VEGF-C/VEGFR-3 signalling in macrophages ameliorates acute lung injury.

BACKGROUND: Successful recovery from acute lung injury requires inhibition of neutrophil influx and clearance of apoptotic neutrophils. However, the mechanisms underlying recovery remain unclear. We investigated the ameliorative effects of vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGFR-3) signalling in macrophages in lipopolysaccharide (LPS)-induced lung injury. METHODS: LPS was intranasally injected into wild-type and transgenic mice. Gain and loss of VEGF-C/VEGFR-3 signalling function experiments employed adenovirus-mediated intranasal delivery of VEGF-C (Ad-VEGF-C vector) and soluble VEGFR-3 (sVEGFR-3) or anti-VEGFR-3 blocking antibodies and mice with a deletion of VEGFR-3 in myeloid cells. RESULTS: The early phase of lung injury was significantly alleviated by the overexpression of VEGF-C with increased levels of bronchoalveolar lavage (BAL) fluid interleukin-10 (IL-10), but worsened in the later phase by VEGFR-3 inhibition upon administration of Ad-sVEGFR-3 vector. Injection of anti-VEGFR-3 antibodies to mice in the resolution phase inhibited recovery from lung injury. The VEGFR-3-deleted mice had a shorter survival time than littermates and more severe lung injury in the resolution phase. Alveolar macrophages in the resolution phase digested most of the extrinsic apoptotic neutrophils and VEGF-C/VEGFR-3 signalling increased efferocytosis via upregulation of integrin αv in the macrophages. We also found that incubation with BAL fluid from acute respiratory distress syndrome (ARDS) patients, but not from controls, decreased VEGFR-3 expression and the efficiency of IL-10 expression and efferocytosis in human monocyte-derived macrophages. CONCLUSIONS: VEGF-C/VEGFR-3 signalling in macrophages ameliorates experimental lung injury. This mechanism may also provide an explanation for ARDS resolution.

Vascular endothelial growth factor receptor-3 is a novel target to improve net ultrafiltration in methylglyoxal-induced peritoneal injury.

Appropriate fluid balance is important for good clinical outcomes and survival in patients on peritoneal dialysis. We recently reported that lymphangiogenesis associated with fibrosis developed in the peritoneal cavity via the transforming growth factor-ß1-vascular endothelial growth factor-C (VEGF-C) pathway. We investigated whether VEGF receptor-3 (VEGFR-3), the receptor for VEGF-C and -D, might be a new target to improve net ultrafiltration by using adenovirus-expressing soluble VEGFR-3 (Adeno-sVEGFR-3) in rodent models of peritoneal injury induced by methylglyoxal (MGO). We demonstrated that lymphangiogenesis developed in these MGO models, especially in the diaphragm, indicating that lymphangiogenesis is a common feature in the peritoneal cavity with inflammation and fibrosis. In MGO models, VEGF-D was significantly increased in the diaphragm; however, VEGF-C was not significantly upregulated. Adeno-sVEGFR-3, which was detected on day 50 after administration via tail vein injections, successfully suppressed lymphangiogenesis in the diaphragm and parietal peritoneum in mouse MGO models without significant effects on fibrosis, inflammation, or neoangiogenesis. Drained volume in the peritoneal equilibration test using a 7.5% icodextrin peritoneal dialysis solution (the 7.5% icodextrin peritoneal equilibration test) was improved by Adeno-sVEGFR-3 on day 22 (P<0.05) and day 50 after reduction of inflammation (P<0.01), indicating that the 7.5% icodextrin peritoneal equilibration test identifies changes in lymphangiogenesis. The solute transport rate was not affected by suppression of lymphangiogenesis. In human peritoneal dialysis patients, the dialysate to plasma ratio of creatinine positively correlated with the dialysate VEGF-D concentration (P<0.001). VEGF-D mRNA was significantly higher in the peritoneal membranes of patients with ultrafiltration failure, indicating that VEGF-D is involved in the development of lymphangiogenesis in peritoneal dialysis patients. These results indicate that VEGFR-3 is a new target to improve net ultrafiltration by suppressing lymphatic absorption and that the 7.5% icodextrin peritoneal equilibration test is useful for estimation of lymphatic absorption.

TGF-ß1 promotes lymphangiogenesis during peritoneal fibrosis.

Peritoneal fibrosis (PF) causes ultrafiltration failure (UFF) and is a complicating factor in long-term peritoneal dialysis. Lymphatic reabsorption also may contribute to UFF, but little is known about lymphangiogenesis in patients with UFF and peritonitis. We studied the role of the lymphangiogenesis mediator vascular endothelial growth factor-C (VEGF-C) in human dialysate effluents, peritoneal tissues, and peritoneal mesothelial cells (HPMCs). Dialysate VEGF-C concentration correlated positively with the dialysate-to-plasma ratio of creatinine (D/P Cr) and the dialysate TGF-ß1 concentration. Peritoneal tissue from patients with UFF expressed higher levels of VEGF-C, lymphatic endothelial hyaluronan receptor-1 (LYVE-1), and podoplanin mRNA and contained more lymphatic vessels than tissue from patients without UFF. Furthermore, mesothelial cell and macrophage expression of VEGF-C increased in the peritoneal membranes of patients with UFF and peritonitis. In cultured mesothelial cells, TGF-ß1 upregulated the expression of VEGF-C mRNA and protein, and this upregulation was suppressed by a TGF-ß type I receptor (TGFßR-I) inhibitor. TGF-ß1-induced upregulation of VEGF-C mRNA expression in cultured HPMCs correlated with the D/P Cr of the patient from whom the HPMCs were derived (P<0.001). Moreover, treatment with a TGFßR-I inhibitor suppressed the enhanced lymphangiogenesis and VEGF-C expression associated with fibrosis in a rat model of PF. These results suggest that lymphangiogenesis associates with fibrosis through the TGF-ß-VEGF-C pathway.

Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice.

We recently reported that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory coreceptor develop autoimmune dilated cardiomyopathy (DCM), with production of high-titer autoantibodies against a heart-specific, 30-kDa protein. In this study, we purified the 30-kDa protein from heart extract and identified it as cardiac troponin I (cTnI), encoded by a gene in which mutations can cause familial hypertrophic cardiomyopathy (HCM). Administration of monoclonal antibodies to cTnI induced dilatation and dysfunction of hearts in wild-type mice. Monoclonal antibodies to cTnI stained the surface of cardiomyocytes and augmented the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. These findings suggest that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca2+ influx in cardiomyocytes.

Calcium channel blockers differentially modulate cytokine production by peripheral blood mononuclear cells.

BACKGROUND: Calcium channel blockers (CCB) are known to modulate immune reactions, so the present study was performed to examine the effects of various CCBs that have shown different effects on transcription factors and on the production of pro-inflammatory cytokines by human peripheral blood mononuclear cells (PBMC). METHODS AND RESULTS: PBMC from healthy volunteers were isolated by Ficoll-paque density centrifugation. To study the effect of CCBs, the PBMC were stimulated with lipopolysaccharide or concanavalin A. After 24 h of incubation, the supernatants were harvested and the interleukin (IL)-1alpha, -1beta, and -6, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma levels were determined by specific enzyme-linked immunosorbent assay. The production of IL-1alpha and -1beta stimulated with lipopolysaccharide was significantly increased in the presence of amlodipine. In contrast, nifedipine and verapamil suppressed the production of IL-1beta, TNF-alpha, and IFN-gamma. Amlodipine and diltiazem significantly increased production of IL-1alpha stimulated with concanavalin A. Nifedipine inhibited production of IL-1alpha, IL-6, and IFN-gamma. Verapamil suppressed production of IFN-gamma. CONCLUSIONS: Differential modulation of cytokine production was seen with various CCBs, and the suppressive effect of nifedipine was most prominent.

Role of substance P in viral myocarditis in mice.

Substance P (SP) is widely expressed in the central nervous system and in peripheral tissues such as myocardial nerves. We examined SP in viral myocarditis in mice induced by encephalomyocarditis virus (EMCV). Localization of SP in the hearts was examined immunohistochemically, and concentrations of SP in hearts and sera were measured by enzyme immunoassay. Substance P levels and density of SP-containing cells in murine hearts on day 6 after EMCV inoculation were decreased compared with those in normal controls. There was a negative correlation between SP levels in the hearts and ratio of heart weight to body weight of the mice at 6 days. Circulating SP levels were decreased in mice on day 6 after EMCV inoculation, and further decreased on day 14. Substance P in hearts and sera is decreased in viral myocarditis in mice, suggesting that SP may play a role in the pathogenesis of viral myocarditis, and that interaction of the neuropeptide nervous system and mast-cell immune system is important in the pathogenesis of viral myocarditis.

Histone acetyltransferase activity of p300 is required for the promotion of left ventricular remodeling after myocardial infarction in adult mice in vivo.

BACKGROUND: Left ventricular (LV) remodeling after myocardial infarction is associated with hypertrophy of surviving myocytes and represents a major process that leads to heart failure. One of the intrinsic histone acetyltransferases, p300, serves as a coactivator of hypertrophy-responsive transcriptional factors such as a cardiac zinc finger protein GATA-4 and is involved in its hypertrophic stimulus-induced acetylation and DNA binding. However, the role of p300-histone acetyltransferase activity in LV remodeling after myocardial infarction in vivo is unknown. METHODS AND RESULTS: To solve this problem, we have generated transgenic mice overexpressing intact p300 or mutant p300 in the heart. As the result of its 2-amino acid substitution in the p300-histone acetyltransferase domain, this mutant lost its histone acetyltransferase activity and was unable to activate GATA-4-dependent transcription. The two kinds of transgenic mice and the wild-type mice were subjected to myocardial infarction or sham operation at the age of 12 weeks. Intact p300 transgenic mice showed significantly more progressive LV dilation and diminished systolic function after myocardial infarction than wild-type mice, whereas mutant p300 transgenic mice did not show this. CONCLUSIONS: These findings demonstrate that cardiac overexpression of p300 promotes LV remodeling after myocardial infarction in adult mice in vivo and that histone acetyltransferase activity of p300 is required for these processes.

Left ventricular pressure-volume relationship in a murine model of congestive heart failure due to acute viral myocarditis.

OBJECTIVES: This study, performed in a murine model of encephalomyocarditis virus myocarditis, used a new Millar 1.4F conductance-micromanometer system for the in vivo determination of the left ventricular (LV) pressure-volume relationship (PVR). BACKGROUND: Viral myocarditis is an important cause of congestive heart failure and may lead to dilated cardiomyopathy. However, the hemodynamic changes associated with its acute phase have not been analyzed in detail. METHODS: Four-week-old DBA/2 mice were inoculated with EMCV (day 0). Serial hemodynamic measurements, compared with uninfected control mice were made on days 0, 1, 3, 4, 5, 7, 9, 12, and 14. RESULTS: On day 1, the hearts of infected mice manifested enhanced contractile function, decreased LV compliance, and abnormal diastolic function with increased LV end-diastolic pressure (EDP). Mean stroke index, ejection fraction (EF), and cardiac index (CI) were significantly higher than in uninfected control mice (p < 0.05). Contractile function decreased from days 4 to 14. On day 7, when hemodynamic abnormalities consistent with heart failure culminated, end-diastolic volume (EDV), EDP, and EDPVR were significantly higher, and CI, EF, end-systolic pressure (ESP), and ESPVR significantly lower in the infected than in control mice. Heart rate remained comparable in both groups. Although heart failure receded between day 9 and day 14, ESPVR, ESP, and EF remained significantly depressed up to day 14, and EDV and EDP remained significantly higher. CONCLUSIONS: These hemodynamic data provide new insights into the pathophysiology of acute viral myocarditis and may be useful in the development of therapeutic interventions.

Carvedilol increases the production of interleukin-12 and interferon-gamma and improves the survival of mice infected with the encephalomyocarditis virus.

OBJECTIVES: This study was designed to examine the effects of carvedilol in a murine model of viral myocarditis induced by encephalomyocarditis virus (EMCV) infection. BACKGROUND: Cytokines play an important role in the pathophysiology of viral myocarditis. Catecholamines influence the production of cytokines via beta-adrenergic receptors, suggesting that beta-adrenergic blockers could modulate the production of cytokines and exert a therapeutic effect in viral myocarditis by blocking the beta-stimulating action of endogenous catecholamines. In clinical trials, the third-generation, nonselective beta-blocker carvedilol was the first among several beta-blockers to reduce mortality in heart failure. However, the effects of carvedilol in acute viral myocarditis and on cytokine production are unknown. METHODS: This study compared the effects of carvedilol, the selective beta(1)-blocker metoprolol, and the nonselective beta-blocker propranolol in a murine model of viral myocarditis induced by EMCV. RESULTS: Carvedilol improved the 14-day survival of the animals, attenuated myocardial lesions on day 7, and increased myocardial levels of interleukin (IL)-12 and interferon (IFN)-gamma, whereas reducing myocardial virus replication. Propranolol also attenuated myocardial lesions, but to a lesser extent, and increased IL-12 and IFN-gamma levels. Metoprolol had no effect in this model. Encephalomyocarditis virus infection increased plasma catecholamine levels. CONCLUSIONS: These results suggest that by blocking the beta(2)-stimulating effects of catecholamines, carvedilol exerts some of its beneficial effects by increasing the production of IL-12 and IFN-gamma. Carvedilol may be effective in patients with viral myocarditis by boosting IL-12 and IFN-gamma production.

Suppression of cytokines and nitric oxide production, and protection against lethal endotoxemia and viral myocarditis by a new NF-kappaB inhibitor.

BACKGROUND: Nuclear factor kappa B (NF-kappaB) is activated by several factors, which increase the inflammatory response, and this activation, in turn, leads to the expression of several genes such as cytokines, and may play an important role in cardiovascular diseases. AIMS: The aim of the study is to examine the effect of SUN C8079, a newly synthesized NF-kappaB inhibitor in vitro and in vivo. METHODS: We examined the effects of SUN C8079 on the transcriptional responses of NF-kappaB, on activation of NF-kappaB in electrophoretic mobility shift assay, and on the gene expressions of tumor necrosis factor (TNF)-alpha and iNOS. We also studied effects of SUN C8079 on lethal endotoxemia and viral myocarditis in mice. RESULTS: SUN C8079 inhibited the lipopolysaccharide (LPS)-induced expression of the genes of TNF-alpha and iNOS by inhibiting the activation of NF-kappaB in vitro. SUN C8079 inhibited the systemic release of TNF-alpha and improved mortality in LPS-treated mice. In addition to protecting mice against lethal endotoxemia, SUN C8079 prevented the development of myocarditis due to the encephalomyocarditis virus (EMCV), and inhibited the expressions of proinflammatory cytokines and the iNOS gene in cardiac tissues. CONCLUSION: These findings suggest that the activation of NF-kappaB plays an important role in the pathogenesis of endotoxemia and viral myocarditis, and that the NF-kappaB inhibitor, SUN C8079, may be therapeutic in these disorders.

Reverse remodeling and improved function by antihypertensive treatment in hypertensive patients with coronary artery disease.

BACKGROUND: Although antihypertensive therapy reduces cardiovascular events, it is unclear whether there are differences in cardiac remodeling and function between treatments with nifedipine retard and angiotensin-converting enzyme inhibitors (ACE-Is). It is also not clear how antihypertensive therapy influences cardiac remodeling and function. METHODS: Hypertensive patients with coronary artery disease were randomly assigned to the nifedipine retard (n = 108) or ACE inhibitors groups (n = 102) and treated for 3 years. The primary endpoints were changes in end-diastolic volume index (EDVI) and end-systolic volume index (ESVI) as indices of cardiac remodeling, whereas the secondary endpoints were changes in ejection fraction (EF), stroke volume index (SVI), cardiac index (CI) and regional wall motion as indices of cardiac function. Left ventriculography was performed at baseline and after 3 years of treatment. Fifty-eight and 61 patients, respectively, were subjected to the final analysis. RESULTS: Comparable changes in remodeling and function were obtained in the nifedipine retard group and the ACE-Is group. Both groups showed a significant reduction of EDVI and ESVI, and a significant increase in EF, SVI, and CI, whereas the decreased regional wall motion significantly improved. In both groups, weak but significant correlations were noted between treatment-induced changes of systolic blood pressure and those of primary and secondary endpoints. CONCLUSION: The above findings show that treatments with nifedipine retard or ACE-Is cause a comparable change in remodeling and cardiac function. Lowering of the blood pressure by either drug leads to reverse remodeling or improvement of cardiac function. In addition to alleviation of coronary artery damage by reducing blood pressure, there is a favorable effect on the left ventricular structure and function. Reducing the blood pressure is critically important for hypertensive patients with coronary artery disease.