Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

eTEP inferior access with tailored retromuscular dissection for small to mid-sized umbilical hernia repair with or without inguinal hernia: early experience.

INTRODUCTION: The minimally invasive surgical repair of combined inguinal and ventral hernias often requires shifting from one approach or plane to another. The traditional enhanced-view totally extraperitoneal Rives-Stoppa repair consists of a large retro muscular dissection that is unjustified for small ventral hernias. Here we describe a modification to the minimally invasive Rives-Stoppa repair using a limited retro muscular dissection based on the ventral defect size for small/medium-sized hernias, with or without combined inguinal hernias. METHODS: From a single surgical team, a retrospective study was performed over a 1-year period. Demographics, hernia characteristics, surgical techniques, intraoperative/postoperative complications, and outcomes were all analyzed and reported. We also included detailed surgical steps, landmarks, pitfalls, and personal tips for this technique. RESULTS: Twenty-four patients underwent a laparoscopic limited retromuscular dissection ventral hernia repair utilizing the eTEP access technique. Eighteen were primary umbilical hernias and six postoperative incisional hernias, and nine were combined ventral and inguinal hernia repairs. Eight of the primary umbilical hernias were EHS classified as medium size, 11 small, and for the incisional hernias, three were classified as M3W1 and two as M3W2. One procedure was converted to TAPP. There were no intraoperative complications. The mean length of stay was 1.25 days (range 1-3). There was one postoperative retromuscular hematoma and no recurrence during the follow-up period. CONCLUSION: eTEP with limited dissection offers a good and safe solution for small to medium size hernias; it provides an efficient solution when an inguinal hernia is to be addressed as well.

A mouse model suggests two mechanisms for thyroid alterations in infantile cystinosis: decreased thyroglobulin synthesis due to endoplasmic reticulum stress/unfolded protein response and impaired lysosomal processing.

Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which are impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin. Cystinotic children develop subclinical and then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns(-/-) mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabeling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, glucose-regulated protein of 78 kDa and protein disulfide isomerase, associated with alternative X-box binding protein-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, activating transcription factor-4 and C/EBP homologous protein, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but lysosome-associated membrane protein-1 immunolabeling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns(-/-) mice showed the following alterations: 1) compensated primary hypothyroidism and accelerated thyrocyte turnover; 2) impaired Tg production linked to ER stress/UPR response; and 3) altered endolysosomal trafficking and iodo-Tg processing. The Ctns(-/-) thyroid is useful to study disease progression and evaluate novel therapies.

Anti-thyroid cancer properties of a novel isoflavone derivative, 7-(O)-carboxymethyl daidzein conjugated to N-t-Boc-hexylenediamine in vitro and in vivo.

The incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) ßmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17ß (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERß-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.

Leptin attenuates follicular apoptosis and accelerates the onset of puberty in immature rats.

Human and rat granulosa cells express receptors to leptin which synergies with glucocorticoid hormones in stimulation of ovarian steroidogenesis. To examine whether leptin affects follicular development and maturation, we injected recombinant ovine leptin (300 ng-10 microg/animal) daily to immature 21 day-old female rats. Non-treated rats reached puberty at 44.5+/-1.6 (n=9) days. In contrast, in leptin treated animals, puberty was reached at 34.5+/-1.6 (n=9) days. Ovarian sections revealed hypertrophy of granulosa cells in leptin treated animals. Moreover, the number of ovulations was 2-fold higher in the treated animals compared to controls (3-4 ovulations versus 7-8 on the first three estrous cycles, P<0.001). Leptin dramatically reduced incidence of follicular apoptosis measured by TUNEL, and was already evident after 7 days of leptin injection (12% of apoptosis in leptin treated group compared to 52% in controls, P<0.001). Maximal protection against apoptosis was achieved at 1-3 microg leptin/animal. The levels of FSH, LH, progesterone and the steroidogenic factors ADX and STAR were elevated earlier in development in the leptin treated animals compared to control animals which is in line with the achievement of early puberty in the leptin treated animals compared to non treated ones. To reveal whether modulation of death and survival genes is involved in leptin attenuation of follicular apoptosis, we examined the expression of the survival gene Bcl-2 and the death gene Bax in Western blots of ovarian homogenates. There was a pronounced elevation in Bcl-2 expression during 7-14 days of leptin injections up to 16.3-fold (P<0.001) compared to Bcl-2 expression in controls. Bax expression was elevated only 3.4 fold (P<0.001), leading to an increase in the Bcl-2/Bax ratio of 4.7 fold (P<0.001). Expression of the tumor suppressor gene p 53 and the oncogene Mdm2 did not change significantly. Our data suggests that leptin may be involved in accelerating follicular maturation by attenuating follicular atresia and increasing the ratio of Bcl-2/Bax.

Nebivolol induces calcium-independent signaling in endothelial cells by a possible beta-adrenergic pathway.

Nebivolol is a highly selective beta1-adrenoreceptor-blocking agent with a peculiar pharmacodynamic profile. It has peripheral acute vasodilating properties that are mediated by modulation of the endogenous production of nitric oxide. In this study we analyzed the different signaling pathways implicated in the response of human umbilical vein endothelial cells to nebivolol. Its effect on endothelial transduction pathways was determined by assaying phospholipase C and A2 activities and cyclic adenosine monophosphate (AMP) production. Variations in intracellular calcium concentration were also measured. Our results showed that nebivolol activates a calcium-independent transduction pathway that implicates an increase in adenylate cyclase and phospholipase A2 activity. Beta1- or beta2-Adrenoreceptor antagonists do not inhibit the action of nebivolol. However, its action on cyclic AMP production is inhibited by bupranolol, a beta1-3-adrenoreceptor antagonist, and S-(-)-cyanopindolol, a selective beta3-adrenoreceptor antagonist. Nebivolol also dose-dependently increased nitrite production. This effect was inhibited by bupranolol, suggesting that the possible action of nebivolol on beta3-adrenoreceptor is involved in its vasodilating properties. This study suggests that nebivolol could behave as a beta3-adrenoreceptor agonist and induce some calcium-independent pathways implicating phospholipase A2 and adenylate cyclase. This agonistic activity of nebivolol seems to be responsible for its endothelium-dependent vasodilating activity.

Increasing endothelial cell permeability improves the efficiency of myocyte adenoviral vector infection.

BACKGROUND: Gene delivery to the myocardium using blood-borne adenoviral vectors is hindered by the endothelium, which represents a barrier limiting the infection rate of underlying myocytes. However, endothelial permeability may be modulated by pharmacological agents. METHODS: In the present study, we modeled the endothelial barrier in vitro using a human umbilical vein endothelial cell (HUVEC) monolayer seeded on a Transwell membrane as a support and diffusion of fluorescent dextrans as a permeability index. We used alpha-thrombin (100 nM) as a pharmacological agent known to increase endothelial permeability and tested the barrier function of the endothelial cell monolayer on adenovector-mediated luciferase gene transfer to underlying isolated cardiac myocytes. RESULTS: A confluent HUVEC monolayer represented a considerable physical barrier to dextran diffusion; it reduced the permeability of the micropore membrane alone to fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights 4, 70, 150 and 2000 kDa by approximately 54, 78, 88 and 98%, respectively. Alpha-thrombin (100 nM) increased the permeability coefficients (P(EC)) by 276, 264, 562 and 4166% for the same dextrans, respectively. A confluent HUVEC monolayer represented a major impediment to adenovector-mediated luciferase gene transfer to cardiac myocytes, largely reducing gene transfer efficiency. However thrombin induced a nine-fold increase in myocyte infection. CONCLUSION: In our model, the endothelial cell monolayer represents a major impediment to myocyte adenovector-mediated gene transfer which can be partially improved by pharmacologically increasing endothelial permeability. The Transwell model is therefore particularly useful for testing the efficiency of pharmacological agents in modulating adenovector passage through the endothelial barrier.

Highly efficient adenovirus-mediated gene transfer to cardiac myocytes after single-pass coronary delivery.

Efficient and homogeneous gene transfer to cardiac myocytes is a major target in myocardial gene therapy. The aim of this study was to determine the conditions permitting efficient, homogeneous, adenovirus-mediated gene transfer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conducted using type 5 adenoviruses carrying the lacZ reporter gene. Adenovirus delivery via coronary arteries was performed on isolated perfused rat hearts, and gene transfer efficiency was analyzed on whole ventricles, freshly isolated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) PFU associated with 1 min of no-flow yielded only 1 +/- 0.5% of positive myocytes. Pretreatment by histamine perfusion (10(-5) M final concentration) increased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessels. The 1-min no-flow period after adenovirus delivery was crucial for efficient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion, rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocytes in situ can be achieved by single-pass intracoronary vector delivery, provided that vascular permeability is first increased and coronary flow is briefly interrupted.

Interleukin-10 inhibits intimal hyperplasia after angioplasty or stent implantation in hypercholesterolemic rabbits.

BACKGROUND: Intimal hyperplasia after stent implantation is the main cause of in-stent restenosis. Activated monocytes play a key role in intimal growth. The anti-inflammatory cytokine interleukin-10 (IL-10) is a potent monocyte deactivator, endogenously produced in the atherosclerotic plaque. We tested the hypothesis that exogenous IL-10 may limit postangioplasty intimal hyperplasia after balloon angioplasty or stenting. METHODS AND RESULTS: Hypercholesterolemic rabbits were treated with recombinant human IL-10 (rhuIL-10) for 3 days after balloon angioplasty or 28 days after stent implantation. High IL-10 serum levels and intense deactivation of circulating monocytic cells, assessed by inhibition of IL-1beta release by lipopolysaccharide-stimulated whole blood, were detected for at least 8 hours after rhuIL-10 intravenous injection (ELISA). Morphometric analyses, performed 28 days after injury, indicated that rhuIL-10 reduced intimal growth by approximately 50% after balloon angioplasty or stenting, resulting in more preserved lumen in stented arteries. Moreover, rhuIL-10 reduced macrophage infiltration by 67% and proliferative activity by 81% in the intima and the media. No toxic effect was detected except minor changes in blood cell count. CONCLUSIONS: The anti-inflammatory cytokine rhuIL-10 reduces postinjury intimal hyperplasia. The potent attenuation of in-stent intimal growth by rhuIL-10 and its favorable toxicity profile suggest that rhuIL-10 may be useful in the prevention of in-stent restenosis.

A monoclonal antibody to oestradiol potentiates the stimulation of the specific activity of the brain type creatine kinase by oestrogen in vivo and in vitro.

We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E2 15 plus oestrogen (E2) potentiated the specific activity of the brain type creatine kinase (CK) response to E2 in the rat tissues or skeletal cells. The enhancing CK response of anti E2 15 plus E2 was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E2 15 blocked the stimulatory CK response to E2. Two other high affinity anti-E2 antibodies, clones 8D9 and 11B6, had no effect in augmenting the response of CK to E2 in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E2 15 plus E2 since the intact anti-E2 in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E2 15 abolished the CK response to E2 in rat tissues and not to anti-E2 15 plus E2 whereas tamoxifen completely blocked the CK response to anti E2 plus E2. Anti E2 15 may therefore serve as a specific carrier in delivering E2 to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E2 effects in vivo and in vitro.

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma.

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

Experimental extension of the time interval between oocyte maturation and ovulation: effect on fertilization and first cleavage.

OBJECTIVE: To test the hypothesis that impaired fertility in human patients with high LH concentrations throughout the follicular phase of the menstrual cycle reflects premature maturation of their oocytes. DESIGN: Previous information that resumption of meiosis is induced by lower hCG concentrations than that required for stimulation of follicular rupture was confirmed and used for establishment of a rat animal model in which oocyte maturation and ovulation can be separated experimentally. In further experiments hypophysectomized, pregnant mare serum gonadotropin (PMSG)-primed, immature female rats injected with 1.1 IU of hCG, a dose found to induce maturation in 72.9% +/- 6% of the rats with no effect on ovulation, were administered with a second injection of an ovulatory dose (4 IU) of hCG, 24 hours later. The ovulated eggs were subjected to IVF. RESULTS: Fertilization and first cleavage in oocytes recovered from our experimental animal model were similar to that observed in control PMSG-primed, either hypophysectomized or intact rats, treated by a single injection of 4 IU of hCG. CONCLUSIONS: The extension of the time interval between oocyte maturation and ovulation in the rat does not result in a lower rate of fertilization or a reduced incidence of cleavage. However, an inferior developmental capacity of these embryos cannot be ruled out.

Glyburide crosses the placenta in vivo in pregnant rats.

Non-insulin-dependent diabetes mellitus (NIDDM) is normally treated by oral hypoglycaemic agents, but their use is excluded during pregnancy because of their potential teratogenic and hypoglycaemic effects on the fetus. This caveat was recently questioned as glyburide was shown to cross an isolated cotyledon in vitro in insignificant amounts. In the present study, placental transport of glyburide in vivo was examined as an indispensable step towards clinical trials. Tritiated glyburide, C14 albumin or C14-labelled diazepam were injected into 13, 9 and 11 pregnant rats, respectively and the radioactivity was measured thereafter in maternal blood and in whole fetal extracts. The ratios between radioactivity in fetal tissue to that in maternal blood for glyburide (0.535 +/- 0.068) were similar to those of diazepam (0.641 +/- 0.057) which readily crosses the placenta. However, they differed significantly from those for albumin (0.048 +/- 0.0004) which does not cross. Moreover, glyburide in fetal tissue consistently reflected its concentration in maternal blood when measured at consecutive intervals after intravenous injection in the mother. In contrast, albumin in fetal tissue was low at all time points regardless of its levels in maternal blood when measured at different times after injection. These data suggest that glyburide crosses the placenta of pregnant rats and should therefore be considered with caution as a hypoglycaemic agent in the treatment of gestational diabetes.

Effect of postnatal treatment with a gonadotropin-releasing hormone antagonist on sexual maturation of male rats.

The role of postnatal pituitary-testicular activity in sexual maturation at puberty was studied in male rats. Rats were injected twice daily with a potent gonadotropin-releasing hormone antagonist (N-Ac-4-Cl-D-Phe1, 4-Cl-D-Phe2, D-Trp3, D-Phe6, D-Ala10-NH2-GnRH) (GnRH-Ant.), 2 mg/kg, on Days 1-15 of life, and killed on Day 48, 56 or 90 of life. The treatment delayed the onset of puberty (monitored by balano-preputial separation) by 8 days (from the age of 48 to 56 days). The weights of testes, seminal vesicles and ventral prostates were reduced by 50-60% on days 48 and 56 of life, but only the testis weights remained suppressed by Day 90. Levels of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), but not those of prolactin (Prl), were elevated 2-to-4-fold in the treated animals at the three ages studied. Serum and testicular testosterone (T) and the receptors for LH and Prl were suppressed in the peripubertal animals (48 and 56 days), but serum T was elevated and the receptor levels were normal in the 90-day group. The testicular FSH receptors were 50% suppressed at all ages studied. Only minor changes were observed in testicular histology when studied at 48 and 56 days. The 85-day-old animals treated with GnRH-Ant. were infertile when mated with females.(ABSTRACT TRUNCATED AT 250 WORDS)