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1. Understanding the
mechanisms of angiogenesis and vascular regression to find out more effective
strategies for antiangiogenic therapies in cancer.
We use
immature testis as a model of very active angiogenesis and testicular waning,
induced by diethylstilbestrol treatment, as a model of vascular regression. We
have observed high expression of VEGF and Ang-2 in developing immature testis,
while the expression of both genes is minimal in adult quiescent mature testis.
Testicular regression is accompanied by high expression of Ang-2 and very low
expression of VEGF. Our observations are in accordance with the proposed
function of Ang-2 inducing angiogenesis in the presence of VEGF and vascular
regression in its absence. We have also reported novel forms of Ang-2: Ang-2B,
with a truncated aminoterminal domain resulting from alternative splicing of the
gene, and Ang-2C that differ from Ang-2A in the absence of the second exon. Both
isoforms codify proteins with a truncated coiled-coil domain and are
differentially expressed in various chicken tissues. To identify the regulatory
mechanisms involved in the differential expression of the endothelial
proapoptotic protein angiopoietin-2 we work in the functional characterization
of the Angiopoietin-2 gene promoter sequences and in the identification of the
5’UTR and 3’UTR regulatory sequences of the Angiopoietin-2 transcripts. We are
mainly interested in the role of hypoxia, NFkB and p53, among other factors. The
results obtained in these studies could provide the basis to find more effective
strategies for antiangiogenic therapies in cancer.
Project BMC2001-3391"Ministerio de
Ciencia y Tecnología"
2.
Understanding the mechanisms of ocular angiogenesis to find out new
pharmacological treatments for diabetic retinopathy.
The primary goal of our project is to study the expression of
VEGF, Angiopoietin-1 and Angiopoietin-2 in a new model of ocular angiogenesis.
Diabetes compromises the survival of vascular cells, which leads to retinal
ischemia and sight-threatening unregulated angiogenesis. VEGF is responsible for
neovascularization and increase in vascular permeability, while angiopoietins
play an essential role modulating apoptosis of endothelial cells and vascular
permeability. We have contributed to these studies with the expression of VEGF
and Angiopoietin-2 in a model of angiogenesis and vascular regression, and with
the genomic structure of the Angiopoietin-2 gene and its alternative splicing
that gives rise to three different isoforms of Angiopoietin-2 mRNA: Ang-2A, Ang-2B
and Ang-2C. Now, our proposal is to investigate the coordinate expression of
VEGF and the angiopoietins in an unexplored model of ocular angiogenesis in the
chick embryo under different experimental conditions. In addition, we work in
the functional characterization of the Angiopoietin-2 gene promoter sequences
and in the identification of the 5’UTR and 3’UTR regulatory sequences of the
Angiopoietin-2 transcripts. The potential impact of our results is based in the
need of new pharmacological treatments for diabetic retinopathy based in
maintaining an adequate balance between VEGF and angiopoietins. Present
treatments are clearly limited for a disease that is a leading cause of
blindness (5% of the population are diabetic and 5% of diabetic progress to
legal blindness).
Project
"Fundació la Marató de
TV3" 1998.
Additional
information (in Catalan)
3. Understanding the mechanisms of coordinate expression of VEGF and angiopoietins to find out more effective strategies for antiangiogenic therapies in inflammatory diseases.
The pain, swelling, and tissue destruction of inflammatory diseases results from many proinflammatory mediators induced by the transcription factor NFkB. This transcription factor also induces angiopoietin-2 expression. While angiopoietin-1 is an anti-permeability and anti-inflammatory protein that is critical for the stabilization of blood vessels, angiopoietin-2, its natural antagonist, disrupts blood vessels structure. We found high expression of vascular endothelial growth factor (VEGF) and angiopoietin-2 in rheumatoid arthritis sinovial tissue. These molecules can be centrally involved in the angiogenic process and in the increase of vascular permeability characteristics of rheumatoid arthritis. In the absence of inflammatory mediators, in quiescent cells, the transcription factor NFkB is bound, in the cytoplasm, to an inhibitor protein, IkB. Upon inflammatory stimulation, the inhibitor is destroyed by the ubiquitin-proteasome pathway and the transcription factor NFkB is released and translocates into the nucleus. Proteasome inhibitors can block the degradation of IkB and, in consequence, the activation of NFkB. Through these mechanisms, proteasome inhibitors have the potential of anti-inflammatory and antiangiogenic properties; however, the stabilization of other transcription factors after the inhibition of the proteasome may increase the expression of VEGF and angiopoietin-2. Using endothelial cell cultures and a new model that allows the study of the coordinate expression of VEGF, angiopoietins and their receptors, we plan to determine the potential anti-inflammatory and antiangiogenic properties of proteasome inhibitors.
Project FIS N-2001-FS1548
4. Mechanisms Involved in Male Fertility: Role of Carbonic Anhydrase II.
Intracellular and extracellular sources of bicarbonate are essential for sperm motility, sperm binding to the zona pellucida and the acrosome reaction. In addition, bicarbonate stimulates cAMP accumulation in spermatids that can be essential for normal differentiation into spermatozoa. Carbonic anhydrase II, catalysing the synthesis of bicarbonate must play a significant role in the production of functional male gametes. We have studied the expression of carbonic anhydrase II (Car2) during mouse spermatogenesis. Both Car2 mRNA and the translated protein were first detectable by day 24, when early spermatids (steps 1–8) become abundant within the seminiferous epithelium. The mRNA and the protein accumulated in adult testis enriched in elongating and elongated spermatids. The protein is also abundant in spermatozoa obtained from the epididymis. We have studied the primary structure of Car2 testicular transcripts that show a 5’ UTR larger and a 3’UTR shorter than the corresponding somatic sequences. Polysomal gradient analysis of carbonic anhydrase II transcripts isolated from adult mouse testis and kidney revealed different translation potential: most of the testicular transcripts were present in the non-polysomal fractions, whereas a considerable fraction of kidney transcripts were polysome-associated. These results suggest that specific transcriptional and post-transcriptional mechanisms regulate the expression of carbonic anhydrase II during mammalian spermatogenesis. Further studies on these mechanisms and on the effect of Car2 inhibitors in the differentiation of spermatids and the function of spermatozoa, may provide new basis for the control of male fertility.
5. Mechanisms Involved in Male Fertility: Role of a new acrosomal protein.
The interaction of sperm and egg is mediated primarily by gamete surface proteins. An essential task in the study of sperm-egg interaction is the characterization of new gamete specific surface proteins. We have characterized a chicken testis specific transcript, with mouse and human homologues, which is highly expressed and polyadenylated in postmeiotic stages of spermatogenesis. We have prepared a polyclonal antibody against a predicted peptide sequence of the protein. The antibody recognizes the recombinant protein obtained from the transcript and has allowed the localization of the protein in the acrosome of spermatozoa by immunofluorescence. Further investigation on the function of this new acrosomal protein may provide new basis for the control of male fertility.
6. Mechanisms Involved in Male Fertility: Resistance/Vulnerability to Apoptosis in Spermatogenic Cells.
An essential feature of the homeostasis of a cell or an organism is the rapid expression of genes whose products play a major role in protecting cells against stress. When cells or whole organisms are exposed to elevated temperatures, they respond by synthesizing a small number of highly conserved proteins, the heat shock proteins (HSP). Using a model of spermatogenic cells that develop at 40-41ºC we have shown that upon heat stress these cells respond in vitro with high expression and polyadenylation of HSP70, ubiquitin and glyceraldehyde-3-phosphate dehydrogenase. New isoforms of the three mRNA are expressed in meiotic and postmeiotic spermatogenic cells. In our model, aspirin, in the range 3-10 mM decreases the expression of HSP70 in unstressed and stressed testicular cells in striking contrast with the effect observed in other tissues as liver. After testicular regression, induced by diethylstilbestrol treatment, constitutive expression of HSP70 decreases. More surprisingly, in the regressed testis the temperature that induces HSP70 in vitro decreases. This induction is completely inhibited in the presence of 6 mM aspirin. Our observations suggest that the decline in the expression of HSP70 upon aspirin treatment may increase the vulnerability of spermatogenic cells to apoptosis.
7. Mechanisms Involved in Male Fertility: Unique Transcriptional and Post-transcriptional Regulation during Spermatogenesis.
Specific expression of many genes during spermatogenesis is accomplished by transcriptional and post-transcriptional regulation. We have studied during spermatogenesis the expression of several genes that undergo alternative initiation, alternative splicing, polyadenylation and alternative use of polyadenylation signals: polyubiquitin genes UbI and UbII, carbonic anhydrase II, glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase B and HSP70. The use of alternative promoters, in addition to opening the possibility for new potential mechanisms of transcription, produces extended 5’UTRs that could stabilize the message and ensure delayed translation. Several mechanisms that block premature translation may be considered: (1) presence of hairpin structures in lengthened 5’UTR; (2) potential binding of transcriptional regulators that may recognize sequences on the testis-specific 5’UTR, and (3) absence of possible destabilizing sequences in the shortened 3’UTR. Delayed translation could be necessary at the end of spermatogenesis when transcription is no longer active. Translational repression also can be an efficient way to localize and concentrate proteins in subcellular domains. Transport of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination has been reported during oogenesis. We are also interested in the mechanisms that regulate the coordinate RNA processing during spermatogenesis. We have studied the expression of a family of RNA-binding proteins referred as STAR (signal transduction and activator of RNA) that link signal transduction to post-transcriptional control. Messages of 7, 6 and 5 kb of the QKI gene, a member of the STAR family, are differentially expressed during spermatogenesis. The STAR protein QKI-6 is a translational repressor. The protein QKI-7 is a potent inducer of apoptosis, while QKI-5 and QKI-6 suppress the apoptosis induced by QKI-7. We have described heterogeneity in the coding region of QKI-5, the predominant form in adult testis, which gives rise to four possible isoforms of the protein.
8. Mechanisms Involved in the Integrity of the Genome of the Male Gamete.
The stability of genetic information of germ line cells is of vital importance for reproduction and development. Undamaged gamete genomes ensure faithful transmission of genetic information to the next generation. One of the most dangerous DNA damages is the double-strand breaks (DSB). DSB are generated to initiate recombination between homologous chromosomes during meiosis. In addition, we have demonstrated that the structural changes that chromatin undergoes in late spermatids are associated with three main events: 1) Histone hyperacetylation, 2) Ubiquitination of histones, and 3) Changes in DNA topology mediated by the activity of DNA topoisomerase II. Topoisomerase II activity transiently breaks both strands of the DNA molecule. When topoisomerase II is inhibited by teniposide, DNA cleavage is maximal in late spermatids in comparison to previous stages of spermatogenesis, including meiotic recombination. Although topoisomerase II is able to both create and ligate the double strand breaks of DNA, other factors may be involved in the re-joining of the strand breaks. We are interested in the relationship between ubiquitin conjugation to nuclear proteins and DNA repair mechanisms during spermatogenesis.
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