Distinct Effects of Inorganic Phosphate on Cell Cycle and Apoptosis in Human Vascular Smooth Muscle Cells.

Inorganic phosphate (Pi) is an essential nutrient to all living organisms. Nevertheless, hyperphosphatemia is now recognized as a risk factor for cardiovascular events and mortality in chronic kidney disease (CKD) patients. To our knowledge, the mechanisms by which elevated Pi alters smooth muscle cell proliferation have been poorly addressed. Therefore, in this study, we investigated the effects of Pi on cell cycle regulation and apoptosis in human aortic smooth muscle cells (HAoSMC). HAoSMC were treated with physiologic (1 mM) or high (2 and 3 mM) Pi concentrations. We showed that Pi not only decreased significantly cell viability (P < 0.001) but also induced apoptosis of HAoSMC. Moreover, Pi treatment blocked G1/S cell cycle progression by increasing cell number in G0/G1 phase up to 82.4 ± 3.4% for 3 mM vs 76.2 ± 3.1% for control (P < 0.01) while decreasing cell number in S phase. Accordingly, this was associated with a decrease protein expression of cyclin E and its associated CDK (CDK2), and phosphorylated retinoblastoma protein. Moreover, we observed an increase of protein expression of cell cycle inhibitors p15, p21, and p27. Interestingly, we also found that induction of cell cycle arrest was partially dependent on phosphate uptake. Our results demonstrated that Pi reduced HAoSMC proliferation by inducing apoptosis and cell cycle arrest. Indeed, we showed for the first time that Pi affected HAoSMC cell cycle by blocking G1/S progression. These findings would be useful for a better understanding of molecular mechanisms involved in vascular complications observed in CKD patients.

ARCHITECT® urine-neutrophil gelatinase-associated lipocalin (u-NGAL) assay as new prognostic marker for clear cell Renal Cell Carcinoma (ccRCC) (preliminary results).

INTRODUCTION: Biomarkers for the diagnosis and monitoring treatment response of kidney cancer are urgently needed. Neutrophil gelatinase-associated lipocalin (NGAL) is a relevant urinary biomarker for the diagnosis of a wide variety of acute and chronic kidney diseases. Its potential utility as a prognostic marker of kidney cancer is largely unknown and, therefore, was the subject of this investigation. MATERIALS AND METHODS: A retrospective study was done on 50 kidney tumor patients (urine samples prospectively collected before nephrectomy between 2004 and 2012, stored at Biobank Resource Center). The specificity, sensitivity and the predictive value of NGAL were determined for progression-free and disease-specific survival after nephrectomy in renal cell carcinoma (particularly, the clear cell renal cell carcinoma (ccRCC)). Urinary NGAL concentration (u-NGAL) was determined by CMIA technique (ARCHITECT® urine NGAL essay/ABBOTT®). RESULTS: Out of the 50 kidney tumor patients, 40 had clear cell carcinoma with a median u-NGAL excretion of 1.4 (IQR: 5.76) ng/mg urinary creatinine (Ucr). u-NGAL was correlated to tumor stage (p = 0.005), and Fuhrman grade (p = 0.0002). Multivariate Cox regression analysis showed a significant association between u-NGAL excretion and clear cell renal cell carcinoma progression free survival and disease specific survival (p = 0.002; p = 0.0001). CONCLUSIONS: Urinary NGAL was significantly associated with the stage and the grade of kidney cancer. u-NGAL excretion could be considered as a potential biomarker to identify ccRCC patients with the more pejorative outcomes.

PPAR alpha inhibits vascular smooth muscle cell proliferation underlying intimal hyperplasia by inducing the tumor suppressor p16INK4a.

Vascular SMC proliferation is a crucial event in occlusive cardiovascular diseases. PPARalpha is a nuclear receptor controlling lipid metabolism and inflammation, but its role in the regulation of SMC growth remains to be established. Here, we show that PPARalpha controls SMC cell-cycle progression at the G1/S transition by targeting the cyclin-dependent kinase inhibitor and tumor suppressor p16(INK4a) (p16), resulting in an inhibition of retinoblastoma protein phosphorylation. PPARalpha activates p16 gene transcription by both binding to a canonical PPAR-response element and interacting with the transcription factor Sp1 at specific proximal Sp1-binding sites of the p16 promoter. In a carotid arterial-injury mouse model, p16 deficiency results in an enhanced SMC proliferation underlying intimal hyperplasia. Moreover, PPARalpha activation inhibits SMC growth in vivo, and this effect requires p16 expression. These results identify an unexpected role for p16 in SMC cell-cycle control and demonstrate that PPARalpha inhibits SMC proliferation through p16. Thus, the PPARalpha/p16 pathway may be a potential pharmacological target for the prevention of cardiovascular occlusive complications of atherosclerosis.

Type III collagen mimetic peptides designed with anti- or pro-aggregant activities on human platelets.

We report the synthesis of collagen related peptides containing the peptide sequence Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys. The alpha-triple helix peptides behave as type III collagen analogues supporting platelet aggregation, while the homotrimer which does not exhibit a triple-helical conformation inhibits type III collagen-induced human platelet aggregation. The incorporation of the octapeptide sequence in type III collagen mimetic peptides may lead to the loss of the anti-thrombotic activity for a pro-thrombotic one.

Comparison of two preventive treatments for patients with recurrent miscarriages carrying a C677T methylenetetrahydrofolate reductase mutation: 5-year experience.

Objective To investigate the effect of anticoagulant treatment on pregnancy outcomes in patients with previous recurrent miscarriages (RM) who carry a methylenetetrahydrofolate reductase ( MTHFR) gene mutation. Methods In this longitudinal retrospective study, patients with RM were treated during pregnancy with either: (i) 100 mg/day aspirin and 5 mg/day folic acid (group 1); or the same protocol plus 0.4 mg/day enoxaparin (group 2). An age-matched group of triparous women without RM or thrombophilia was used as the control group (group 3). Results This study enrolled 246 women with RM (123 per treatment group) and age-matched controls ( n = 117). The delivery rate was significantly lower in group 1 than group 2 (46.3% versus 79.7%, respectively). The miscarriage rate was significantly lower in group 2 compared with group 1 (20.3% versus 51.2%, respectively). In the control group 3, the delivery rate was 86.3% and the miscarriage rate was 12.8%. Conclusion Treatment with low-dose aspirin, enoxaparin and folic acid was the most effective therapy in women with RM who carried a C677T MTHFR mutation.

Antiangiogenesis mediates cisplatin-induced peripheral neuropathy: attenuation or reversal by local vascular endothelial growth factor gene therapy without augmenting tumor growth.

BACKGROUND: Toxic neuropathies induced by cisplatin and other chemotherapeutic agents are important clinical problems because of their high incidence, their lack of effective treatment, and the fact that neuropathy represents a dose-limiting factor for these therapies. The pathogenic basis for toxic neuropathies induced by chemotherapeutic agents has not been completely elucidated. METHODS AND RESULTS: We investigated the hypothesis that experimental toxic neuropathy results from an antiangiogenic effect of these drugs, resulting in destruction of the vasa nervorum, and accordingly that the neuropathy could be prevented or reversed by locally administered VEGF gene transfer without augmenting tumor growth. In an animal model of cisplatin-induced neuropathy, nerve blood flow was markedly attenuated, and there was a profound reduction in the number of vasa nervorum associated with marked endothelial cell apoptosis, resulting in a severe peripheral neuropathy with focal axonal degeneration characteristic of ischemic neuropathy. After intramuscular gene transfer of naked plasmid DNA encoding VEGF-1 in animals with an established neuropathy, vascularity and blood flow returned to levels similar to those of control rats, peripheral nerve function was restored, and histological nerve architecture was normalized. Gene therapy administered in parallel with cisplatin chemotherapy completely attenuated endothelial cell apoptosis and inhibited destruction of nerve vasculature, deterioration of nerve function, and axonal degeneration. In a rat tumor model, VEGF gene transfer administered locally did not alter tumor growth or vascularity. CONCLUSIONS: These findings implicate microvascular damage as the basis for toxic neuropathy induced by cisplatin and suggest that local angiogenic gene therapy may constitute a novel prevention or treatment for this disorder without augmenting tumor growth or vascularization.

Antithrombotic effect of the type III collagen-related octapeptide (KOGEOGPK) in the mouse.

Platelet adhesion to subendothelial types I and III collagens exposed upon vascular injury plays a crucial role in hemostasis and thrombosis. We previously identified a KOGEOGPK sequence (O for hydroxyproline) within type III collagen interacting with platelets, and demonstrated a strong inhibitory effect of the KOGEOGPK peptide on human platelet interactions with type III collagen in vitro. In the present study, we tested the antithrombotic effect of KOGEOGPK in vivo. In a mouse model of pulmonary thromboembolism induced by intravenous injection of type III collagen and epinephrine, prior administration of 80 mg/kg KOGEOGPK reduced by 50% the size of thrombi embolized in lungs, compared to vehicle-treated mice (p<0.0001). In a mouse model of photochemically induced lesion of caecum venules and arterioles, intravenous injection of 80 mg/kg KOGEOGPK decreased by 76% the occurrence of arteriole occlusion 45 min after vascular injury (p<0.05). A moderate antithrombotic effect of KOGEOGPK was also observed in the injured venules. In addition, intracardiac injection of KOGEOGPK had no effect on the tail bleeding time. These findings demonstrate a substantial contribution of platelet interactions with the type III collagen-related KOGEOGPK sequence in thrombus formation in vivo with preferential involvement in arterial thrombosis.

Synthesis, Physicochemical Studies, Molecular Dynamics Simulations, and Metal-Ion-Dependent Antiproliferative and Antiangiogenic Properties of Cone ICL670-Substituted Calix[4]arenes.

Iron chelators, through their capacity to modulate the iron concentration in cells, are promising molecules for cancer chemotherapy. Chelators with high lipophilicity easily enter into cells and deplete the iron intracellular pool. Consequently, iron-dependent enzymes, such as ribonucleotide reductase, which is over-expressed in cancer cells, become nonfunctional. A series of calix[4]arene derivatives substituted at the lower rim by ICL670, a strong FeIII chelator, have been synthesized. Physicochemical properties and antiproliferative, angiogenesis, and tumorigenesis effects of two calix[4]arenes mono- (5a) or disubstituted (5b) with ICL670 have been studied. These compounds form metal complexes in a ratio of one to two ligands per FeIII atom as shown by combined analyses of the protometric titration curves and ESIMS spectra. The grafting of an ICL670 group on a calix[4]arene core does not significantly alter the acid-base properties, but improves the iron-chelating and lipophilicity properties. The best antiproliferative and anti-angiogenic results were obtained with calix[4]arene ligand 5a, which possesses the highest corresponding properties. Analyses of molecular dynamics simulations performed on the two calix[4]arenes provide three-dimensional structures of the complexes and proved 5a to be the most stable upon complexation.

Prospective evaluation of the familial prevalence of the brugada syndrome.

The familial prevalence of Brugada syndrome (BrS) in a consecutive series of patients was prospectively determined. BrS is genetically determined with autosomal dominant transmission. The familial prevalence of the BrS is unknown. A detailed pedigree of each family of patients with BrS was assembled and permission was obtained to invite relatives for electrocardiography and an ajmaline challenge. Sixty-two of 98 patients participated in the study and were included over a 6-year period. SCN5A genotyping was performed in 56 of these 62 patients (90%). Electrocardiograms (ECGs) of 488 relatives (mean age 38 ± 20 years, 45% men) were recorded and 270 of these relatives agreed to undergo an ajmaline challenge. Spontaneous type 1 BrS ECG was found in 4 of 488 relatives (0.8%). In the group of relatives in whom ajmaline challenge was performed (n = 270), the finding was positive in 79 subjects (29%). SCN5A genotyping identified 5 other affected relatives. As a result, the total number of affected relatives was 88. Standard 12-lead ECG was normal in 64 of the 88 affected relatives (73%). Mean percentage of affected relatives per family was 27 ± 32% (95% confidence interval 19 to 35). Familial forms of BrS were observed in 41 of the 62 families (66%) and no SCN5A mutations were found in sporadic forms. In conclusion, after active family screening affected relatives were found in almost 1/3 of subjects. BrS appeared to be a familial disease in 2/3 of subjects.

Polymorphism of the codon 129 of the prion protein (PrP) gene and neuropathology of cerebral ageing.

We studied whether codon 129 polymorphism of the PrP gene modulates the presence of tau- and Abeta-associated lesions among 188 patients over 70 years of age without evidence of dementia. Val allele carriers, either heterozygotes or homozygotes, were more frequently affected by Abeta-associated lesions than non Val allele carriers, whereas there were no differences for tau-positive neurones. Val allele carriers also had more focal and diffuse Abeta deposits. This association was most significant in the highest Braak's stages for neurofibrillary tangles (>/=III). In this group, cases with at least one Val allele had nearly twice as many Abeta-associated lesions. The most affected areas were the entorhinal cortex, TF-TH and the superior temporal cortex, where odds ratios for focal Abeta deposits ranged from 3.5 to 4.6.