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Biomedical subjects

Guillermo Villegas

Publications and source records attributed to Guillermo Villegas.

6 recordsLinked to original sources
PubMed2006

Semaphorin 3C regulates endothelial cell function by increasing integrin activity.

Class 3 semaphorins (sema 3) are secreted guidance proteins. Sema 3A expressed by endothelial cells controls vascular morphogenesis through integrin inhibition. Sema 3C is required for normal cardiovascular patterning. Here we examined the potential role of sema 3C as regulator of endothelial cell function in vitro using mouse glomerular endothelial cells (MGEC). We determined that MGEC express sema 3C mRNA and protein and its receptors mRNA. Recombinant sema 3C induced MGEC proliferation 18 +/- 2% above control, as assessed by bromodeoxyuridine (BrdU) incorporation, and reduced starvation-induced apoptosis by 46 +/- 3%, as indicated by an in situ marker of activated caspase 3. Sema 3C increased MGEC adhesion to fibronectin 79 +/- 13% and to collagen 55 +/- 12% as compared with control. Sema 3C-induced MGEC adhesion was prevented by integrin blocking antibodies and involved beta1 integrin serine phosphorylation. Sema 3C-induced MGEC adhesion and proliferation were similar to those induced by vascular endothelial growth factor (VEGF)-A. Sema 3C induced a 44 +/- 11% increase in MGEC directional migration and stimulated MGEC capillary-like network formation on collagen I gels. Collectively, our data indicate that sema 3C promotes glomerular endothelial cell proliferation, adhesion, directional migration, and tube formation in vitro by stimulating integrin phosphorylation and VEGF120 secretion, functions that are similar to VEGF-A and opposite to sema 3A.

Animals↗
PubMed2006

Autocrine VEGF-A system in podocytes regulates podocin and its interaction with CD2AP.

Vascular endothelial growth factor (VEGF-A) signaling is required for endothelial cell differentiation, vasculogenesis, angiogenesis, and vascular patterning. During kidney morphogenesis, podocyte VEGF-A guides endothelial cells toward developing glomeruli. Podocyte VEGF-A expression continues throughout life but its function after completion of development remains unclear. Here, we examined the expression of VEGF-A and its receptors VEGFR1, VEGFR2, NP1, and NP2 in conditionally immortalized mouse podocytes cultured in undifferentiated and differentiated conditions using RT-PCR and Western analysis. VEGF-A secretion was assessed by ELISA and Western analysis. Upon podocyte differentiation, VEGF-A protein expression and secretion increased threefold. Differentiated podocytes expressed eightfold higher VEGFR2 mRNA levels than undifferentiated podocytes, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels were similar. We examined the regulation and function of the VEGF-A system by exposing differentiated podocytes to recombinant VEGF(165) (20 ng/ml) or control media for 24 h. VEGF(165) induced a twofold increase in VEGFR2 mRNA and protein levels, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels remained unchanged. VEGF(165) induced VEGFR2 phosphorylation. VEGF(165) reduced podocyte apoptosis approximately 40%, whereas anti-VEGFR2 neutralizing antibody enhanced it twofold. We determined that VEGF-A signaling regulates slit diaphragm proteins by inducing a dose-response podocin upregulation and increasing its interaction with CD2AP. The data indicate that podocytes in culture have a functional autocrine VEGF-A system that is regulated by differentiation and ligand availability. VEGF-A functions in podocytes include promoting survival through VEGFR2, inducing podocin upregulation and increasing podocin/CD2AP interaction.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Autocrine and paracrine functions of vascular endothelial growth factor (VEGF) in renal tubular epithelial cells.

BACKGROUND: VEGF secreted by organ parenchymal cells controls vascularization by recruiting endothelial cells and supporting their proliferation. In the developing kidney VEGF-expressing epithelial cells also express VEGF receptors. We showed that VEGF stimulates tubulogenesis in addition to promoting vascularization in metanephric explants. Since explants are grown in serum-free media and are not perfused, we hypothesized that VEGF secreted by renal epithelia may induce their proliferation in an autocrine manner and chemoattract endothelial cells. METHODS: To test these hypotheses, we analyzed VEGF-mediated responses in vitro using several renal epithelial cell lines [immortalized rat proximal tubular cells (IRPT), transformed mouse proximal tubular cells (tsMPT), and normal rat kidney cells (NRK-52E)] expressing VEGF receptors (VEGFR). RESULTS: We demonstrated that VEGFR-2 phosphorylates upon human recombinant VEGF (rhVEGF) exposure, indicating that VEGFR-2 is the signaling receptor. All three cell lines secreted VEGF into the media as indicated by enzyme-linked immunosorbent assay (ELISA) and Western blotting. We showed that these tubular epithelial cells chemoattract endothelial cells when cocultured in vitro and that the chemoattraction is abolished by anti-VEGF neutralizing antibody. rhVEGF (10 ng/mL) induced a mitogenic effect similar to 10% fetal bovine serum (FBS) as assessed by H(3)-thymidine incorporation and elicited 30% decrease in apoptosis as determined by annexin V-fluorescein isothiocyanate (FITC) staining. CONCLUSION: These in vitro studies indicate that (1) tubular epithelial cells chemoattract endothelial cells in a paracrine fashion by secreting VEGF, and (2) VEGF stimulates proliferation and promotes survival of renal epithelial cells in an autocrine manner via VEGFR-2. Taken together, our results suggest that VEGF supports the growth of renal epithelia in addition to mediating kidney vascularization.

Animals↗
PubMed2003

[Design of an impedance plethysmograph for reactive hyperemia evaluation in the forearm].

Impedance pletismography is based on the evaluation of the voltage change that occur as a consequence of blood flow variations in a particular tissue section. Current applied for the procedure should be alternating, weak and of high frequency to avoid electrically excitable cells stimulation, such as muscles and nerves. Blood volume changes can be measured by this method and has been applied in the diagnosis of deep venous thrombosis of limbs. An impedance meter was designed and built to be used in the forearm of patients. It consists of a voltage stabilizer TPS 76150 with simultaneous ECG recording (DII lead). Injected signal had 50 KHz (Wien's bridge) and registered through two operational amplifiers LM 3080 (1 mA). Signal was demodulated and amplified. Electrodes used were made of silver strips with conductance improved by conductivity gel, fixed by elastic rubber strips. Circuit wiring and equisition software was developed at the Bioengineering Department of Simón Bolívar University. Fifteen healthy subjects, age range 18-30 years old, were submitted to noninvasive forearm blood flow evaluation with the already described electronic device. Brachial artery occlusions were made with a cuff at subdiastolic, supradiastolic and suprasystolic blood pressures, for 60 and 90 seconds and then this occlusion was abruptly released. The curve was displayed and recorded in a portable PC (laptop). During suprasystolic occlusion the impedance observed curve showed a progressive increased until stabilization. When occlusion was released a sudden peak appeared which corresponds to reactive hyperemia of the forearm. This peak has been associated to the secretion of endothelial vasodilatory substances. In conclusion, this device is suitable to be used in clinical settings for the evaluation of reactive hyperemia and potentially useful in diagnosis of deep veins occlusions.

Adult↗
PubMed2002

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney.

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.

Animals↗
PubMed2002

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney.

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.

Humans↗