9-cis-retinoic acid enhances fatty acid-induced expression of the liver fatty acid-binding protein gene.

The role of retinoic acids (RA) on liver fatty acid-binding protein (L-FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9-cis-Retinoic acid (9-cis-RA) specifically enhanced L-FABP mRNA levels in a time- and dose-dependent manner. The higher induction was found 6 h after addition of 10(-6) M 9-cis-RA in the medium. RA also enhanced further both L-FABP mRNA levels and cytosolic L-FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR), which are known to be activated, respectively, by 9-cis-RA and long chain fatty acid (LCFA), co-operated to bind specifically the peroxisome proliferator-responsive element (PPRE) found upstream of the L-FABP gene. Our result suggest that the PPAR-RXR complex is the molecular target by which 9-cis-RA and LCFA regulate the L-FABP gene.

Control of the proliferation of activated CD4+ T cells by connexins.

As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.

Effect of selective phosphodiesterase inhibitors on response of ovine pulmonary arteries to prostaglandin E2.

Several adenosine 3',5'-cyclic monophosphate (cAMP)-hydrolyzing phosphodiesterase isozymes are present in the pulmonary vasculature. The present study was designed to determine the effect of selective inhibitors of phosphodiesterase subtypes on prostaglandin E2 (PGE2)-induced relaxation of isolated fourth-generation pulmonary arteries of newborn lambs. PGE2 and forskolin caused pulmonary arteries to relax and induced an increase in the intracellular cAMP content in the vessels. The relaxation and change in cAMP content were augmented by milrinone and rolipram, inhibitors of phosphodiesterase type 3 (PDE3) and type 4 (PDE4), respectively. The augmentation in relaxation and the increase in cAMP content caused by milrinone plus rolipram was greater than the sum of the responses caused by either of the inhibitors alone. 8-Methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine, an inhibitor of phosphodiesterase type 1, had no effect on relaxation and change in cAMP induced by PGE2 and forskolin. Acetylcholine alone had no effect on cAMP content in the vessels but augmented the relaxation and the increase in cAMP induced by PGE2 and forskolin in arteries with endothelium. This effect was not observed in arteries without endothelium or in arteries with endothelium treated with NG-nitro-L-arginine. These results suggest that PDE3 and PDE4 are the primary enzymes hydrolyzing cAMP of pulmonary arteries of newborn lambs and that an inhibition of both PDE3 and PDE4 would result in a greater effect than that caused by inhibition of either one of the subtype isozymes alone. Furthermore, endothelium-derived nitric oxide may enhance cAMP-mediated relaxation by inhibition of PDE3.

Changes in sleeping and basal energy expenditure and substrate oxidation induced by short term thyroxin administration in man.

The present study was designed to explore the thermogenic effect of thyroid hormone administration and the resulting changes in nitrogen homeostasis. Normal male volunteers (n = 7) received thyroxin during 6 weeks. The first 3-week period served to suppress endogenous thyroid secretion (180 micrograms T4/day). This dose was doubled for the next 3 weeks. Sleeping energy expenditure (respiratory chamber) and BMR (hood) were measured by indirect calorimetry, under standardized conditions. Sleeping heart rate was continuously recorded and urine was collected during this 12-hour period to assess nitrogen excretion. The changes in energy expenditure, heart rate and nitrogen balance were then related to the excess thyroxin administered. After 3 weeks of treatment, serum TSH level fell to 0.15 mU/L, indicating an almost complete inhibition of the pituitary-thyroid axis. During this phase of treatment there was an increase in sleeping EE and sleeping heart rate, which increased further by doubling the T4 dose (delta EE: +8.5 +/- 2.3%, delta heart rate +16.1 +/- 2.2%). The T4 dose, which is currently used as a substitutive dose, lead to a borderline hyperthyroid state, with an increase in EE and heart rate. Exogenous T4 administration provoked a significant increase in urinary nitrogen excretion averaging 40%. It is concluded that T4 provokes an important stimulation of EE, which is mostly mediated by an excess protein oxidation.

The lymphoproliferative defect in CTLA-4-deficient mice is ameliorated by an inhibitory NK cell receptor.

T-cell responses are regulated by activating and inhibiting signals. CD28 and its homologue, cytotoxic T-lymphocyte antigen 4 (CTLA-4), are the primary regulatory molecules that enhance or inhibit T-cell activation, respectively. Recently it has been shown that inhibitory natural killer (NK) cell receptors (NKRs) are expressed on subsets of T cells. It has been proposed that these receptors may also play an important role in regulating T-cell responses. However, the extent to which the NKRs modulate peripheral T-cell homeostasis and activation in vivo remains unclear. In this report we show that NK cell inhibitory receptor Ly49A engagement on T cells dramatically limits T-cell activation and the resultant lymphoproliferative disorder that occurs in CTLA-4-deficient mice. Prevention of activation and expansion of the potentially autoreactive CTLA-4(-/-) T cells by the Ly49A-mediated inhibitory signal demonstrates that NKR expression can play an important regulatory role in T-cell homeostasis in vivo. These results demonstrate the importance of inhibitory signals in T-cell homeostasis and suggest the common biochemical basis of inhibitory signaling pathways in T lymphocytes.

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1.

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance.

Site of the action of a synthetic atrial natriuretic peptide evaluated in humans.

The renal site of the natriuretic effect of human, atrial natriuretic peptide (hANP) was studied using clearance techniques in eight salt-loaded normal volunteers undergoing maximal water diuresis. Lithium was used as a marker of proximal sodium reabsorption. According to a two-way, single blind, crossover design, hANP (Met12-(3-28)-eicosahexapeptide, (2 micrograms/min) or its vehicle (Ve) were infused for two hours, followed by a two-hour recovery period. Blood pressure, heart rate and insulin clearance remained unchanged. During hANP infusion, the filtration fraction increased slightly from 19.6 to 24.3% (P less than 0.001), fractional water excretion rose transiently at the beginning of the infusion. Fractional excretion of sodium increased markedly from 2.2% to 7.4% (P less than 0.001) but remained unchanged with Ve. ANP increased fractional excretion of lithium slightly from 46 to 58% (P less than 0.01), while it remained stable at 47% during Ve. The distal tubular rejection fraction of sodium calculated from sodium and lithium clearances rose markedly from 4.7 to 13% (P less than 0.001) and returned to 6.2% at the end of the recovery period. Thus, under salt loading and water diuresis conditions, hANP infusion did not alter GFR, but reduced proximal reabsorption of sodium, and markedly enhanced the fraction of sodium escaping distal tubular reabsorption, suggesting that hANP-induced natriuresis is due, for an important part, to inhibition of sodium reabsorption in the distal nephron.

Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.

Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide.

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1-5 mM), or NO donor (DETA NONOate 10-100 microM). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 microM had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 microM decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.

Evaluating in vivo delivery of riboflavin with coulomb-controlled iontophoresis for corneal collagen cross-linking: a pilot study.

PURPOSE: To evaluate the efficacy of coulomb-controlled iontophoresis (CCI) for delivery of riboflavin prior to corneal collagen cross-linking (CXL). METHODS: The eyes of 20 8-week-old Lewis rats, subject to epithelium-ON (epi-ON, n = 20 eyes) or epithelium-OFF (epi-OFF, n = 20 eyes) conditions, were used to evaluate the in vivo delivery of two riboflavin solutions: 0.1% riboflavin-20% dextran T500 solution (riboflavin-dextran) and 0.1% riboflavin 5'-phosphate (riboflavin-phosphate). After systemic intramuscular anesthesia, 0.25 mL of the photosensitizing agent was delivered by either instillation or CCI (2.11 mA/cm(2) for 4 or 10 minutes) into either epithelial condition. The CCI probe on the eye without current served as control. Confocal microscopy of flat-mounted corneas was used to analyze intracorneal penetration and fluorometry was used to quantify riboflavin in the aqueous within 30 minutes of treatment. RESULTS: Instillation and CCI allowed for uniform delivery of riboflavin-dextran throughout the stroma after epithelial debridement. Transepithelial delivery of riboflavin-dextran was not efficacious. Riboflavin-phosphate was successfully delivered in both epithelium conditions. Complete saturation of the cornea was achieved using CCI after removing the epithelium, the epi-ON case allowed for limited diffusion. Increasing the time from 4 to 10 minutes greatly increased the amount of riboflavin detected in the cornea and aqueous humor. CONCLUSIONS: Coulomb-controlled iontophoresis is an effective technique for transepithelial delivery of riboflavin-phosphate into the cornea. This drug delivery method would allow clinicians to significantly shorten the time required for the CXL procedure, with or without epithelial debridement. Whether efficient crosslinking can be achieved through an intact epithelium remains to be demonstrated.

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway.

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.

PPARgamma in placental angiogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in diverse biological processes including adipocyte differentiation, glucose homeostasis, and inflammatory responses. Analyses of PPARγ knockout animals have been so far preempted by the early embryonic death of PPARγ-/- embryos as a consequence of the severe alteration of their placental vasculature. Using Sox2Cre/PPARγL2/L2 mice, we obtained fully viable PPARγ-null mice through specific and total epiblastic gene deletion, thereby demonstrating that the placental defect is the unique cause of PPARγ-/- embryonic lethality. The vasculature defects observed in PPARγ-/- placentas at embryonic d 9.5 correlated with an unsettled balance of pro- and antiangiogenic factors as demonstrated by increased levels of proliferin (Prl2c2, PLF) and decreased levels of proliferin-related protein (Prl7d1, PRP), respectively. To analyze the role of PPARγ in the later stage of placental development, when its expression peaks, we treated pregnant wild-type mice with the PPARγ agonist rosiglitazone. This treatment resulted in a disorganization of the placental layers and an altered placental microvasculature, accompanied by the decreased expression of proangiogenic genes such as Prl2c2, vascular endothelial growth factor, and Pecam1. Together our data demonstrate that PPARγ plays a pivotal role in controlling placental vascular proliferation and contributes to its termination in late pregnancy.

The biomechanic origin of sprint performance enhancement after one-week creatine supplementation.

In order to test whether an improvement of maximal sprinting speed after creatine (Cr) supplementation was due to the increase of stride frequency (SF), stride length (SL) or both, 7 subjects ran 4 consecutive sprints after 1 week of placebo or Cr supplementation. SF and SL were assessed by a triaxial accelerometer. Compared to the placebo, Cr induced an increase of running speed (+1.4% p < 0.05) and SF (+1.5%, p < 0.01), but not of SL. The drop in performance following repeated sprints was partially prevented by Cr. In conclusion, exogenous Cr enhanced sprinting performance by increasing SF. This result may be related to the recent findings of shortening in muscular relaxation time after Cr supplementation.

Histone deacetylase inhibitors impair antibacterial defenses of macrophages.

Histone deacetylases (HDACs) control gene expression by deacetylating histones and nonhistone proteins. HDAC inhibitors (HDACi) are powerful anticancer drugs that exert anti-inflammatory and immunomodulatory activities. We recently reported a proof-of-concept study demonstrating that HDACi increase susceptibility to bacterial infections in vivo. Yet, still little is known about the effects of HDACi on antimicrobial innate immune defenses. Here we show that HDACi belonging to different chemical classes inhibit at multiple levels the response of macrophages to bacterial infection. HDACi reduce the phagocytosis and the killing of Escherichia coli and Staphylococcus aureus by macrophages. In line with these findings, HDACi decrease the expression of phagocytic receptors and inhibit bacteria-induced production of reactive oxygen and nitrogen species by macrophages. Consistently, HDACi impair the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and inducible nitric oxide synthase. These data indicate that HDACi have a strong impact on critical antimicrobial defense mechanisms in macrophages.