Alejandro Zentella-Dehesa

Vascular permeability changes involved in tumor metastasis.

Cancer cell extravasation resembles the leukocyte recruitment during inflammation. Evidence suggests that cancer cells need to weaken the interendothelial junctions in order to cross the endothelial barrier. Several tumor-derived vasoactive compounds have been pointed out to drive this increase in vascular permeability: VEGF, Angptl4, CCL2, SDF-1, etc. Therefore, tumor cells have a wide repertoire of soluble factors to increase vascular permeability in order to colonize new tissues. Tumor soluble factors activate different signaling pathways to induce interendothelial junction disassembly, one common element is Src kinase. Here we summarize the relevant current knowledge about vascular permeability changes involved in tumor metastasis.

Reversal of multidrug resistance by morning glory resin glycosides in human breast cancer cells.

Reversal of multidrug resistance (MDR) by thirty resin glycosides from the morning glory family (Convolvulaceae) was evaluated in vinblastine-resistant human breast carcinoma cells (MCF-7/Vin). The effects of these amphipathic compounds on the cytotoxicity and P-glycoprotein (P-gp)-mediated MDR were estimated with the sulforhodamine B colorimetric assay. Active noncytotoxic compounds exerted a potentiation effect of vinblastine susceptibility by 1- to over 1906-fold at tested concentrations of 5 and 25 μg/mL. Murucoidin V (1) enhanced vinblastine activity 255-fold when incorporated at 25 μg/mL and also, based on flow cytometry, significantly increased the intracellular accumulation of rhodamine 123 with the use of reserpine as a positive control for a MDR reversal agent. Incubation of MCF-7/Vin cells with 1 caused an increase in uptake and notably lowered the efflux rate of rhodamine 123. Decreased expression of P-glycoprotein by compound 1 was detected by immunofluorescence flow cytometry after incubation with an anti-P-gp monoclonal antibody. These results suggest that resin glycosides represent potential efflux pump inhibitors for overcoming MDR in cancer therapy.

Locus on Chromosome 6p Linked to Elevated HDL Cholesterol Serum Levels and to Protection Against Premature Atherosclerosis in a Kindred With Familial Hypercholesterolemia

Abstract— Heterozygous familial hypercholesterolemia (FH) is a highly atherogenic genetic disorder leading to premature coronary heart disease (CHD), usually before 60 years of age. We studied an extended multigenerational kindred with FH linked to chromosome 1p32 in which atherosclerotic complications were either delayed or prevented in individuals with elevated HDL cholesterol (HDL-C) levels or hyperalphalipoproteinemia (HA). Premature CHD was observed in FH individuals without HA. The study of this family established that the HA trait in the family also followed an autosomal dominant mode of inheritance with a pattern of segregation independent from FH. We identified a locus on chromosome 6 linked to elevated HDL-C levels (HA) in this family. Haplotype analysis refined the localization to a 7.32-cM interval (73 to 80 cM from pter) flanked by markers D6S1280 and D6S1275. Parametric 2-point and multipoint analyses yielded maximum LOD scores of 3.05 and 3.17, respectively. This finding was confirmed with a nonparametric multipoint score of 3.78 (P =0.0009). We propose that this locus, linked to elevated HDL-C levels, confers protection against premature CHD within an FH context.

Placental blood leukocytes are functional and phenotypically different than peripheral leukocytes during human labor

Rupture of the fetal membranes during human labor is associated with an inflammatory process localized to the maternal-fetal interface. There is evidence that specific leukocytes subsets are attracted to the choriodecidua, and that after homing they condition a local inflammatory microenvironment, possibly being directly involved in rupture of the membranes. In this study our aim was to compare the phenotypes and function of leukocytes located in the placental intervillous blood with peripheral leukocytes obtained before or after labor, including expression of modulators of inflammation in these cells. Flow cytometry revealed that the proportion of CD14(+) cells is increased in intervillous blood, suggesting the participation of monocytes/macrophages during labor. Real time qRT-PCR showed that at term gestation and particularly during labor, placental blood leukocytes adopt a different expression pattern of pro-inflammatory cytokines than leukocytes in peripheral blood, including IL-1beta and IL-1RA. During labor, both placental and peripheral leukocytes increase their secretion of matrix metalloproteinase-9. Moreover, we showed that placental leukocytes respond differently than peripheral leukocytes to bacterial lipopolysaccharide, secreting differential amounts of TNF-alpha, IL-1beta and IL-6. Finally, a preliminary proteomic characterization of placental leukocytes revealed a significantly higher number of individual proteins than in peripheral leukocytes. Our results support the existence of selective subsets of leukocytes recruited to the maternal-fetal interface that may participate in the triggering of parturition.

NF-κB translocation and endothelial cell activation is potentiated by macrophage-released signals co-secreted with TNF-α and IL-1β

Abstract.Objective and Methods: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-κB in the presence of CM by studying the degradation rate of individual IκB isoforms.Results: We show that, as expected, CM induced NF-κB translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF-α and IL1β naturally present in the CM. However, both the amplitude of NF-κB translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF-α and IL-1β, either separately or together. Our results show that CM induced a faster degradation of the IκB-β and IκB-ɛ isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-κB activity.Conclusions: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF-α mediated endothelial activation.

A novel ARH splice site mutation in a Mexican kindred with autosomal recessive hypercholesterolemia

Autosomal recessive hypercholesterolemia (ARH) is characterized by elevated LDL serum levels, xanthomatosis, and premature coronary artery disease. Three loci have been described for this condition (1p35, 15q25-q26 and 13q). Recently, the responsible gene at the 1p35 locus, encoding an LDL receptor adaptor protein (ARH) has been identified. We studied a Mexican ARH family with two affected siblings. Sequence analysis of the ARH gene (1p35 locus) revealed that the affected siblings are homozygous for a novel mutation (IVS4+2T>G) affecting the donor splice site in intron 4, whereas both the parents and an unaffected sister are heterozygous for this mutation. The IVS4+2T>G mutation results in a major alternative transcript derived from a cryptic splice site, which carries an in-frame deletion of 78 nucleotides in the mature mRNA. The translation of this mRNA yields a mutant protein product (ARH-26) lacking 26 amino acids, resulting in the loss of β-strands β6 and β7 from the PTB domain. This is the first case where a naturally occurring mutant with an altered PTB domain has been identified.

Effect of vitamin E and C supplementation on oxidative damage and total antioxidant capacity in lead-exposed workers.

The molecular response of the antioxidant system and the effects of antioxidant supplementation against oxidative insult in lead-exposed workers has not been sufficiently studied. In this work, antioxidants (vitamin E 400 IU+vitamin C 1g/daily) were supplemented for one year to 15 workers exposed to lead (73 μg of lead/dl of blood) and the results were compared with those on 19 non-lead exposed workers (6.7 μg of lead/dl). Lead intoxication was accompanied by a high oxidative damage and an increment in the erythrocyte antioxidant response due to increased activity of catalase and superoxide dismutase. Antioxidant supplementations decreased significantly the oxidative damage as well as the total antioxidant capacity induced by lead intoxication with reduction of the antioxidant enzyme activities. We conclude that antioxidant supplementation is effective in reducing oxidative damage and induces modifications in the physiopathological status of the antioxidant response in lead-exposed workers.

Glycine suppresses TNF-alpha-induced activation of NF-κB in differentiated 3T3-L1 adipocytes

Glycine strongly reduces the serum levels of pro-inflammatory cytokines and increases the levels of anti-inflammatory cytokines. Recently, glycine has been shown to decrease the expression and secretion of pro-inflammatory adipokines in monosodium glutamate-induced obese (MSG/Ob) mice. It has been postulated that these effects may be explained by a reduction in nuclear factor kappa B (NF-κB) activation. NF-κB is a transcription factor, which is crucial to the inflammatory response. Hasegawa et al. (2011 and 2012) recently reported a glycine-dependent reduction in NF-κB levels. Here, we have investigated the role of glycine in the regulation of NF-κB in differentiated 3T3-L1 adipocytes. The results revealed that pretreatment with glycine interfered with the activation of NF-κB, which has been shown to be stimulated by tumor necrosis factor-alpha (TNF-α). Glycine alone stimulated NF-κB activation in an unusual way such that the inhibitor κB-β (IκB-β) degradation was more significant than that of the inhibitor κB-α (IκB-α) and led to NF-κB complexes comprised of p50 and p65 subunits; IκB-ε degradation did not affect by glycine. These findings suggest that glycine could be used as an alternative treatment for chronic inflammation, which is a hallmark of obesity and other comorbidities, and is characterized by an elevated production of pro-inflammatory cytokines.

Expression and function of utrophin associated protein complex in stretched endothelial cells: dissociation and activation of eNOS.

Several studies have emphasized the relevance of dystrophin-associated protein complex (DAPC) to maintain the vascular function. Previously we postulated the presence of an utrophin associated protein complex (UAPC) in endothelium from umbilical cord vessels. In the present work, we demonstrate that utrophin (UTR) indeed forms a complex, with beta-dystroglycan (DG), epsilon-sarcoglycan (SG), caveolin-1 (cav-1), and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) by co-immunoprecipitation analysis. Additionally, we observed an increment in the protein levels of epsilon-SG, beta-DG, UTR and cav-1 after mechanical stretching. Interestingly, this stimulus also induced eNOS up-regulation, activation and release from the UAPC, and led to a significant increase in nitric oxide (NO) production. Finally, we propose that UAPC in HUVECs may play an important role in the regulation of vascular tone.

Caveolae and non-caveolae lipid raft microdomains of human umbilical vein endothelial cells contain utrophin-associated protein complexes.

Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity.