cerebral malaria infection from day 5p.i. and became moribund on day 6p.i. This pathology was not seen in PbA-infected, perforin-deficient (pfp-/-) mice. From days 5-6p.i. onwards there was a significant increase in mRNA for granzyme B and CD8, but not CD4, in brain tissue from PbA-infected C57BL/6 and pfp-/- mouse brains. Perforin mRNA was strongly increased in the brains of PbA-infected C57BL/6 mice on day 6p.i. Immunohistochemistry revealed increased perforin staining and elevated numbers of CD8(+) cells within the cerebral microvessels in PbA-infected C57BL/6 at days 5 and 6p.i. compared with uninfected animals. At day 6p.i., there were TUNEL-positive cells and activated caspase-3 positive cells of endothelial morphology in the CNS of PbA-infected C57BL/6 mice. The TUNEL-positive cells were greatly reduced in pfp-/- mice. These results suggest that CD8(+)T lymphocytes induce apoptosis of endothelial cells via a perforin-dependent process, contributing to the fatal pathogenic process in murine cerebral malaria.     

Recombinant human IFN-alpha inhibits cerebral malaria and reduces parasite burden in mice

Most C57BL/6 mice infected i.p. with Plasmodium berghei ANKA (PbA) die between 7 and 14 days with neurologic signs, and the remainder die later (>15 days) with severe anemia. Daily i.p. injections of a recombinant human IFN-alpha (active on mouse cells) prevented death by cerebral malaria (87% deaths in the control mice vs 6% in IFN-alpha-treated mice). The mechanisms of this IFN-alpha protective effect were multiple. IFN-alpha-treated, PbA-infected mice showed 1) a marked decrease in the number of PbA parasites in the blood mediated by IFN-gamma, 2) less sequestered parasites in cerebral vessels, 3) reduced up-regulation of ICAM-1 expression in brain endothelial cells, 4) milder rise of blood levels of TNF, 5) increased levels of IFN-gamma in the blood resulting from an increased production by splenic CD8+ T cells, and 6) fewer leukocytes (especially CD8+ T cells) sequestered in cerebral vessels. On the other hand, IFN-alpha treatment did not affect the marked anemia observed in PbA-infected mice. Survival time in IFN-alpha-treated mice was further increased by performing three blood transfusions over consecutive days.     

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.     

Parasite burden and CD36-mediated sequestration are determinants of acute lung injury in an experimental malaria model

Although acute lung injury (ALI) is a common complication of severe malaria, little is known about the underlying molecular basis of lung dysfunction. Animal models have provided powerful insights into the pathogenesis of severe malaria syndromes such as cerebral malaria (CM); however, no model of malaria-induced lung injury has been definitively established. This study used bronchoalveolar lavage (BAL), histopathology and gene expression analysis to examine the development of ALI in mice infected with Plasmodium berghei ANKA (PbA). BAL fluid of PbA-infected C57BL/6 mice revealed a significant increase in IgM and total protein prior to the development of CM, indicating disruption of the alveolar-capillary membrane barrier-the physiological hallmark of ALI. In contrast to sepsis-induced ALI, BAL fluid cell counts remained constant with no infiltration of neutrophils. Histopathology showed septal inflammation without cellular transmigration into the alveolar spaces. Microarray analysis of lung tissue from PbA-infected mice identified a significant up-regulation of expressed genes associated with the gene ontology categories of defense and immune response. Severity of malaria-induced ALI varied in a panel of inbred mouse strains, and development of ALI correlated with peripheral parasite burden but not CM susceptibility. Cd36(-/-) mice, which have decreased parasite lung sequestration, were relatively protected from ALI. In summary, parasite burden and CD36-mediated sequestration in the lung are primary determinants of ALI in experimental murine malaria. Furthermore, differential susceptibility of mouse strains to malaria-induced ALI and CM suggests that distinct genetic determinants may regulate susceptibility to these two important causes of malaria-associated morbidity and mortality.     

Biochemical properties of endothelin converting enzyme in renal epithelial cell lines.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme (ECE) in non-vascular cells (renal epithelial cell lines, MDCK and LLC-PK1). ECEs derived from these epithelial cells were very similar to the endothelial ECE in the following biochemical properties: 1) The optimum pH was 7.0; 2) the Km value for big ET-1 was approximately 30 microM; 3) the enzyme was potently inhibited by EDTA, o-phenanthroline and phosphoramidon; and 4) the enzyme did not convert big ET-2 or big ET-3. These data suggest that phosphoramidon-sensitive ECE is involved in the processing of big ET-1 to ET-1 in the renal tubule.     

Induction of the oxLDL receptor LOX-1 by endothelin-1 in human endothelial cells.

In this study, we analyzed the effect of endothelin-1 (ET-1) on expression of the lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 LOX-1 and on oxLDL uptake in primary cultures of human umbilical vein endothelial cells (HUVEC). LOX-1 mRNA was quantified by standard-calibrated competitive RT-PCR, LOX-1 protein expression by Western analysis and endothelial oxLDL uptake using DiI-labeled oxLDL. ET-1 induces LOX-1 mRNA expression, reaching its maximum after 1 h (160 +/- 14% of control, 100 nM ET-1, P < 0.05). This increased ET-1-mediated LOX-1 mRNA expression could be inhibited by endothelin receptor B antagonist BQ-788. In addition, ET-1 stimulates LOX-1 protein expression and oxLDL uptake in HUVEC. The augmented oxLDL uptake by ET-1 is mediated by endothelin receptor B, but not by protein kinases. These data support a new pathophysiological mechanism how locally and systemically increased ET-1 levels could promote LOX-1-mediated oxLDL uptake in human endothelial cells and the development and progression of endothelial dysfunction and atherosclerosis.     

Endothelin-stimulated Ca(2+)signaling and endothelin receptor expression are decreased by parathyroid hormone treatment in UMR-106 osteoblastic osteosarcoma cells

Modulation of endothelin (ET-1)-induced [Ca(2+)](i)transients and receptor expression by parathyroid hormone (PTH) was studied in UMR-106 osteoblastic osteosarcoma cells. Ca(2+)signaling was assessed with Fura-2, and ET receptor mRNA expression was determined using ET(A)- and ET(B)-specific primers and RT-PCR amplification. ET-1 binding in UMR-106 cell membranes was also measured. PTH pretreatment for 8 h decreased the [Ca(2+)](i)transients elicited by ET-1 and by the ET(B)-selective agonist sarafotoxin 6c (S6c). When ET(B)receptors were desensitized by pretreatment with S6c or blocked with the ET(B)-selective antagonist BQ-788, the remaining ET(A)component of the signal was also decreased by PTH pretreatment. In contrast, [Ca(2+)](i)transients elicited by PGF(2alpha)and ionomycin were increased following PTH pretreatment, indicating that the effect of PTH to decrease ET-1-stimulated transients was selective. PTH pretreatment also decreased [(125)I]ET-1 binding and ET(A)and ET(B)mRNA, with maximal effects at approximately 8 h. ET-1 was not detectable in medium from either control or PTH treated UMR-106 cultures, suggesting that the decreased expression of ET receptors was not due to enhanced ET production and subsequent homologous desensitization. The downregulation of ET receptors in osteoblasts by PTH pretreatment may serve as a homeostatic mechanism in bone.     

Characterization of the contractile and relaxant action of the endothelin-1 precursor, big endothelin-1, in the isolated rat basilar artery

The presence of functional endothelin converting enzyme (ECE) activity in basilar artery ring segments was investigated by measuring the contractile and relaxant effects of big endothelin (ET)-1. Under resting tension conditions cumulative application of big ET1-1 elicited a concentration-related contraction with the concentration-effect curve (CEC) shifted to the right against ET-1 by a factor of 31 and 29 in segments with the endothelium intact or mechanically removed, respectively. Preincubation with the ET(A) receptor antagonist, BQ123, induced an apparently parallel rightwards shift without affecting the maximum contraction. This shift was more pronounced for ET-1 than for big ET-1. With the putative ECE inhibitor phosphoramidon (10(-3) M) in the bath a small rightwards shift of the CEC for big ET-1 was observed in control segments and a more marked one in de-endothelialized segments. In segments precontracted with prostaglandin (PG) F(2alpha) big ET-1 induced a significant although transient relaxation whereas ET-1 did not. However, in the presence of BQ123 both ET-1 and big ET-1 elicited concentration-related relaxation with a significantly higher maximum effect obtained with big ET-1. The potency was 13 fold higher for ET-1, which is markedly less than that found for contraction. The results, therefore, suggest 1) the presence of functional ECE-activity in the rat basilar artery wall, and 2) differences in the functional ECE activity located in the endothelium and media.     

The endothelin system in human and monkey ovaries: in situ gene expression of the different components

The endothelin system is composed of three endothelin isoforms (ET-1, ET-2, and ET-3), the endothelin receptors ETA and ETB, and the endothelin-converting enzyme (ECE). Besides having a major vasoactive role, endothelins have roles in different cell types at a local level. We investigated the presence of the different components of the endothelin system in primate ovaries. Human ovaries and gonadotropin-stimulated monkey ovaries were studied using immunohistochemistry for endothelin, and in situ hybridization with probes for ET-1, ET-2, ET-3, ETA and ETB receptors, and ECE. ET-1 and ETA receptors were detected in endothelial cells and vascular smooth muscle cells, respectively, in stromal vessels adjacent to follicles and corpora lutea. ETB receptors and ET-1 were found in the endothelial cells of capillaries of corpora lutea. ECE was present in internal theca cells of secondary, de Graaf, atretic follicles, and in luteinized granulosa cells of the corpora lutea. The endothelin system components are present in or around the follicles of human and monkey ovaries. Although the components are not expressed in the same cell types, they are synthesized, mainly in follicles, by cells that are in close proximity. Thus, the endothelin system could act in a paracrine manner. ECE expression in steroid-producing cells changes its compartmentalization during follicle maturation.     

Cardiac effects of endothelin-1 (ET-1) and related C terminal peptide fragment: increased inotropy or contribution to heart failure?

The contrasting pattern of cardiac inotropy induced by human peptide endothelin-1 (ET-1) has not been satisfactorily explained. It is not clear whether ET-1 is primarily responsible for increased myocardial ET-1 expression and release with resultant inotropic effects, or for the induction of myocardial hypertrophy and heart failure. There are at least two subtypes of endothelin receptors (ET(A) and ET(B)) and the inotropic effects of ET-1 differ depending on the receptor involved. Along with some other groups, we reported significant subtype-ET(B) endothelin receptor down-regulation in human cardiac cells preincubated with endothelin agonists (Drímal et al. 1999, 2000). The present study was therefore designed to clarify the subtype-selective mechanisms underlying the inotropic response to ET-1 and to its ET(B)-selective fragment (8-21)ET-1 in the isolated rat heart. The hearts were subjected to (1-21)ET-1 and to (8-21)ET-1, or to 30 min of stop-flow ischemia followed by 40 min of reperfusion, both before and after selective blockade of endothelin receptors.The present study revealed that both peptides, ET-1 and its (8-21)ET-1 fragment, significantly reduced coronary blood flow in nmolar and higher concentrations. The concomitant negative inotropy and chronotropy were marked after ET-1, while the infusion of the ET-1(8-21) fragment produced a slight but significant positive inotropic effect. Among the four endothelin antagonists tested in continuous infusion only the non-selective PD145065 and ET(B1/B2) selective BQ788 (in molar concentrations) slightly reduced the early contractile dysfunction of the heart induced by ischemia, whereas ET(A)-selective PD155080 partially protected the rat heart on reperfusion.     

Cyclooxygenase is regulated by ET-1 and MAPKs in peripheral lung microvascular smooth muscle cells

We examined the hypothesis that the potent vasoconstrictor endothelin (ET)-1 regulates both its own production and production of the vasodilator prostaglandins PGE(2) and prostacyclin in sheep peripheral lung vascular smooth muscle cells (PLVSMC). Confluent layers of PLVSMC were exposed to 10 nM ET-1; expression of the prepro (pp)-ET-1, cyclooxygenase (COX)-1, and COX-2 genes was examined by RT-PCR and Western analysis. Intracellular levels of ET-1 were measured by ELISA with and without addition of the protein synthesis inhibitor brefeldin A (50 microg/ml). Prostaglandin levels were measured by gas chromatography-mass spectrometry. Through use of ET(A) and ET(B) antagonists (BQ-610 and BQ-788, respectively), the contribution of the ET receptors to COX-1 and -2 expression and ppET-1 gene expression was examined. The contribution of phosphorylated p38 and p44/42 MAPK on COX-1 and COX-2 expression was also examined with MAPK inhibitors (p38, SB-203580 and p44/42, PD-98056). ET-1 resulted in transient increases in ppET-1, COX-1, and COX-2 gene and protein expression and release of 6-keto-PGF(1alpha) and PGE(2) (P < 0.05). Both internalization of ET-1 and synthesis of new peptide contributed to an increase in intracellular ET-1 (P < 0.05). Although increased ppET-1 was regulated by both ET(A) and ET(B), COX-2 expression was upregulated only by ET(A); COX-1 expression was unaffected by either antagonist. ET-1 treatment resulted in transient phosphorylation of p38 and p44/42 MAPK; inhibitors of these MAPKs suppressed expression of COX-2 but not COX-1. Our data indicate that local production of ET-1 regulates COX-2 by activation of the ET(A) receptor and phosphorylation of p38 and p44/42 MAPK in PLVSMC.     

Characterization of phosphoramidon-sensitive metalloproteinases with endothelin-converting enzyme activity in porcine lung membrane

Endothelin-1 (ET-1), a 21 amino-acid potent vasoconstrictor peptide, is produced from the biologically inactive intermediate big ET-1 via an endoproteolytic cleavage between Trp-21 and Val-22 by endothelin converting enzyme (ECE). cDNA sequence analysis predicts that the two other members of the endothelin family, ET-2 and ET-3, are also generated from the corresponding intermediates called big ET-2 and big ET-3, respectively. The metalloproteinase inhibitor phosphoramidon inhibited the conversion of big ET-1 into mature ET-1 both in vivo and in cultured endothelial cells, suggesting that ECE may be a neutral metalloproteinase. In this study, we solubilized and partially purified ECE from the membrane fraction of porcine lung. Using gel filtration chromatography, we separated two distinct ECE activities, designated M1 (apparent molecular mass approx. 300 kDa) and M2 (approx. 65 kDa). Optimum pH for the cleavage of big ET-1 by M1 and M2 was 7.0 and 7.5, respectively. M1 efficiently converted human big ET-1(1–38) to ET-1, but not human big ET-2(1–37) or human big ET-3(1–41)-amide. In contrast, M2 converted both big ET-1 and big ET-2, but not big ET-3. M1 was inhibited by phosphoramidon (IC₅₀ approx. 1 μM) but not by thiorphan or bacitracin. In contrast, M2 was inhibited by much lower concentrations of phosphoramidon (IC₅₀ approx. 0.3 nM), as well as by thiorphan and bacitracin. ECE activity in M1 was able to bind to a concanavalin A-agarose column and was eluted by α-methyl-d-glucoside, indicating that the ECE is glycosylated. From these results, M1 and M2 from the porcine lung membrane are similar to the candidate of ECE in endothelial cells and neutral endopeptidase in kidney (EC 3.4.24.11), respectively. Taken in conjunction with the previous finding that neither thiorphan nor bacitracin affected the conversion of endogenously synthesized big ET-1 in cultured endothelial cells, we conclude that physiologically relevant ECE found in the endothelial cells is more similar to M1 than to M2.     

IL-33-Mediated Protection against Experimental Cerebral Malaria Is Linked to Induction of Type 2 Innate Lymphoid Cells,M2 Macrophages and Regulatory T Cells

Cerebral malaria (CM) is a complex parasitic disease caused by Plasmodium sp. Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to the development of cerebral pathology. Using the blood-stage PbA (Plasmodium berghei ANKA) model of infection, we show here that administration of the pro-Th2 cytokine, IL-33, prevents the development of experimental cerebral malaria (ECM) in C57BL/6 mice and reduces the production of inflammatory mediators IFN-γ, IL-12 and TNF-α. IL-33 drives the expansion of type-2 innate lymphoid cells (ILC2) that produce Type-2 cytokines (IL-4, IL-5 and IL-13), leading to the polarization of the anti-inflammatory M2 macrophages, which in turn expand Foxp3 regulatory T cells (Tregs). PbA-infected mice adoptively transferred with ILC2 have elevated frequency of M2 and Tregs and are protected from ECM. Importantly, IL-33-treated mice deleted of Tregs (DEREG mice) are no longer able to resist ECM. Our data therefore provide evidence that IL-33 can prevent the development of ECM by orchestrating a protective immune response via ILC2, M2 macrophages and Tregs.     

Cardiac myofibroblasts isolated from the site of myocardial infarction express endothelin de novo

Recently it was demonstrated that treatment with a nonselective endothelin (ET) receptor antagonist significantly reduces myocardial infarct size, which suggests a major role for ET in tissue repair following myocardial infarction (MI). Tissue repair and remodeling found at the site of MI are mainly attributed to myofibroblasts (myoFbs), which are phenotypically transformed fibroblasts that express alpha-smooth muscle actin. It is unclear whether myoFbs generate ET peptides and consequentially regulate pathophysiological functions de novo through expression of the ET-1 precursor (prepro-ET-1), ET-converting enzyme-1 (ECE-1), a metalloprotease that is required to convert Big ET-1 to ET-1 and ET receptors. To address these intriguing questions, we used cultured myoFbs isolated from 4-wk-old MI scar tissue. In cultured cells, we found: 1) expression of mRNA for ET precursor gene (ppET1), ECE-1, and ETA and ETB receptors by semiquantitative RT-PCR; 2) phosphoramidon-sensitive ECE-1 activity, which converts Big ET-1 to biologically active peptide ET-1; 3) expression of ETA and ETB receptors; 4) elaboration of Big ET-1 and ET-1 peptides in myoFb culture media; and 5) upregulation of type I collagen gene expression and synthesis by ET, which was blocked by bosentan (a nonselective ETA- and ETB receptor blocker). These studies clearly indicated that myoFbs express and generate ET-1 and receptor-mediated modulation of type I collagen expression by ET-1. Locally generated ET-1 may contribute to tissue repair of the infarcted heart in an autocrine/paracrine manner.     

ET-1 receptor gene expression and distribution in L1 and L2 cells from hypertensive sheep pulmonary artery

We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (ETA and ETB) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative real-time RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of ETB than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of ET(B) in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [125I]-labeled ET-1 at 1 h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with ETA (BQ-610) and ETB (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in ETB receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.